presentation in nucleus

• DNA Replication
• Active Transport
• Cellular Receptors
• Endocytosis and Exocytosis
• Enzyme Inhibition
• Enzyme Kinetics
• Protein Structure
• Transcription of DNA
• Translation of DNA
• Anaerobic Respiration
• Electron Transport Chain
• Gluconeogenesis
• Calcium Regulation
• External Balance of Potassium
• Internal Balance of Potassium
• Sodium Regulation
• Cell Membrane
• Endoplasmic Reticulum
• Golgi Apparatus
• Mitochondria
• Blood Vessels
• Cellular Adaptations
• Epithelial Cells
• Muscle Histology
• Structure of Glands
• Control of Stroke Volume
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• Cardiac Cycle
• Cardiac Pacemaker Cells
• Conduction System
• Contraction of Cardiac Muscle
• Ventricular Action Potentials
• Blood Flow in Vessels
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• Cardiac Muscle
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• Transport of Carbon Dioxide in the Blood
• Ventilation-Perfusion Matching
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• Respiratory Regulation of Acid-Base Balance
• Responses of The Respiratory System to Stress
• Regulation of Saliva
• Secretion of Saliva
• Gastric Acid Production
• Gastric Mucus Production
• Digestion and Absorption
• Histology and Cellular Function of the Small Intestine
• Absorption in the Large Intestine
• Large Intestinal Motility
• Bilirubin Metabolism
• Carbohydrate Metabolism in the Liver
• Lipid Metabolism in the Liver
• Protein and Ammonia Metabolism in the Liver
• Storage Functions of the Liver
• Bile Production
• Function of The Spleen
• Exocrine Pancreas
• Somatostatin
• Proximal Convoluted Tubule
• Loop of Henle
• Distal Convoluted Tubule and Collecting Duct
• Storage Phase of Micturition
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• Renin-Angiotensin-Aldosterone System
• Urinary Regulation of Acid-Base Balance
• Water Filtration and Reabsorption
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• Pain Pathways
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• Descending Tracts
• Lower Motor Neurones
• Muscle Stretch Reflex
• Upper Motor Neurones
• Aqueous Humour
• Ocular Accommodation
• Thyroid Gland
• Parathyroid Glands
• Adrenal Medulla
• Zona Glomerulosa
• Zona Fasciculata
• Zona Reticularis
• Endocrine Pancreas
• The Hypothalamus
• Anterior Pituitary
• Posterior Pituitary
• White Blood Cells – Summary
• Barriers to Infection
• Infection Recognition Molecules
• Phagocytosis
• The Complement System
• Antigen Processing and Presentation
• Primary and Secondary Immune Responses
• T Cell Memory
• Acute Inflammation
• Autoimmunity
• Chronic Inflammation
• Hypersensitivity Reactions
• Immunodeficiency
• Types of Immunity
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• Blood Groups
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• Erythropoiesis
• Iron Metabolism
• Mononuclear Phagocyte System

Original Author(s): Aarushi Khanna Last updated: 29th October 2023 Revisions: 20

• 1.1 Nuclear envelope
• 1.2 Nuclear lamina
• 1.3 Chromatin
• 1.4 Nucleolus

The nucleus is a membrane bound organelle found in the majority of eukaryotic cells. It is the largest organelle of the eukaryotic cell, accounting for around 10% of its volume. It houses the genome, and through gene expression , it co-ordinates the activities of the cell.

In this article, we will consider the structure and function of the nucleus.

The nucleus is a relatively large and spherical membrane-bound organelle . The nucleus itself is comprised of distinct components, and understanding their structure allows a deeper understanding of their function.

Fig 1 – The nucleus

Nuclear envelope

The nucleus is completely surrounded by the nuclear envelope.  This consists of both an  inner  and  outer membrane which run parallel to each other. The envelope is perforated by small gaps known as the nuclear pores.  These pores are around 100nm wide in true diameter, however due to the presence of central regulatory proteins the true size of the gap is around 9nm .

This small size controls the passage of molecules into and out of the nucleus. Larger molecules such as larger proteins and nucleic acid are unable to pass through these pores, and so the function of the nuclear envelope is to selectively separate the contents of the nucleus from that of the cytoplasm.

Nuclear lamina

Mechanical support for the nucleus is provided by the nuclear  lamina. This is a protein mesh, which is more organised on the internal surface on the nucleus than on the cytoplasmic surface.

Chromatin describes DNA that is complexed with proteins. The primary protein components of chromatin are histones,  which are highly basic proteins that associate readily with DNA. Histones combined with DNA form nucleosomes , which are the subunit of chromatin. Specifically, a nucleosome describes a segment of DNA associated with 8 histone proteins. By associating with histones, DNA is more compact and able to fit into the nucleus.

Chromatin can exist as either euchromatin or heterochromatin. Euchromatin is the form of chromatin present during gene expression, and has a characteristic ‘ beads on a string’  appearance. It is activated by  acetylation.  In contrast, heterochromatin  is the ‘inactive’ form, and is densely packed. On electron microscopy, euchromatin stains lighter than heterochromatin which reflects their relative densities.

Fig 2 – Schematic diagram of euchromatin and heterochromatin

The nucleolus is the site of ribosome and ribosomal RNA production. On microscopy, it appears as a large dense spot within the nucleus. After a cell divides, a nucleolus is formed when chromosomes are brought together into nucleolar organising regions. During cell division, the nucleolus disappears.

The information above can be simplified into three key functions:

• Cell compartmentalisation: The presence of a selectively permeable nuclear envelope separates the contents of the nucleus from that of the cytoplasm.
• Gene expression:  Gene expression first requires transcription , which is the process by which DNA is transcribed into mRNA. As the nucleus is the site of transcription, proteins within the nucleus play a key role in regulating the process.
• Processing of pre-mRNA:  Newly synthesised mRNA molecules are known as pre-mRNA. Before they exit the nucleus, they undergo a process known as  post-transcriptional  modification where molecules are added or removed from the structure.

Fig 3 – Nucleus with the cisternae of a continuous endoplasmic reticulum highlighting its main features.

Chromatin can exist as either euchromatin or heterochromatin. Euchromatin is the form of chromatin present during gene expression, and has a characteristic ' beads on a string'  appearance. It is activated by  acetylation.  In contrast, heterochromatin  is the 'inactive' form, and is densely packed. On electron microscopy, euchromatin stains lighter than heterochromatin which reflects their relative densities.

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Cell nucleus

Author: Rachel Baxter, MSc • Reviewer: Uruj Zehra, MBBS, MPhil, PhD Last reviewed: September 19, 2023 Reading time: 11 minutes

The cell nucleus is the most noticeable organelle within the  eukaryotic cell , and perhaps the most important and defining feature of the eukaryotic cells. Most of the genetic material (DNA) is contained in the nucleus, while a small amount of it is found in mitochondria. The majority of human cells have a single nucleus, although there are several cell types that have multiple nuclei (e.g. osteoclasts ) or don't have a nucleus at all ( erythrocytes ).

Since the structure of the nucleus is an important milestone for understanding citology and histology , this article will discuss the structure and function of the cell nucleus in an easy to read mode. 

Shape and appearance

Nuclear envelope, nuclear lamina, chromosomes, nucleoplasm, laminopathies, nucleus abnormalities, nucleolus abnormalities.

The nucleus is normally around 5-10 μm in diameter in many multicellular organisms, and the largest organelle in the cell. The smallest nuclei are approximately 1 μm in diameter and are found in yeast cells.

Mostly the shape of the nucleus is spherical or oblong . Usually cells have one nucleus but many at times there are multinucleated cells. Multinucleation in cells may be due to karyokinesis (when cell undergoes nuclear division) or when cells fuse to form syncytium, like in mature muscle cells .

The nucleus has very important roles to play. As it contains genetic material , it coordinates cell activities like protein synthesis and cell division . Anatomically the nucleus is made up of several components: nuclear envelope, nuclear lamina, nucleolus , chromosomes, nucleoplasm are some of these components. 

All of these components work together in order for the nucleus to accomplish all of its functions. Namely, these functions are: 

• control of the genetical information of the cell and thus the heredity characteristics of an organism,
• control of the protein and enzyme synthesis
• control of cell division and cell growth
• storage of DNA, RNA and ribosome
• regulation of the transcription of the mRNA to protein
• production of ribosomes

When a cell is histologically stained , the nucleus normally appears as a large, dark organelle, mostly at or near the centre of a cell . 

Test your knowledge on the structure and components of the eukaryotic cell with our quiz:

Structure of the nucleus

As its name suggests, the nuclear envelope surrounds the nucleus, separating it from the cell's cytoplasm. It is a double membrane . Each membrane is a phospholipid bilayer associated with proteins, and the two membranes are divided by 20 to 40 nm of space. The two membranes of the nuclear envelope are often referred to as the inner and outer nuclear membranes. The outer membrane is continuous with the cell’s endoplasmic reticulum, and therefore the space between the inner and outer nuclear membranes links to the lumen of the endoplasmic reticulum. Like the endoplasmic reticulum, the outer nuclear membrane has ribosomes attached to it. Contrastingly, the inner membrane of the nuclear envelope is attached to proteins that are specific to the nucleus, and therefore found nowhere else.

Studying histology will be a lot easier once you master the examining of the histology slides. Check out our histology guide on how to learn this important skill and make your student life a lot easier!

The nuclear envelope is perforated with tiny nuclear pores with diameters of around 100 nm. The inner and outer membranes of the envelope are continuous around the pores. Each pore is lined with a structure of 50 to 100 different proteins known as the nuclear pore complex . These pore complexes regulate the movements of macromolecules, RNAs and proteins into and out of the nucleus. This movement of molecules is known as nuclear transport . Small molecules can move passively through the pores, but larger molecules, including RNAs and many proteins, are too large for this and must move actively . During this active process, they are selectively recognised and transported in one specific direction. The traffic of RNAs and proteins through the nuclear pore complex is particularly important, as they play a role in gene expression.  

The inner nuclear membrane is internally lined by protein filaments meshwork organised in a net-like fashion, called nuclear lamina. The proteins that make up the nuclear lamina are known as lamins , which are intermediate filament proteins. These support the nuclear envelope, ensuring that the overall shape and structure of the nucleus is maintained.

In addition to lamins there is another set of membrane proteins called lamina associated proteins , which help to mediate the interaction between the lamina and inner nuclear membrane. The nuclear lamina, along with protein fibers called the nuclear matrix , is also thought to aid in the organisation of genetic material, allowing it to function more efficiently.

The DNA of a cell is found within the nucleus. It is organised into units known as chromosomes, each containing a long DNA molecule which is associated with various proteins. The DNA coils around protein complexes called nucleosomes , formed of proteins called histones , making it easier for the chromosome to fit inside the nucleus.

The mass of DNA and proteins inside a chromosome is referred to as chromatin . When a cell is not dividing, it is difficult to see the chromosomes within a cell, even when it is stained. However, when DNA prepares and begins to divide, the chromosomes can be visualised more clearly. During the metaphase of mitosis, the chromosomes become visible as they prepare to divide by aligning with one another. The chromosomes are copied, forming sister chromosomes known a chromatids . Human cell nuclei contain 46 chromosomes, although gamete nuclei contain 23. The whole of the nucleus is not filled by chromatin material, in fact, there are  chromatin free regions called interchromosomal domains containing poly RNAs.

When a nucleus is not dividing, a structure called a nucleolus becomes visible. In fact, it is the most prominent structure within the nucleus. Usually there is only a single nucleolus present, but some nuclei have multiple nucleoli. It is a mass of granules and fibers attached to chromatin.

The nucleolus is important because it is the site of ribosomal RNA (rRNA) production . Inside the nucleolus, rRNA molecules are combined with proteins to form ribosomes . The nucleolus is involved in rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus is not surrounded by a membrane, but it has a unique density , separating it from the surrounding nucleoplasm, and allowing it to be visualised under a microscope. As well as being involved in ribosomal biogenesis, the nucleolus is thought to have other roles, as it contains a number of proteins unrelated to rRNA and ribosome synthesis. It is thought be play a role in activities such as DNA damage repair, cell cycle regulation and RNA editing.

Nucleoplasm is similar to the cytoplasm of a cell, in that it is semi-liquid, and fills the empty space in the nucleus. It is a form of protoplasm and surrounds the chromosomes and nucleoli inside the nucleus. It also has various proteins and enzymes dissolved within it.

Nuclear bodies can be found in the nucleoplasm, and these include structures such as Cajal bodies, Gemini bodies, and Polycomb bodies. Cajal bodies are between 0.3-1.0 µm in diameter, and can be found in proliferating cells such as embryonic and cancerous cells, as well as in cells which have a high metabolic rate, such as neurons . Sometimes referred to as coiled bodies , Cajal bodies are bound to nucleoli by specialised proteins called coilin proteins . Having these proteins concentrated within Cajal bodies improves the efficiency of nuclear processes such as the modification and assembly of UsnRNPs, which can become spliceosomes .

Adjacent to Cajal bodies, Gemini bodies or “Gems” can be found. These comprise Gemin 2 protein and motor neuron s gene product (SMN), which are involved in the assembly and maturation of snRNPs.

Solidify your knowledge about the nucleus and structure of the eukaryotic cell with our study unit.

Clinical notes

Mutations in the genes that code for lamins in the nucleus can lead to a number of rare genetic disorders, normally due to a change in the abundance of lamins in the nuclei. Collectively these diseases are known as laminopathies. These diseases include autosomal dominant Emery-Dreifuss muscular dystrophy, Dunnigan-type familial partial lipodystrophy, as well as developmental and aging disorders.

Additionally, certain blood disorders can lead to abnormalities in the nuclei, meaning that analysis of the shape and structure of nuclei in blood cells can lead to diagnoses. For example, Wilson’s disease leads to an increase in glycogen in the nuclei, whilst acute myeloid leukaemia causes nuclei to become cup-shaped.

Moreover, abnormalities in the nucleoli can lead to some forms of rare hereditary disease , as well as degenerative diseases such as Huntington’s and Alzheimer’s. Several diseases can also result from changes in the nuclear envelope. These include cardiomyopathy and muscular dystrophy.

References:

• G. M. Cooper: The Cell: A Molecular Approach, 2nd edition, Sinauer Associates (2000)
• G. Morris: The cajal body. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research (2008), volume 1783, issue 11, pp.2108-2115.
• H. Worman and G. Bonne: “Laminopathies”: a wide spectrum of human diseases. Experimental cell research (2007), volume 313, issue 10, pp.2121-2133.
• J. B. Reece, L. A Urry, M. L. Cain et al.: Campbell Biology, International Edition, Pearson (2011), p. 148
• Y. Mao, B. Zhang, and D. Spector: Biogenesis and function of nuclear bodies. Trends in Genetics (2011), volume 27, issue 8, pp. 295-306.
• Y. W. Lam, L. Trinkle-Mulcahy, A. I. Lamond: The nucleolus. Journal of Cell Science (2005), volume 118, p. 1335-1337

Illustrator:

• Mitosis - Photo credit  kat m research via Visual Hunt / CC BY-SA

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Microbe Notes

Nucleus: Definition, Structure, Parts, Functions, Diagram

The cell nucleus​ is a membrane-bound structure that contains the cell’s hereditary information and controls the cell’s growth and reproduction.

It is the command center of a eukaryotic cell and is commonly the most prominent organelle in a cell accounting for about 10 percent of the cell’s volume.

In general, a eukaryotic cell has only one nucleus. However, some eukaryotic cells are enucleated cells (without a nucleus), for example, red blood cells (RBCs); whereas, some are multinucleate (consists of two or more nuclei), for example, slime molds .

The nucleus is separated from the rest of the cell or the cytoplasm by a nuclear membrane.

As the nucleus regulates the integrity of genes and gene expression, it is also referred to as the control center of a cell.

Table of Contents

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Nucleus Structure

The structure of a nucleus encompasses the nuclear membrane, nucleoplasm, chromosomes, and nucleolus.

Nuclear Membrane

• The nuclear membrane is a double-layered structure that encloses the contents of the nucleus. The outer layer of the membrane is connected to the endoplasmic reticulum.
• Like the cell membrane, the nuclear envelope consists of phospholipids that form a lipid bilayer.
• The envelope helps to maintain the shape of the nucleus and assists in regulating the flow of molecules into and out of the nucleus through nuclear pores. The nucleus communicates with the remaining of the cell or the cytoplasm through several openings called nuclear pores.
• Such nuclear pores are the sites for the exchange of large molecules (proteins and RNA) between the nucleus and cytoplasm.
• A fluid-filled space or perinuclear space is present between the two layers of a nuclear membrane.

Nucleoplasm

• Nucleoplasm is the gelatinous substance within the nuclear envelope.
• Also called karyoplasm, this semi-aqueous material is similar to the cytoplasm and is composed mainly of water with dissolved salts, enzymes, and organic molecules suspended within.
• The nucleolus and chromosomes are surrounded by nucleoplasm, which functions to cushion and protect the contents of the nucleus.
• Nucleoplasm also supports the nucleus by helping to maintain its shape. Additionally, nucleoplasm provides a medium by which materials, such as enzymes and  nucleotides  (DNA and RNA subunits), can be transported throughout the nucleus. Substances are exchanged between the cytoplasm and nucleoplasm through nuclear pores.
• Contained within the nucleus is a dense, membrane-less structure composed of RNA and proteins called the nucleolus.
• Some of the eukaryotic organisms have a nucleus that contains up to four nucleoli.
• The nucleolus contains nucleolar organizers, which are parts of chromosomes with the genes for ribosome synthesis on them. The nucleolus helps to synthesize ribosomes by transcribing and assembling ribosomal RNA subunits. These subunits join together to form a ribosome during protein synthesis.
• The nucleolus disappears when a cell undergoes division and is reformed after the completion of cell division.

Chromosomes

• The nucleus is the organelle that houses chromosomes.
• Chromosomes consist of DNA, which contains heredity information and instructions for cell growth, development, and reproduction.
• Chromosomes are present in the form of strings of DNA and histones (protein molecules) called chromatin. 
• When a cell is “resting” i.e. not dividing, the chromosomes are organized into long entangled structures called chromatin.
• The chromatin is further classified into heterochromatin and euchromatin based on the functions. The former type is a highly condensed, transcriptionally inactive form, mostly present adjacent to the nuclear membrane. On the other hand, euchromatin is a delicate, less condensed organization of chromatin, which is found abundantly in a transcribing cell.

Besides the nucleolus, the nucleus contains a number of other non-membrane-delineated bodies. These include Cajal bodies, Gemini of coiled bodies, polymorphic interphase karyosome association (PIKA), promyelocytic leukemia (PML) bodies, paraspeckles, and splicing speckles.

Nucleus Functions

The nucleus provides a site for genetic transcription that is segregated from the location of translation in the cytoplasm, allowing levels of gene regulation that are not available to prokaryotes. The main function of the cell nucleus is to control gene expression and mediate the replication of DNA during the cell cycle.

• It controls the hereditary characteristics of an organism.
• The organelle is also responsible for protein synthesis, cell division, growth, and differentiation.
• Storage of hereditary material, the genes in the form of long and thin DNA (deoxyribonucleic acid) strands, referred to as chromatin.
• Storage of proteins and RNA (ribonucleic acid) in the nucleolus.
• The nucleus is a site for transcription in which messenger RNA (mRNA) are produced for protein synthesis.
• During the cell division, chromatins are arranged into chromosomes in the nucleus.
• Production of ribosomes (protein factories) in the nucleolus.
• Selective transportation of regulatory factors and energy molecules through nuclear pores.

Nucleus Structure Free Worksheet

Nucleus FAQs

Where is the nucleus found.

Nucleus is found in the center of the cell.

Where is the Nucleolus found?

The nucleolus is found within the nucleus.

• Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Chapter 8, The Nucleus. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9845/
• Lammerding J. Mechanics of the nucleus.  Compr Physiol . 2011;1(2):783-807. doi:10.1002/cphy.c100038.
• Pederson T. The nucleus introduced.  Cold Spring Harb Perspect Biol . 2011;3(5):a000521. Published 2011 May 1. doi:10.1101/cshperspect.a000521.
• Guo T, Fang Y. Functional organization and dynamics of the cell nucleus.  Front Plant Sci . 2014;5:378. Published 2014 Aug 12. doi:10.3389/fpls.2014.00378.
• Zwerger M, Ho CY, Lammerding J. Nuclear mechanics in disease.  Annu Rev Biomed Eng . 2011;13:397-428. doi:10.1146/annurev-bioeng-071910-124736.
• Gerbi SA. The Nucleus.  Nucleus . 2011;2(2):84-86. doi:10.4161/nucl.2.2.15006.
• Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution & Ecology (1 ed.). S .Chand and company Ltd.
• Smith, C. M., Marks, A. D., Lieberman, M. A., Marks, D. B., & Marks, D. B. (2005). Marks’ basic medical biochemistry: A clinical approach. Philadelphia: Lippincott Williams & Wilkins.
• Alberts, B. (2004). Essential cell biology. New York, NY: Garland Science Pub.

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The Cell Nucleus

Definition, Structure, and Function

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The cell nucleus​ is a membrane-bound structure that contains a cell's hereditary information and controls its growth and reproduction. It is the command center of a eukaryotic cell and is usually the most notable cell organelle in both size and function.

The key function of the nucleus is to control cell growth and multiplication. This involves regulating gene expression, initiating cellular reproduction, and storing genetic material necessary for all of these tasks. In order for a nucleus to carry out important reproductive roles and other cell activities, it needs proteins and ribosomes.

Protein and Ribosome Synthesis

The nucleus regulates the synthesis of proteins in the cytoplasm through the use of messenger RNA (mRNA). Messenger RNA is a transcribed DNA segment that serves as a template for protein production. It is produced in the nucleus and travels to the cytoplasm through the nuclear pores of the nuclear envelope, which you'll read about below. Once in the cytoplasm, ribosomes and another RNA molecule called transfer RNA work together to translate mRNA in order to produce proteins.

Physical Characteristics

The shape of a nucleus varies from cell to cell but is often depicted as spherical. To understand more about the role of the nucleus, read about the structure and function of each of its parts.

Nuclear Envelope and Nuclear Pores

The cell nucleus is bound by a double membrane called the nuclear envelope . This membrane separates the contents of the nucleus from the cytoplasm , the gel-like substance containing all other organelles. The nuclear envelope consists of phospholipids that form a lipid bilayer much like that of the cell membrane. This lipid bilayer has nuclear pores that allow substances to enter and exit the nucleus, or transfer from the cytoplasm to the nucleoplasm.

The nuclear envelope helps to maintain the shape of the nucleus. It is connected to the endoplasmic reticulum (ER) in such a way that the internal chamber of the nuclear envelope is continuous with the lumen, or inside, of the ER. This also allows the transfer of materials as well.

The nucleus houses chromosomes containing DNA. DNA holds heredity information and instructions for cell growth, development, and reproduction. When a cell is "resting", or not dividing, its chromosomes are organized into long entangled structures called chromatin .

Nucleoplasm

Nucleoplasm is the gelatinous substance within the nuclear envelope. Also called karyoplasm, this semi-aqueous material is similar to cytoplasm in that it is composed mainly of water with dissolved salts, enzymes, and organic molecules suspended within. The nucleolus and chromosomes are surrounded by nucleoplasm, which cushions and protects nuclear contents.

Like the nuclear envelope, the nucleoplasm supports the nucleus to hold its shape. It also provides a medium by which materials, such as enzymes and nucleotides  (DNA and RNA subunits), can be transported throughout the nucleus to its various parts.

Contained within the nucleus is a dense, membrane-less structure composed of RNA and proteins called the nucleolus . The nucleolus contains nucleolar organizers, the parts of chromosomes carrying the genes for ribosome synthesis. The nucleolus helps to synthesize ribosomes by transcribing and assembling ribosomal RNA subunits. These subunits join together to form ribosomes during protein synthesis.

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Cell Nucleus

Reviewed by: BD Editors

Cell Nucleus Definition

The cell nucleus is a large organelle in eukaryotic organisms which protects the majority of the DNA within each cell. The nucleus also produces the necessary precursors for protein synthesis. The DNA housed within the cell nucleus contains the information necessary for the creation of the majority of the proteins needed to keep a cell functional. While some DNA is stored in other organelles, such as mitochondria , the majority of an organism’s DNA is located in the cell nucleus. The DNA housed in the cell nucleus is extremely valuable, and as such the cell nucleus has a variety of important structures to help maintain, process, and protect the DNA.

Cell Nucleus Structure

A cell nucleus is surrounded by a double membrane, known as the nuclear envelope . This membrane covers and protects the DNA from physical and chemical damage. In doing so, the membrane creates a separate environment to process the DNA in. The outer membrane is in contact with the cytoplasm, and connects in some places to the endoplasmic reticulum . The inner membrane connects to the nuclear lamina . This nuclear framework inside the cell nucleus helps it maintain its shape. There is also evidence that this scaffolding of proteins helps form a matrix to transport and distribute products within and out of the nucleus. Nuclear pores create passages through the nuclear membrane, and allow products of the cell nucleus to enter the cytoplasm or endoplasmic reticulum. The pores also allow some specific macromolecules and chemicals from the cytoplasm to pass back into the cell nucleus. These macromolecules are needed to synthesize DNA and RNA, and are needed for the creation of new proteins and macromolecules within the cell nucleus. In a stained nucleus, a dark spot can be seen. This spot is the nucleolus . Within the nucleolus, the several different parts of ribosomes are produced and exported. These structures can be seen in the following image.

While the cell nuclei of plants and animals differ in subtle ways, their main purpose and general activities remain the same. The cell nucleus is responsible for producing two main products to support the efforts of each cell. The first, messenger RNA , or mRNA, is the product of transposing a gene coding for a specific protein from the DNA structure to the RNA structure. This shorter mRNA strand can exit the nucleus and enter the cytoplasm. When a ribosome picks up this mRNA, it will translate this mRNA into the language of proteins and create a long strand of amino acids. This strand will then be folded into a functional protein, which may serve one of a thousand different roles. Examples of the differences between plant and animal cell nuclei can be seen below.

Function of Cell Nucleus

Animal cell nucleus.

This generic animal cell has all the components that every animal cell has. The cell nucleus can be seen on the left side of the cell. It is the large purple circle. Remember that this is a cross-section view, and in reality the nucleus would be more of a sphere. In animal cells it usually takes a spherical shape if there is enough room within the cell. The nucleus is surrounded by the endoplasmic reticulum, which is covered in spots by ribosomes. When the animal cell divides, the nucleus breaks up, and the nuclear envelope falls apart. The nuclear envelope is then reassembled around each new nucleus after the chromosomes have been divided.

Plant Cell Nucleus

Above is a generic plant cell. Notice how it has a rigid shape, due to the presence of a cell wall. Further, a large central vacuole occupies the majority of the cell, pushing all the other constituents to the sides of the cell. The nucleus here is orange, shown with a chunk taken out to expose the interior. Like animal cell nuclei, this cell nucleus will retain a spherical shape if there is enough room. Oftentimes in plant cells, the central vacuole expands with water to apply pressure to the cell walls. This pressure forces the nucleus into a more flattened, oblong shape. As with animal cell nuclei, this cell nucleus will break down during cell division. Unlike animal cells, plant cells must build new cell walls between dividing cells. The two new nuclei must be moved away from the metaphase plate , or the nuclei may become damaged by the formation of the cell wall.

Other Examples of Cell Nuclei

Besides these two simple examples of cell nuclei, there are countless variations to these two general schemes in nature. Some cells merge together, creating large cells with multiple cell nuclei in each cell. Many organism have cells with more than one nucleus, including humans. Human muscle cells are multi-nucleated. Other organisms, like some fungi, exist with most or all of their cells being multi-nucleated. In some organisms, the process of cell division does not include the breakdown of the nuclear envelope. Instead, microtubules extend through the cell nucleus and directly manipulate the chromosomes and work to divide the nucleus. Evolutionarily, it is assumed that early organisms that developed nuclei had clear advantages over those without. Over the course of millennia, different strategies for managing and maintaining the cell nucleus have evolved. While the nucleus may seem like a more advanced form of life, don’t forget that prokaryotes , like bacteria and other single-celled life forms, are still some of the most abundant on the planet. That being said, the cell nucleus has evolved as a highly successful strategy in multi-cellular forms of life.

Nelson, D. L., & Cox, M. M. (2008). Principles of Biochemistry . New York: W.H. Freeman and Company.

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Cell Nucleus: Definition, Structure, and Functions

What is a nucleus.

The nucleus is a double membrane-bound organelle located centrally only in a eukaryotic cell , enclosing the   DNA, the genetic material. It is the most important and defining feature of all higher organisms, including plant and animal cells, whose main function is to control and coordinate the functioning of the entire cell. 

The word ‘nucleus’ (plural: nuclei) is derived from the Latin word ‘ nucleus ‘, meaning ‘kernel’ or ‘ seed ’.

Structure and Characteristics

The largest and most prominent organelle in the cell, the nucleus, accounts for almost 10% of the volume of the entire cell. In mammalian cells, the average diameter of the nucleus is approximately 6 µm in size. Mostly the shape of the nucleus is found to be either spherical or oblong. Eukaryotes usually contain a single nucleus, however erythrocytes and platelets are without a nucleus and osteoclasts of bones have many of them. The color of the nucleus is usually grayish but can differ depending on the type of the cell.

Parts and Their Functions

Anatomically, the nucleus of all plant and animal cell is made up of several components that are listed below. All of these components work together in order for the nucleus to accomplish its purpose as the ‘controlling center’ of the cell.

1) Nuclear Envelope and Nuclear Pores

Surrounding the nucleus, the nuclear envelope is made of a phospholipid bilayer, similar to cell membranes, and contains tiny openings called nuclear pores over them. The two membranes are often referred to as the inner and outer nuclear membranes with a fluid-filled region called perinuclear space in between. The perinuclear space has a thickness of 20 to 40 nm. The outer membrane is attached to ribosomes and is continuous with the cell’s endoplasmic reticulum , a system that helps to package, transport, and export substances outside the cell.

• Nuclear envelope separates the nuclear content from the cytoplasm an is selectively permeable in nature
• Nuclear pores regulate the flow of molecules into and out of the nucleus

2) Nuclear Lamina

They are meshwork of protein filaments organized in a net-like fashion that line below the inner nuclear membrane. The proteins that make up the nuclear lamina are known as lamins, which are intermediate filament proteins.

• Supports the nuclear envelope, maintaining the overall shape and structure of the nucleus

3) Chromatin

It is a complex of genetic material (DNA or RNA) and proteins found in a resting or non-dividing cell nucleus. The chromatin is classified into two types, heterochromatin and euchromatin, based on functions. The heterochromatin is a functionally inactive form of chromatin, found near the nuclear envelope. On the contrary, euchromatin is a mild, less condensed form that is in functionally active state. An organized chromatin material that is highly condensed and paired is known as the  chromosome . 

• Contains hereditary information and instructions necessary for controlling processes such as metabolism, cell growth, and cell division
• Helps in gene expression where DNA molecules make an RNA copy, a process called transcription which is later converted to proteins by a process called translation

4) Nucleoplasm

Also known as karyoplasm, it is found inside the nucleus, and is a gelatinous substance similar to the cytoplasm, being composed mainly of water with dissolved salts, enzymes, and suspended organic molecules.

• Protects the nuclear content by providing a cushion around the nucleolus and the chromosome
• Supports the nucleus to hold its shape
• Provides a medium through which enzymes and fragments of genetic materials (DNA or RNA), can be transported throughout the nucleus

5) Nucleolus

It looks like a dark spot within the nucleus and is a dense, membrane-less structure composed of RNA and proteins along with granules and fibers that remain attached to chromatin. The nucleolus contains multipleregions called nucleolar organizers that are the segments of chromosomes that contain the genes for ribosomal RNA. The nucleolus disappears when a cell undergoes division and is reformed after the completion of cell division.

• Synthesize ribosomes that are involved in protein synthesis

Ans. The nucleus was the first organelle to be discovered by Antonie van Leeuwenhoek during his study involving microorganisms, which was further described in detail by Robert Brown in 1831.

Ans . Prokaryotic cells, including bacteria and archaea , do not have a true nucleus; instead, they have a membrane-less nucleoid region that holds their free-floating DNA.

Ans . Both archaebacteria and eubacteria being prokaryotic organisms lack all membrane-bound organelles, including the nucleus.

Ans . Like all eukaryotic cells, protists have a characteristic central compartment called the nucleus, which houses their genetic material.

Ans . Being eukaryotes, fungi, and amoeba has a membrane-bound nucleus within their cell.

Ans . White blood cells also known as leucocytes have a distinct nucleus that differentiates them from other blood cells.

Ans . The DNA, which is the genetic material of the cell, is a polymer of nucleotides, found within the nucleus.

Ans . Eukaryotic DNA never leaves the nucleus but is copied into RNA molecules, which may then travel out of the nucleus.

Ans . A nucleus is a membrane-bound organelle that houses DNA, the genetic material of eukaryotes whereas nucleoid is an irregularly shaped region that houses the genetic material of prokaryotes.

Ans . Nucleus is a membrane-bound organelle that houses DNA, the genetic material of a eukaryotic cell whereas nucleolus is a sub-organelle found within the nucleus containing RNA and is responsible for ribosome synthesis.

• Nucleus – Britannica.com
• Nucleus- Definition, Structure, Functions and Diagram – Microbenotes.com
• The Cell Nucleus – Thoughtco.com
• Cell Nucleus – Kenhub.com
• Nucleus – Biologyonline.com
• The Structure and Functions of a Cell Nucleus Explained – Biologywise.com

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• Biology Article
• The Nucleus

Nucleus - Structure and Function

Table of Contents

• Important Questions

What is a Nucleus?

The most integral component of the cell is the nucleus (plural: nuclei). It is derived from a Latin word which means “ kernel of a nut ”.

Nucleus Definition:

A nucleus is defined as a double-membraned eukaryotic cell organelle that contains the genetic material.

A nucleus diagram highlighting the various components. Moreover, only eukaryotes have the nucleus, prokaryotes have the nucleoid

As stated above, the nucleus is found only in eukaryotes and is the defining characteristic feature of eukaryotic cells . However, some cells, such as RBCs do not possess a nucleus, though they originate from a eukaryotic organisms.

More to Explore:   Difference Between Nucleus and Nucleoid

Structure Of Nucleus

• Typically, it is the most evident organelle in the cell.
• The nucleus is completely bound by membranes.
• It is engirdled by a structure referred to as the nuclear envelope.
• The membrane distinguishes the cytoplasm from the contents of the nucleus
• The cell’s chromosomes are also confined within it.
• DNA is present in the Chromosomes, and they provide the genetic information required for the creation of different cell components in addition to the reproduction of life.

Also Read:  Nucleolus

Nucleus Function

Following are the important nucleus function:

• It contains the cell’s hereditary information and controls the cell’s growth and reproduction.
• The nucleus has been clearly explained as a membrane-bound structure that comprises the genetic material of a cell.
• It is not just a storage compartment for DNA, but also happens to be the home of some important cellular processes.
• First and foremost, it is possible to duplicate one’s DNA in the nucleus. This process has been named DNA Replication and produces an identical copy of the DNA.
• Producing two identical copies of the body or host is the first step in cell division, where every new cell will get its own set of instructions.
• Secondly, the nucleus is the site of transcription. Transcription creates different types of RNA from DNA. Transcription would be a lot like creating copies of individual pages of the human body’s instructions which may be moved out and read by the rest of the cell.
• The central rule of biology states that DNA is copied into RNA, and then proteins.

Also Read: Nuclear membrane

Discover more about the Nucleus, its features and functions, or any other related topics by registering at BYJU’S Biology.

Further Reading:

• Animal Cell
• Eukaryotic Cells
• Difference between Prokaryotic and Eukaryotic Cells

Frequently Asked Questions

What is the nucleus.

The nucleus is a double-membraned organelle that contains the genetic material and other instructions required for cellular processes. It is exclusively found in eukaryotic cells and is also one of the largest organelles.

Outline the structure of the Nucleus.

• A double-membraned organelle known as the nuclear membrane/envelope engirdles the nucleus.
• The nucleolus is found within the nucleus, occupying 25% per cent of the volume.
• Thread-like, dense structures known as chromatins are found within the nucleus containing proteins and DNA.
• The mechanical strength for the nucleus is provided by the nuclear matrix, a network of fibres and filaments which performs functions similar to the cytoskeleton.

Highlight the functions of the nucleus.

The nucleus has 2 primary functions:

• It is responsible for storing the cell’s hereditary material or the DNA.
• It is responsible for coordinating many of the important cellular activities such as protein synthesis, cell division, growth and a host of other important functions.

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3.3 The Nucleus and DNA Replication

Learning objectives.

By the end of this section, you will be able to:

• Describe the structure and features of the nuclear membrane
• List the contents of the nucleus
• Explain the organization of the DNA molecule within the nucleus
• Describe the process of DNA replication

The nucleus is the largest and most prominent of a cell’s organelles ( Figure 3.3.1 ). The nucleus is generally considered the control center of the cell because it stores all of the genetic instructions for manufacturing proteins. Interestingly, some cells in the body, such as muscle cells, contain more than one nucleus ( Figure 3.3.2 ), which is known as multinucleated. Other cells, such as mammalian red blood cells (RBCs), do not contain nuclei at all. RBCs eject their nuclei as they mature, making space for the large numbers of hemoglobin molecules that carry oxygen throughout the body ( Figure 3.3.3 ). Without nuclei, the life span of RBCs is short, and so the body must produce new ones constantly.

External Website

View the University of Michigan WebScope at http://141.214.65.171/Histology/Basic%20Tissues/Muscle/058thin_HISTO_83X.svs/view.apml to explore the tissue sample in greater detail.

View the University of Michigan WebScope at http://virtualslides.med.umich.edu/Histology/EMsmallCharts/3%20Image%20Scope%20finals/139%20-%20Erythroblast_001.svs/view.apml to explore the tissue sample in greater detail.

Inside the nucleus lies the blueprint that dictates everything a cell will do and all of the products it will make. This information is stored within DNA. The nucleus sends “commands” to the cell via molecular messengers that translate the information from the DNA. Each cell in your body (with the exception of germ cells) contains the complete set of your DNA. When a cell divides, the DNA must be duplicated so that each new cell receives a full complement of DNA. The following section will explore the structure of the nucleus and its contents, as well as the process of DNA replication.

Organization of the Nucleus and its DNA

Like most other cellular organelles, the nucleus is surrounded by a membrane called the nuclear envelope . This membranous covering consists of two adjacent lipid bilayers with a thin fluid space in between them. Spanning these two bilayers are nuclear pores. A nuclear pore is a tiny passageway for the passage of proteins, RNA, and solutes between the nucleus and the cytoplasm. Proteins called pore complexes lining the nuclear pores regulate the passage of materials into and out of the nucleus.

Inside the nuclear envelope is a gel-like nucleoplasm with solutes that include the building blocks of nucleic acids. There also can be a dark-staining mass often visible under a simple light microscope, called a nucleolus (plural = nucleoli). The nucleolus is a region of the nucleus that is responsible for manufacturing the RNA necessary for construction of ribosomes. Once synthesized, newly made ribosomal subunits exit the cell’s nucleus through the nuclear pores.

The genetic instructions that are used to build and maintain an organism are arranged in an orderly manner in strands of DNA. Within the nucleus are threads of chromatin composed of DNA and associated proteins ( Figure 3.3.4 ). Along the chromatin threads, the DNA is wrapped around a set of histone proteins. A nucleosome is a single, wrapped DNA-histone complex. Multiple nucleosomes along the entire molecule of DNA appear like a beaded necklace, in which the string is the DNA and the beads are the associated histones. When a cell is in the process of division, the chromatin condenses into chromosomes, so that the DNA can be safely transported to the “daughter cells.” The chromosome is composed of DNA and proteins; it is the condensed form of chromatin. It is estimated that humans have almost 22,000 genes distributed on 46 chromosomes.

DNA Replication

In order for an organism to grow, develop, and maintain its health, cells must reproduce themselves by dividing to produce two new daughter cells, each with the full complement of DNA as found in the original cell. Billions of new cells are produced in an adult human every day. Only very few cell types in the body do not divide, including nerve cells, skeletal muscle fibers, and cardiac muscle cells. The division time of different cell types varies. Epithelial cells of the skin and gastrointestinal lining, for instance, divide very frequently to replace those that are constantly being rubbed off of the surface by friction.

A DNA molecule is made of two strands that “complement” each other in the sense that the molecules that compose the strands fit together and bind to each other, creating a double-stranded molecule that looks much like a long, twisted ladder. Each side rail of the DNA ladder is composed of alternating sugar and phosphate groups ( Figure 3.3.5 ). The two sides of the ladder are not identical, but are complementary. These two backbones are bonded to each other across pairs of protruding bases, each bonded pair forming one “rung,” or cross member. The four DNA bases are adenine (A), thymine (T), cytosine (C), and guanine (G). Because of their shape and charge, the two bases that compose a pair always bond together. Adenine always binds with thymine, and cytosine always binds with guanine. The particular sequence of bases along the DNA molecule determines the genetic code. Therefore, if the two complementary strands of DNA were pulled apart, you could infer the order of the bases in one strand from the bases in the other, complementary strand. For example, if one strand has a region with the sequence AGTGCCT, then the sequence of the complementary strand would be TCACGGA.

DNA replication is the copying of DNA that occurs before cell division can take place. After a great deal of debate and experimentation, the general method of DNA replication was deduced in 1958 by two scientists in California, Matthew Meselson and Franklin Stahl. This method is illustrated in Figure 3.3.6 and described below.

Stage 1: Initiation. The two complementary strands are separated, much like unzipping a zipper. Special enzymes, including helicase , untwist and separate the two strands of DNA.

Stage 2: Elongation. Each strand becomes a template along which a new complementary strand is built. DNA polymerase brings in the correct bases to complement the template strand, synthesizing a new strand base by base. A DNA polymerase is an enzyme that adds free nucleotides to the end of a chain of DNA, making a new double strand. This growing strand continues to be built until it has fully complemented the template strand.

Stage 3: Termination. Once the two original strands are bound to their own, finished, complementary strands, DNA replication is stopped and the two new identical DNA molecules are complete.

Each new DNA molecule contains one strand from the original molecule and one newly synthesized strand. The term for this mode of replication is “semiconservative,” because half of the original DNA molecule is conserved in each new DNA molecule. This process continues until the cell’s entire genome , the entire complement of an organism’s DNA, is replicated. As you might imagine, it is very important that DNA replication take place precisely so that new cells in the body contain the exact same genetic material as their parent cells. Mistakes made during DNA replication, such as the accidental addition of an inappropriate nucleotide, have the potential to render a gene dysfunctional or useless. Fortunately, there are mechanisms in place to minimize such mistakes. A DNA proofreading process enlists the help of special enzymes that scan the newly synthesized molecule for mistakes and corrects them. Once the process of DNA replication is complete, the cell is ready to divide. You will explore the process of cell division later in the chapter.

Watch this video to learn about DNA replication. DNA replication proceeds simultaneously at several sites on the same molecule. What separates the base pair at the start of DNA replication?

Chapter Review

The nucleus is the command center of the cell, containing the genetic instructions for all of the materials a cell will make (and thus all of its functions it can perform). The nucleus is encased within a membrane of two interconnected lipid bilayers, side-by-side. This nuclear envelope is studded with protein-lined pores that allow materials to be trafficked into and out of the nucleus. The nucleus contains one or more nucleoli, which serve as sites for ribosome synthesis. The nucleus houses the genetic material of the cell: DNA. DNA is normally found as a loosely contained structure called chromatin within the nucleus, where it is wound up and associated with a variety of histone proteins. When a cell is about to divide, the chromatin coils tightly and condenses to form chromosomes.

There is a pool of cells constantly dividing within your body. The result is billions of new cells being created each day. Before any cell is ready to divide, it must replicate its DNA so that each new daughter cell will receive an exact copy of the organism’s genome. A variety of enzymes are enlisted during DNA replication. These enzymes unwind the DNA molecule, separate the two strands, and assist with the building of complementary strands along each parent strand. The original DNA strands serve as templates from which the nucleotide sequence of the new strands are determined and synthesized. When replication is completed, two identical DNA molecules exist. Each one contains one original strand and one newly synthesized complementary strand.

Interactive Link Questions

Review questions, critical thinking questions.

Explain in your own words why DNA replication is said to be “semiconservative”?

DNA replication is said to be semiconservative because, after replication is complete, one of the two parent DNA strands makes up half of each new DNA molecule. The other half is a newly synthesized strand. Therefore, half (“semi”) of each daughter DNA molecule is from the parent molecule and half is a new molecule.

Why is it important that DNA replication take place before cell division? What would happen if cell division of a body cell took place without DNA replication, or when DNA replication was incomplete?

During cell division, one cell divides to produce two new cells. In order for all of the cells in your body to maintain a full genome, each cell must replicate its DNA before it divides so that a full genome can be allotted to each of its offspring cells. If DNA replication did not take place fully, or at all, the offspring cells would be missing some or all of the genome. This could be disastrous if a cell was missing genes necessary for its function and health.

This work, Anatomy & Physiology, is adapted from Anatomy & Physiology by OpenStax , licensed under CC BY . This edition, with revised content and artwork, is licensed under CC BY-SA except where otherwise noted.

Images, from Anatomy & Physiology by OpenStax , are licensed under CC BY except where otherwise noted.

Access the original for free at https://openstax.org/books/anatomy-and-physiology/pages/1-introduction .

Anatomy & Physiology Copyright © 2019 by Lindsay M. Biga, Staci Bronson, Sierra Dawson, Amy Harwell, Robin Hopkins, Joel Kaufmann, Mike LeMaster, Philip Matern, Katie Morrison-Graham, Kristen Oja, Devon Quick, Jon Runyeon, OSU OERU, and OpenStax is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License , except where otherwise noted.

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nucleus , in biology , a specialized structure occurring in most cells (except bacteria and blue-green algae ) and separated from the rest of the cell by a double layer, the nuclear membrane . This membrane seems to be continuous with the endoplasmic reticulum (a membranous network) of the cell and has pores, which probably permit the entrance of large molecules. The nucleus controls and regulates the activities of the cell (e.g., growth and metabolism ) and carries the genes , structures that contain the hereditary information. Nucleoli are small bodies often seen within the nucleus. The gel-like matrix in which the nuclear components are suspended is the nucleoplasm .

Because the nucleus houses an organism’s genetic code , which determines the amino acid sequence of proteins critical for day-to-day function, it primarily serves as the information centre of the cell. Information in DNA is transcribed, or copied, into a range of messenger ribonucleic acid ( mRNA ) molecules, each of which encodes the information for one protein (in some instances more than one protein, such as in bacteria). The mRNA molecules are then transported through the nuclear envelope into the cytoplasm, where they are translated, serving as templates for the synthesis of specific proteins. For more information on these processes, see transcription ; translation .

A cell normally contains only one nucleus. Under some conditions, however, the nucleus divides but the cytoplasm does not. This produces a multinucleate cell ( syncytium ) such as occurs in skeletal muscle fibres. Some cells—e.g., the human red blood cell —lose their nuclei upon maturation. See also cell .

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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

Chapter 8 the nucleus.

The presence of a nucleus is the principal feature that distinguishes eukaryotic from prokaryotic cells. By housing the cell's genome, the nucleus serves both as the repository of genetic information and as the cell's control center. DNA replication, transcription, and RNA processing all take place within the nucleus, with only the final stage of gene expression (translation) localized to the cytoplasm.

By separating the genome from the cytoplasm, the nuclear envelope allows gene expression to be regulated by mechanisms that are unique to eukaryotes. Whereas prokaryotic mRNAs are translated while their transcription is still in process, eukaryotic mRNAs undergo posttranscriptional processing (e.g., splicing) before being transported from the nucleus to the cytoplasm. The presence of a nucleus thus allows gene expression to be regulated by posttranscriptional mechanisms, such as alternative splicing. By limiting the access of proteins to the genetic material, the nuclear envelope also provides novel opportunities for the control of gene expression at the level of transcription. For example, the expression of some eukaryotic genes is controlled by the regulated transport of transcription factors from the cytoplasm into the nucleus—a form of transcriptional regulation unavailable to prokaryotes. The separation of the genome from the site of mRNA translation thus plays a central role in eukaryotic gene expression.

• The Nuclear Envelope and Traffic between the Nucleus and Cytoplasm
• Internal Organization of the Nucleus
• The Nucleolus
• The Nucleus during Mitosis
• References and Further Reading
• Cite this Page Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Chapter 8, The Nucleus.

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Cell Nucleus Powerpoint Template

Cell Nucleus

Transcript: Cell Membrane Cell Nucleus The Cell Nucleus is the brain of the cell. It controls all functions of the cell. Without a nucleus a cell is brain dead The ER packages proteins Mitocondrea Ribosomes make protein for the cell. They are very rough and rigid as shown below Cell Wall The Mitocondrea is the power source of a cell. It provides the cell energy to carry out its functions Ribosomes The membrane of a cell is like our immune system. It controls all of the things that go in and out of the cell. Both the plant and animal cell have this feature The Cell Wall protects the plant cell from harmful things. Its kinda like a membrane. Only plant cells contain these It works like a mail room endoplasmic reticulum (ER)

cell nucleus

Transcript: The nucleus controlls the cells just like the mayor tells us what to do Think of it as the "Control Center" or "Headquarters" of the cell. The nucleus of the cell contains much of the DNA of the cell, and it regulates the activities of that cell, whatever those activities are. Basically it controls everything that happens inside the cell, it's like a boss of the cell. Read more: http://wiki.answers.com/Q/What_does_a_nucleus_do#ixzz1a2dxiLMN The nucleus is always located inside of the cell, but there is no rigid point in the cell in which the nucleus stays Read more: http://wiki.answers.com/Q/Where_in_the_cell_is_the_nucleus_located#ixzz1Yn07uNsx the control of the cell which contains the cells chromosmal d.n.a the function the control these are some pictrures of cells

Transcript: Cell Nucleus DNA CELL NUCLEUS Nucleus means kernel in Latin. It's kinda like the brain of a cell. It has an outside layer that protects it from the rest of the cell. It controls everything that goes on in the cell.

Transcript: When the cell begins to divide, the small and inconspicuous chromatin throughout the cell condenses into 46 thick and noticable chromosomes. Deep inside the nucleus is the nucleolus. It can account for as much as 25% of the nucleus' mass and is where ribosonal RNA is used to create ribosomes. Once a strand of mRNA is transcribed from a strand of DNA, it leaves the nucleus through a nuclear pore. The cell nucleus is the command center of the eukaryotic cell. It is responsible for instructing the other parts of the cell on what proteins to make and where to put them. The nucleus is the boss of all the other organelles. It tells them what to do and when to do it. Spread throughout the nucleus are strands of chromatin which contain DNA, the blueprint of life. DNA is the code for all proteins used in the body. Rough Endoplasmic Reticulum Ribosome The job of the nucleus is to transcribe messenger RNA from instructions from DNA. Cell Nucleus The mRNA then travels to ribosomes usually located in the rough endoplasmic reticulum which surrounds the nucleus. The nucleus is surrounded by a double membrane called a nuclear envelope. Other ribosomes are located in the nuclear envelope, in mitochondria or free floating in the cytoplasm.

Transcript: Nucleolus The Nuclear Envelope, or Nuclear Membrane, is composed of two lipid-bi layers and surrounds the Nucleus. The semi-permeable membrane allows for certain materials to enter or exit the cell Nucleus through Nuclear Pores. The cell nucleus is in the middle of the cell, it provides and stores information for the cell. The nucleus is surrounded by the nuclear envelope. Nucleus Overview Cell Nucleus The largest structure in the Nucleus in Eukaryotic cells, mainly used to construct and synthesize ribosomes DNA Nuclear Envelope Cites DNA in cells is contained inside of the nucleus. In Eukaryotes, DNA encodes for the genome. DNA can also be located in mitochondria and plastids. RNA, or Messenger or RNA that is in the Nucleus is used to transmit information from the DNA to ribosomes where they specify the sequence of proteins in amino acids. RNA

Transcript: The nucleus takes up 10% of a a cell's volumen. Making it the most prominent organelle. It is surrounded by a two-layer membrane called the nuclear envelope. The envelope has pores. Inside the nucleus you find the Nucleoplasm, DNA, and the Nucleolus. Cell Mitosis has 4 stages: Prophase, Metaphase, anaphase, and telophase. Before mitosis the cell copies it's chromosomomes making 2 connected coppies called sister chromatids. Nuclear envelope The DNA determines the apperance of the organism. It also tells the cells within the organism how to function and organize themselfs. Outter Membrane Metaphase: The chromosomes are done lining up at the at the metaphase plate. At this point the 2 kinetochores from each chromosome attaches to microtubules from opposite poles. Nucleosomes are smaller sections of chromatins. Under a microscope they look like beads. They are composed of 8 histone proteins which form a histone octamer. The DNA double-helix than wraps around the histone octamer to form the bead like structures. They are typiclly 11 nm in diameter. The nuclear envelope is made of an inner and outter nuclear membrane. The inner membrane has proteins that lash to the lamina, which is a protein framework that gives the nucleus its structure. Chromatins are nucleosomes wrapped into a spiral called a solenoid. This structure is latter supported with more histone patricles. Chromatins are further condensed to form chromosomes. Chromatins are also inportant in cell division Nucleoplasm Chromosomes Nucleosomes The nuclear pores regulate the passage of macromelecules into and out of the nucleus, but gives free movement to particles like ATP. Cell Division Sources Telophase: Two nuclei reappear one for each cell. The chromosomes decondense and become more stringy. Nuclear Pores Cell Nucleus Chromatins DNA http://micro.magnet.fsu.edu/cells/nucleus/nucleus.html https://www.genome.gov/26524120 http://www.nature.com/scitable/definition/nucleosome-nucleosomes-30 http://www.nature.com/scitable/definition/chromatin-182 http://study.com/academy/lesson/nuclear-envelope-definition-function-structure.html https://www.khanacademy.org/science/biology/cellular-molecular-biology/mitosis/a/phases-of-mitosis The nucleoplasm mainly serves as a suspension substance for the organelles within the nucleous. It also helps to maintain the shape of the nucleus. It is mainly composed of water and a mixture of ions and molecules. The outter membrane is connected to the endoplsmic reticulum. The outer membrane is covered in ribosomes. Prophase: During this phase the nucleoulus disappears within the cell. The chromatins condense more tightly into chromosomes. The chromosomes start to line up. In the cytoplasm spindle fibers made of microtubules are formed. Appearance Anaphase: The sister chromatids are separated into opposite poles of the cell. Now each is considered its own chromosome. Microtubules not attached to chromosomes push appart making the cell bigger. All of this processes are powered by motor proteins. Most humans have 23 pairs of chromosomes, 46 in total. The chromosomes determine the characteriscs of the organism. They also contain information on cell division and organisation. The chromosomes contain about 6 feet of DNA. They are also important for the production of proteins and RNA. Appereance of chromosomes and Chromatins The nuclear envelope separates the contents of the nucleous from the rest of the cell. It is made if lipids like the cell membrane Inner Membrane

Nucleus Cell

Transcript: Nifty Nucleus What is the Nucleus? The Nucleus is the most important part of the Cell. It is a membrane enclosed organelle found in Eukaryotic Cells. The Nucleus contains most of the Cell's genetic material know as 'DNA' (deoxiribonucleic acid). LOCATION The nucleus is usually found in the centre of animal cells, but in plant cells it may not be the same due to the presence of "vacuoles" which are spaces within the cytoplasm containing fluid THE FUNCTION The function of the nucleus is to maintain and protect the genes, and to also control the activities in the cell! Nucleus surroundings The nucleus consists of a nuclear membrane, nucleoplasm, nucleolus and and chromosones. The membrane is a double-layered structure that encloses the contents of the nucleus and the nucleolus is found inside the nucleus What is the Nucleus made of? The nucleus containing DNA is made of proteins surrounded by a membrane. - First organelle discovered - The nucleus has a double membrane - Not all Prokarayotic Cells contain a nucleus By: Priyanka Sharma 11B Interesting Facts www.buzzle.com http://www.cellsalive.com/cells/nucleus.htm Year 11 Biology book Bibliogrpahy http://en.wikipedia.org/wiki/Cell_nucleus Nature of Biology http://www.biology-questions-and-answers.com/cell-nucleus.html The nucleus is surrounded by cytoplasm which is made up of a jelly like substance called "cytosol." This is to protect the nucleus The nucleus in every cell varies in sizes. In animals the nucleus is the largest organelle in the cell, in diameter it is about 6 micrometers which takes up about 10% of the cell.

Transcript: Introduction A highly especialized organelle that serves as information procesing. Nucleus Where is? Eukaryotic Cells Chromatin and Chromosome Chromatin and Chromosome Nucleolus The Nucleolus Nuclear Envelope The Nuclear Envelope Nuclear Pores Nuclear Pores

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• Published: 27 September 2024

Dopamine-mediated formation of a memory module in the nucleus accumbens for goal-directed navigation

• Kanghoon Jung   ORCID: orcid.org/0000-0002-5381-5161 1 , 2 , 3 , 4 ,
• Sarah Krüssel   ORCID: orcid.org/0000-0003-1168-5458 1 , 2 ,
• Sooyeon Yoo   ORCID: orcid.org/0000-0003-4718-900X 1 ,
• Myungmo An 2 ,
• Benjamin Burke 1 ,
• Nicholas Schappaugh 1 , 2 ,
• Youngjin Choi   ORCID: orcid.org/0000-0003-2239-2503 1 ,
• Zirong Gu 5   nAff6 ,
• Seth Blackshaw 1 ,
• Rui M. Costa 4 , 5 &
• Hyung-Bae Kwon   ORCID: orcid.org/0000-0001-5683-3720 1 , 2  

Nature Neuroscience ( 2024 ) Cite this article

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• Cellular neuroscience
• Spatial memory

Spatial memories guide navigation efficiently toward desired destinations. However, the neuronal and circuit mechanisms underlying the encoding of goal locations and its translation into goal-directed navigation remain unclear. Here we demonstrate that mice rapidly form a spatial memory of a shelter during shelter experiences, guiding escape behavior toward the goal location—a shelter—when under threat. Dopaminergic neurons in the ventral tegmental area and their projection to the nucleus accumbens (NAc) encode safety signals associated with the shelter. Optogenetically induced phasic dopamine signals are sufficient to create a place memory that directs escape navigation. Converging dopaminergic and hippocampal glutamatergic inputs to the NAc mediate the formation of a goal-related memory within a subpopulation of NAc neurons during shelter experiences. Artificial co-activation of this goal-related NAc ensemble with neurons in the dorsal periaqueductal gray was sufficient to trigger memory-guided, rather than random, escape behavior. These findings provide causal evidence of cognitive circuit modules linking memory with goal-directed action.

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The ability of animals to navigate their surroundings with a specific goal is a crucial skill that allows them to fulfill basic needs such as hunger, thirst and discomfort avoidance 1 , 2 . Through experience of an environment, animals rapidly develop spatial memories tied to these goals, which are later accessed for future navigation 3 . These goal-oriented memories are thought to arise from animals’ current motivational needs and guide their movements through a mental representation of the environment called a ‘cognitive map’ 4 until the desired goal is attained. The brain structure subserving the goal memory can thus be expected to receive information about the spatial context and the animals’ current needs, to provide the necessary navigational control 5 . Decades of spatial mapping studies in the hippocampal–entorhinal system have provided key elements of this structure, such as spatial coding in place, grid and head-direction cells 6 , 7 , 8 , 9 , 10 . Representations of spatial goals have been reported in the hippocampus, which in turn influences spatial coding in the entorhinal cortex 11 , 12 . However, the absence of a direct link between goal representation and locomotive control in the hippocampal–entorhinal system opens the possibility of a separate goal-oriented system outside these circuits 13 , 14 .

The nucleus accumbens (NAc) has long been recognized as a crucial interface for action selection, integrating cognitive and affective information 15 , 16 . System-level studies have suggested that hippocampal inputs convey spatial-contextual information to the NAc 17 , and dopamine (DA) inputs from the ventral tegmental area (VTA) to the NAc encode the value of expected rewards and mediate motivation during learning by reward prediction 18 , 19 . The function of the NAc as a ‘goal system’ is further suggested by electrophysiological data showing goal-related firing in NAc neurons 20 , 21 and computational studies in which spatial learning of goal locations could be simulated based on neural network models of hippocampal formation and NAc circuits 22 , 23 . Nevertheless, the neuronal mechanisms underpinning the formation of goal-memory ensembles and the extent of their contributions to memory-guided navigation remain elusive.

Here, we identified a neural substrate of DA-mediated spatial memory modules in the NAc. Our findings demonstrate that VTA dopaminergic (VTA DA ) signals encode relief states during escape to shelter, and that goal-encoding NAc ensembles emerge with shelter experience in a DA-mediated manner. We observed distinct contributions of VTA DA and glutamatergic inputs from the ventral hippocampus (vHPC Glu ) to the development of location-selective NAc activity. Notably, silencing the activity of the shelter-related NAc ensemble disrupts shelter-directed navigation, whereas reactivating this ensemble facilitates memory-guided navigation. Our findings highlight the role of DA in the formation of spatial memory modules in the NAc, which subsequently guide goal-directed actions based on motivational demands.

Escape to shelter requires escape drive and shelter memory

We used an escape behavior paradigm where mice freely explored a circular arena with a shelter and surrounding visual cues before responding to a threat by escaping to the shelter (Fig. 1a ). This paradigm reliably measured escape navigation (Fig. 1b–d , Extended Data Fig. 1 and Supplementary Videos 1 – 3 ). When faced with a threat, mice quickly escaped to the shelter. Over time, their preference for the shelter increased, and they navigated to it without effortfully attending to their environment 3 , 24 . Mice escaped to the previous shelter location even when it was removed or changed after the shelter acclimation period, whereas mice without shelter acclimation exhibited disrupted shelter-directed escape (Fig. 1b and Extended Data Fig. 1p,q ), suggesting that shelter experiences are necessary to form spatial memory of the shelter for guiding escape behavior. To interrogate the neural mechanisms governing shelter-directed escape, we triggered the flight response by optogenetically activating neurons in the dorsal periaqueductal gray (dPAG; Fig. 1e ), a region responsible for initiating escape 25 . Initially, in the absence of shelter, dPAG stimulation caused mice to flee in random directions (Fig. 1f,g ). However, after shelter acclimation, the same dPAG stimulation directed them either to the shelter or its previous shelter location if removed (Supplementary Video 4 ). These results indicate that the escape drive triggered by dPAG activation recruits shelter memory, subsequently determining the escape destination.

a , Top: schematic for testing escape behavior. Shelter is in a circular arena with four visual cues. Threat stimuli are visual (looming disk expansion) or auditory (high-frequency tone). Bottom: experiment timeline. 7-min acclimation followed by testing escape responses to threats. b , Video frames. Top: acclimation period. Bottom: threat-evoked escape trajectories. Left: shelter acclimation followed by testing. Middle: acclimation without shelter followed by shelter introduction at testing start. Right: shelter removed after acclimation. c , Left: example threat-evoked escape traces (5 trials) from a mouse that experienced the shelter during the acclimation period showing movement (gray) and head direction (blue), and its normalized trajectory relative to shelter. Right: average traces of speed, distance to shelter and goal angle. d , Reaching percentages to shelter in shelter-acclimated and shelter-unacclimated conditions, and to previous shelter location in shelter-removed conditions ( n  = 12, shelter acclimated; n  = 12, shelter unacclimated; n  = 12, shelter removed; F 2,33  = 13.53, P  = 5.11 × 10 −5 , one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test). e , Schematic of virus injection (AAV5-hSyn-hChR2-EYFP) into dPAG neurons and its expression. f , Movement traces of escapes triggered by optogenetic activation of dPAG neurons with blue light (473 nm): no shelter (left), shelter acclimation (middle) and shelter removed after acclimation period (right). Scale bars, 10 cm. g , Average speed (left) and distance to shelter (right) per condition, with the optogenetic activation period (3 s) shaded in blue. h , Top: design for inducing artificial shelter-like memory via optogenetic activation of VTA DA neurons in DAT-Cre mice. Bottom: confocal image of VTA DA neurons expressing ChR2-mCherry. Phasic (25 Hz) or tonic (4 Hz) photostimulation protocols (5 ms, 20 pulses, 60 trains) when mice were in a stimulation zone with no shelter ( n  = 9 mice). i , Distance to stimulation zone upon threat delivery. j , Normalized escape trajectories. Blue circle indicates the stimulation zone. k , Polar distributions of head-stimulation zone angle for each condition. l , Comparison of average head-stimulation zone angle during escape between two conditions ( n  = 9 mice, 27 trials for phasic and 23 trials for tonic; t 48  = −4.18, P  = 1.21 × 10 −4 , two-sided paired Student’s t -test). m , Comparison of reaching percentages to the stimulation zone ( n  = 9 mice, t 8  = 7.56, P  = 6.53 × 10 −5 , two-sided paired Student’s t -test). *** P  < 0.001. Error bars or shaded error bars indicate the s.e.m. For detailed statistical information, see Supplementary Table 1 .

Source data

Vta da signals encode relief states during escape to shelter.

To investigate whether shelter experience correlates with the activity of VTA DA neurons 26 , we monitored VTA DA activity using fiber photometry as mice explored and responded to threats (Extended Data Fig. 2a ). VTA DA activity notably increased upon shelter entry (Extended Data Fig. 2b–d ). In this aversive context where mice sought safety, VTA DA activity displayed a distinctive pattern—rising, declining, then peaking during shelter stay (Extended Data Fig. 2f ). Additionally, there was a surge in VTA DA activity upon threat termination, possibly indicating stress relief 27 . The average VTA DA activity was notably higher when mice were sheltered compared to when they were not, and higher after threat stimuli than during ongoing threat (Extended Data Fig. 2f–h ). These combined findings suggest that VTA DA activity participates in encoding relief signals in a context-specific manner related to location and threat state, contributing to a state representation tied to place.

Next, we examined if optogenetic activation of VTA DA neurons could replicate the goal-memory formation induced by shelter experience, focusing on DA release triggered by laser frequency and duration (Extended Data Fig. 3 ). Without a shelter, we activated VTA DA neurons when mice entered a designated stimulation zone (Fig. 1h ). VTA DA neurons exhibit two distinct firing modes: high-frequency phasic and low-frequency tonic, each linked to distinct roles in reward learning and motivated behavior 28 . We used either phasic or tonic stimulation for zone-locked excitation. After 60 rounds of zone-locked excitation, we subjected the mice to threat stimuli to see if they would escape to the stimulation zone. Mice receiving phasic stimulation efficiently directed their escapes to the stimulation zone upon threat delivery, whereas those receiving tonic stimulation did not (Fig. 1i–m ). This result suggests that mimicking shelter experience through the phasic firing pattern of VTA DA neurons is sufficient to create a ‘shelter-like’ memory that later guides escape behavior.

Goal-encoding NAc ensemble emerges with shelter experience

To understand the circuit mechanism by which the goal memory is formed through shelter experience, we investigated the role of the NAc in encoding shelter experience. Anatomically, the NAc is predominantly innervated by VTA DA and hippocampal neurons 15 . Functionally, NAc meditates goal-directed actions by the association between actions and behavioral outcomes 29 . Using viral tracing and in vivo fiber photometry recording with optogenetic activation, we confirmed that VTA DA and vHPC Glu inputs innervate the NAc medial shell and validated their anatomical and functional connectivity by monitoring DA and Ca 2+ in the NAc upon VTA and vHPC photoactivation, respectively (Extended Data Fig. 3 and Supplementary Video 5 ).

To assess the relevance of NAc activity to memory-guided escape, we injected a chemogenetic silencer (AAV 5 -hSyn-hM4Di(Gi)-mCherry) into the NAc medial shell (Fig. 2a ) and tested escape behavior in an arena where the shelter was removed after acclimation. Chemogenetic inhibition of NAc activity disrupted escape to the previous shelter location under threat (Fig. 2b ). To determine if this reduction in reaching percentage to shelter reflected decreased anxiety/fear or impaired memory formation, we examined behavioral characteristics such as freezing/darting or exploring/grooming during threat stimuli. Mice with clozapine N -oxide (CNO) injection showed normal freezing/darting in the majority of trials, similarly to control mice with saline, indicating that the reduced reaching percentage to shelter was not due to a loss of anxiety or fear (Fig. 2c ). To specify whether NAc was important for forming the shelter goal memory or just the escape behavior, we used optogenetics to bilaterally inhibit the NAc during shelter acclimation. Photoinhibition of NAc neurons during the acclimation period later impaired escape behavior to the previous shelter location upon threat stimuli (Fig. 2d ), suggesting that the NAc is required for goal-memory formation.

a , Top: schematics and timeline for chemogenetic inactivation of the NAc. Bottom: a confocal image of NAc neurons expressing hM4Di-mCherry. b , Left: flight traces upon threat in NAc::hM4Di mice. Right: reaching percentages to previous shelter location ( n  = 7 mice for saline, n  = 7 mice for CNO; t 12  = 5.715, P  = 9.68 × 10 − 5 , two-sided Student’s t -test). c , Response types (freeze/darting and exploring/grooming) to threat stimuli in NAc::hM4Di mice with saline or CNO ( n  = 7 mice, χ 2  = 0.002, P  = 0.963, two-sided Chi-square test). d , Left: schematic and image of NAc neurons with AAV 5 -hSyn-eNpHR3.0-EYFP (NAc::eNpHR3.0) for optogenetic inactivation. Right: reaching percentages to previous shelter location for photoinactivation (on) and no photoactivation (off; n  = 5 mice; t 8  = 2.4462, P  = 0.04, two-sided Student’s t -test). e , Left: dual-color fiber photometry setup for simultaneous DA and neuronal activity measurements in the NAc with AAV 9 -CAG-dLight1.1 and AAV 1 -Syn-NES- jRGECO1a, respectively ( n  = 5 mice). Right: confocal images of expressions. DM, dichroic mirror; DAQ, data acquisition unit. Scale bar, 10 μm. f , Temporospatial patterns of Ca 2+ (left), DA (middle) and their conjunctive (right) signals in the NAc during acclimation. x – y plane indicates the animal’s positions. z -values indicate signal intensity (color coded). Circle indicates shelter location. F jRGECO1a and F dLight1.1 , normalized transient fluorescent changes for jRGECO1a and dLight1.1, respectively. g , i , Average distance to shelter (gray) and speed (blue) during shelter approach ( g ) or shelter leaving ( i ) and average traces of DA release (green) and Ca 2+ activity (orange) in the NAc, aligned to shelter entry ( g ) or shelter exit ( i ). h , j , Changes in Ca 2+ transients (top) and DA concentrations (bottom) in the NAc, aligned to shelter entry ( h ) ( n  = 5 mice; one-way repeated-measures ANOVA with Greenhouse–Geisser correction, F 2.69,198.92  = 2.812, P  = 0.0463 for Ca 2+ ; F 2.76,204.31  = 19.14, P  = 2.26 × 10 − 10 for DA) and shelter exit ( j ) ( n  = 5 mice; one-way repeated-measures ANOVA with Greenhouse–Geisser correction, F 2.71,311.47  = 0.125, P  = 0.931 for Ca 2+ ; F 2.62,301.42  = 0.808, P  = 0.476 for DA). k , In vivo Ca 2+ microendoscopic imaging in NAc neurons; confocal image of GCaMP6s expression ( n  = 7 mice). Scale bar, 500 μm. l , Neuronal activity in the NAc during a session. Top: distance to shelter. Red lines indicate threat onsets. Bottom: z -score of the change in the fluorescence response (Δ F/F ) of NAc neurons sorted by response preference (outside preferring (magenta), nonspecific (gray) and shelter preferring (green)). m , Representative map and population statistics of NAc neurons by response preference. n , Cumulative distribution of SPI plotted over time. o , Experience-dependent dynamics of average event rates of NAc neurons with shelter experience. p , Left: time-related changes in SPI of individual NAc neurons for control and CNO conditions. Right: the difference of SPI between the initial 10 min to the last 10 min by preference types ( n  = 7 mice; t 6  = −5.532, P  = 1.47 × 10 − 3 for outside preferring; t 6  = −0.109, P  = 0.917 for nonspecific; t 6  = 6.396, P  = 6.88 × 10 −4 for shelter preferring; one-sided Student’s t -test). * P  < 0.05, ** P  < 0.01, *** P  < 0.001; NS, not significant. Box plots show the mean (square), the median (horizontal line) and percentile ranges (25th–75th, box; 10th–90th, whiskers). Error bars or shaded areas indicate the s.e.m. For detailed statistical information, see Supplementary Table 1 .

We next sought to examine the temporal dynamics of local neuronal activity and DA in the NAc using a dual-color fiber photometry recording with a red-shifted Ca 2+ indicator (AAV 1 -Syn-NES-jRGECO1a) and a DA sensor (AAV 9 -CAG-dLight1.1), respectively (Fig. 2e ). Both signals were enhanced at the shelter (Fig. 2f ). We observed a decrease in NAc activity and a slight DA dip during shelter approach, reaching maximum speed at −1.1 s from the shelter entry. Both signals peaked upon shelter entry, with NAc activity peaking at 137 ms and DA concentration at 430 ms after entry (Fig. 2g ). DA signals in the NAc varied with action and state: increasing during shelter approach but not during shelter exit (Fig. 2g–j and Extended Data Fig. 4a ). A state-dependent activity pattern was observed during shelter experience and during threat (Extended Data Fig. 4b–e ).

To understand how goal representations are formed in the NAc, we injected a Ca 2+ indicator (AAV 1 -Syn-GCaMP6s), implanted a gradient index (GRIN) lens into the NAc medial shell ( n  = 5 mice, 140.8 ± 23.8 neurons per animal) and performed microendoscopic Ca 2+ imaging while mice underwent the escape behavior task (Fig. 2g and Extended Data Fig. 5a ). Neural activity was dynamic and location dependent (Fig. 2l ). Shelter-preferring or outside-preferring neurons were classified by tuning to either the inside or outside of the shelter, respectively (Fig. 2m ; n  = 5 mice, n  = 704 cells, 19.32% shelter-preferring cells, 61.51% outside-preferring cells, 19.18% nonspecific cells). We characterized neurons’ spatial tuning (Fig. 2n and Extended Data Fig. 5b,c ) and computed their spatial information (SI). Quantitative analysis revealed that NAc neurons most likely encoded SI (0.27 ± 0.008, experimental data; 0.13 ± 0.003, surrogate data; Extended Data Fig. 5c ). Most of NAc neurons (95.5% of neurons) were sparsely active across the arena (Extended Data Fig. 5d,e ). Next, we examined whether NAc neuronal place-tuning changes over time. Preferential responses of NAc neurons to either shelter or outside became more distinctive with shelter experience (Fig. 2o,p and Extended Data Fig. 5j,k ). The average shelter preference index (SPI), defined as the log of the ratio of activity rates between inside and outside of shelter, gradually decreased with shelter experience (overall, −0.06 ± 0.02 at 5 min; −0.27 ± 0.03 at 50 min, P  < 0.001, two-sided Student’s t -test). In contrast, the average SPI from the top 10% of the population increased (top 10%, 0.89 ± 0.04 at 5 min; 1.15 ± 0.05 at 50 min, P  < 0.001, two-sided Student’s t -test), indicating that activity in the shelter-associated subpopulation increased with experience (Fig. 2n ). Both populations of shelter-preferring and outside-preferring neurons increased with experiences, whereas nonspecific neurons decreased (Fig. 2o,p ). Tracing individual neurons over time showed that the SPI of shelter-preferring neurons increased with shelter experience, whereas that of outside-preferring neurons decreased (Fig. 2p ). Thus, these results suggest that shelter experiences facilitate the formation of shelter-specific neuronal ensembles, driving the differentiation of neuronal responses to shelter and non-shelter conditions in the NAc population.

DA signaling regulates the emergence of location-specific activity in NAc

The NAc is predominantly innervated by VTA DA neurons and hippocampal neurons 15 . We next examined how these inputs influence shelter-associated representation in the NAc. To assess whether shelter-memory formation is mediated by DA release in the NAc, we bilaterally injected the neurotoxin 6-hydroxydopamine (6-OHDA; 9 µg) into the NAc medial shell to lesion dopaminergic axon terminals (Fig. 3a ). Dopaminergic axon terminals stained by tyrosine hydroxylase (TH) were largely reduced by 6-OHDA injections (Fig. 3b ). This loss of DA signaling in the NAc impaired escape behaviors both when shelter was present and removed after shelter acclimation (Fig. 3c ). Additionally, we examined the roles of dopamine receptor subtypes by suppressing dopamine receptor D1 (Drd1) or dopamine receptor D2 (Drd2) through intracranial injection of antagonists SCH23390 or haloperidol, in the NAc medial shell. Drd1 suppression substantially impaired escape to shelter under threat, while Drd2 suppression caused only mild deficits (Extended Data Fig. 6a ). To further explore the role of NAc synaptic plasticity in learning shelter location, we selectively knocked out α-Ca 2+ /calmodulin-dependent protein kinase II (αCaMKII), a synaptic protein molecule that underpins Drd1-mediated plasticity 30 in the NAc medial shell (Extended Data Fig. 6b ). The αCaMKII knockout resulted in impairment of escape behavior to shelter under threat (Extended Data Fig. 6c ), suggesting the necessity of NAc synaptic plasticity for learning shelter location.

a , Schematic and timeline of 6-OHDA lesions in the NAc medial shell (left) and confocal images of the NAc with TH and DAPI staining (right). Scale bar, 500 μm. b , TH/DAPI ratio comparison between control and lesion groups (control: 12 hemisphere sections, 6 mice; 6-OHDA: 12 hemisphere sections, 6 mice; t 22  = 19.45, P  = 2.39 × 10 −15 , two-sided Student’s t -test). c , Reaching percentages to previous shelter location (left, shelter removed; WT control, mean ± s.e.m: 71.63% ± 13.3%, n  = 6; 6-OHDA, mean ± s.e.m: 22.83% ± 5.95%, n  = 6 mice; t 10  = 3.350, P  = 0.0074, two-sided Student’s t -test) and to shelter location (right, shelter present; WT control, mean ± s.e.m: 82.07% ± 4.28%, n  = 12; 6-OHDA, mean ± s.e.m: 23.21% ± 9.79%, n  = 6 mice; t 16  = 6.47, P  = 7.68 × 10 −6 , two-sided Student’s t -test). d , Schematic of 6-OHDA lesion strategy and in vivo Ca 2+ imaging in NAc neurons. e , Neuronal activity in the NAc with 6-OHDA lesions during a session. Top: distance to shelter. Red lines indicate threat onsets. Bottom: z -score of Δ F/F of NAc neurons sorted by response preference (outside preferring (magenta), nonspecific (gray) and shelter preferring (green)). f , Left: neuronal preference proportions in the NAc with 6-OHDA lesions. Right: comparison of the proportions between 6-OHDA and control groups (WT control, n  = 7 mice; 6-OHDA, n  = 7 mice; t 12  = 10.52, P  = 2.06 × 10 −7 for outside preferring, t 12  = −7.75, P  = 5.20 × 10 −6 for nonspecific, t 12  = −1.17, P  = 0.263 for shelter preferring, two-sided Student’s t -test). g , Cumulative distribution of SPIs of NAc neurons with 6-OHDA lesions plotted over time. h , Left: time-related SPI changes of individual NAc neurons with 6-OHDA lesions. Right: comparisons of the magnitude of SPI change over a session from individual mice by preference types ( n  = 7 mice; t 12  = −5.088, P  = 2.67 × 10 −4 for outside preferring, t 12  = −2.38, P  = 3.45 × 10 −2 for nonspecific, t 12  = 4.80, P  = 4.36 × 10 −4 for shelter preferring, two-sided Student’s t -test). * P  < 0.05, *** P  < 0.001. Box plots show the mean (square), the median (horizontal line) and percentile ranges (25th–75th, box; 10th–90th, whiskers). Error bars or shaded areas indicate the s.e.m. For detailed statistical information, see Supplementary Table 1 .

To test if location-specific neuronal responses are mediated by DA, we performed microendoscopic Ca 2+ imaging in mice with dopaminergic axon terminals lesioned by 6-OHDA injection (Fig. 3d,e ). The 6-OHDA lesion reduced the fraction of outside-preferring neurons (23%) compared to control (61.51%; Fig. 2m ), while the fraction of shelter-preferring neurons (22%) remained similar to that of control (19.32%), suggesting that DA contributes to shaping the geometric structure of environmental context (Fig. 3f ). Additionally, we observed an increase over time in the fraction of shelter-preferring neurons in mice with 6-OHDA lesions (Fig. 3g ). However, the magnitude of SPI changes for individual shelter-preferring and outside-preferring neurons was lower than that of control (Fig. 3h ). These results indicate that 6-OHDA lesions disrupt the emergence of location-selective responses in NAc populations.

Inhibiting vHPC Glu -NAc disrupts development of shelter-preferring NAc populations

To next examine whether shelter-memory formation is mediated by vHPC inputs, we bilaterally silenced vHPC Glu neurons projecting to the NAc (vHPC Glu -NAc) by injecting a retrograde Cre-dependent inhibitory DREADD virus (AAVrg-DIO-hM4Di-mCherry) into the NAc and tested escape behavior in an arena where the shelter was removed after acclimation (Fig. 4a,b ). Mice with CNO-induced chemogenetic inactivation of vHPC Glu -NAc neurons showed deficits in escaping to the previous shelter location upon threat compared to control groups (hM4Di + saline, control + saline, control + CNO; Fig. 4c,d and Extended Data Fig. 6d,e ), consistent with previous studies on the necessary role of vHPC Glu inputs to the NAc in place–reward associations 17 , 31 .

a , Schematics for chemogenetic silencing of vHPC glutamatergic neurons projecting to the NAc (vHPC Glu -NAc) via retrograde virus. b , Top: confocal image of DREADD expression in vHPC Glu -NAc neurons. Sub, subiculum. Bottom: behavioral experiment timeline. c , Normalized movement traces for flights upon threat. d , Reaching percentages to previous shelter location ( n  = 6 mice for vHPC Glu -NAc::hM4Di; n  = 7 mice control; F 3,25  = 16.88, P  = 6.46 × 10 −6 , one-way ANOVA followed by a Bonferroni post hoc test). e , Schematic of vHPC Glu -NAc inactivation and in vivo Ca 2+ imaging in NAc neurons. f , Neuronal activity in the NAc with inactivation of vHPC Glu -NAc neurons during a session. Top: distance to shelter. Red lines indicate threat onsets. Bottom: z -score of Δ F/F of NAc neurons sorted by response preference (outside preferring (magenta), nonspecific (gray) and shelter preferring (green)). g , Left: neuronal preference proportions in the NAc with vHPC Glu -NAc inactivation. Right: comparison of the proportions between control and CNO groups ( n  = 5 mice; t 4  = −3.64, P  = 0.022 for outside preferring, t 4  = 2.62, P  = 0.059 for nonspecific, t 4  = 3.07, P  = 0.037 for shelter preferring, two-sided paired Student’s t -test). h , Cumulative distribution of SPIs of NAc neurons with vHPC Glu -NAc inactivation plotted over time. i , Left: time-related changes in SPI of individual NAc neurons. Right: comparisons of the magnitude of SPI change over a session from individual mice by preference types ( n  = 5 mice; t 4  = 2.89, P  = 0.044 for outside preferring, t 4  = 0.96, P  = 0.393 for nonspecific, t 4  = 0.36, P  = 0.737 for shelter preferring, two-sided paired Student’s t -test). * P  < 0.05, *** P  < 0.001. Error bars or shaded areas indicate the s.e.m. For detailed statistical information, see Supplementary Table 1 .

To further investigate whether vHPC inputs affect location-specific responses of NAc cells, we performed microendoscopic Ca 2+ imaging while we bilaterally silenced vHPC Glu -NAc neurons with CNO (vHPC-CNO) or control vehicle (vHPC-control; Fig. 4e,f ). The vHPC-CNO condition showed a lower fraction of shelter-preferring neurons (6%) compared to control (14%), and a higher fraction of outside-preferring neurons (65%) compared to control (47%; Fig. 4g ), suggesting the role of vHPC Glu -NAc neurons in forming shelter-preferring neuronal populations. While the control condition increased the population fractions of both shelter-preferring and outside-preferring neurons over time, the vHPC-CNO condition only increased the population fractions of outside-preferring neurons (Fig. 4h ). The SPI of individual shelter-preferring neurons increased and of outside-preferring neurons decreased over time in both conditions (Fig. 4i ), showing the emerging location specificity of individual neurons with shelter experience. Furthermore, the decrease in SPI for outside-preferring neurons in the vHPC-CNO condition was more pronounced than in control. These findings suggest that silencing vHPC Glu -NAc neurons disrupts the experience-dependent increase in shelter-preferring neuronal populations.

Silencing NAc Shelter ensemble disrupts escape navigation to shelter

To determine if shelter-tuned neuronal ensembles enable shelter-directed navigation, we labeled these neurons by using Cal-Light, an optogenetic system that allows selective tagging and subsequent controlling over active neuronal ensembles in a user-defined time window using light (Fig. 5a ) 32 . When mice explored the arena with a shelter, we labeled shelter-tuned NAc neurons (NAc Shelter ) for a test group and nonspecific NAc neurons (NAc Random ) for control by delivering blue light into NAc when mice were inside the shelter or at random locations outside the shelter, respectively (Fig. 5b–d ).

a , Illustration of Ca 2+ -gated and light-gated gene expression system (Cal-Light) for selective tagging of active neurons by delivering blue light in a user-defined time window to induce gene expressions in the labeled population. b , Schematics for behavioral experiments to silence shelter-associated NAc ensembles (NAc Shelter ). Left: labeling session with blue light to the NAc at shelter entry. 48 h were given for TRE-NpHR-EYFP expressions in NAc Shelter . Right: probe trials with amber light (589 nm) to the NAc to inhibit labeled neurons (NAc Shelter ::NpHR) during escape behavior by threat stimulus. c , Top: schematics for ensemble labeling conditions (blue light at shelter entry (left; NAc Shelter ) and at random outside locations on the arena (right; NAc Random )). Bottom: percentages of NAc Cal-Light-labeled neuronal populations (tdT − /EYFP − (black), tdT − /EYFP + (green), tdT + /EYFP − (red) and tdT + /EYFP + (yellow)). d , Labeling density of tdT + /EYFP + cells in NAc medial shell for each condition ( n  = 5 mice for NAc Shelter , n  = 4 mice for NAc Random ; t 7  = 0.51, P  = 0.63, two-sided Student’s t -test). Error bars indicate the s.e.m. e – g , Escape behavior across conditions, where each threat was presented alone (left) or with concurrent 589-nm light delivery to the NAc (right). NAc Shelter ::NpHR inactivation condition ( n  = 10 mice; e ). NAc Random ::NpHR inactivation condition ( n  = 4 mice; f ). NAc Shelter ::EGFP labeling condition ( n  = 8 mice; g ). Top: distance to the shelter. Middle: normalized flight traces. Bottom: polar histograms of head-shelter direction distribution. h , Quantification of head-shelter angle during flight upon threat stimulus ( F 5,89  = 5.74, P  = 1.24 × 10 −4 , two-way ANOVA followed by a Fisher’s least significant difference (LSD) post hoc test). i , Shelter-reaching percentage ( F 5,38  = 5.95, P  = 3.71 × 10 −4 , two-way ANOVA followed by a Fisher’s LSD post hoc test). Box plots show the mean (square), the median (horizontal line) and percentile ranges (25th–75th, box; 10th–90th, whiskers). Error bars indicate the s.e.m. * P  < 0.05, ** P  < 0.01, *** P  < 0.001. For detailed statistical information, see Supplementary Table 1 .

Forty-eight hours after labeling, we examined whether inhibiting the NAc Shelter affected escape behavior to shelter. For probe trials, mice explored the arena again, with a 7-min acclimation period with the shelter (positioned in the same location as during labeling) before undergoing the inactivation test. Inhibiting NAc Shelter during threat presentation disrupted the escape response to the shelter (Fig. 5e ). Similarly, while optogenetic activation of dPAG neurons alone prompted mice to escape to the shelter, inhibiting NAc Shelter while activating dPAG neurons impaired the heading accuracy to shelter during escape (Extended Data Fig. 7 ). Inhibiting NAc Random had no such effects on escape behavior to shelter (Fig. 5f ). Escape directionality and shelter-reaching percentages were largely decreased with NAc Shelter inhibition, but not with NAc Random inhibition (Fig. 5h,i ). Post hoc analyses showed similar labeling quantities in NAc Shelter and NAc Random , indicating that behavioral differences were not due to the number of cells manipulated (Fig. 5c,d and Extended Data Fig. 8 ). In another control condition with EGFP expression in NAc Shelter , mice exhibited normal escape behavior to shelter (Fig. 5g ), confirming that disruption of escape behavior was mediated by specific inhibition of the shelter-associated ensemble, and not by inhibition of arbitrary neuronal subsets or visual interference from laser lights. These results suggest that the shelter-associated ensemble plays a necessary role in guiding spatial navigation during escape.

Reactivating NAc Shelter ensemble facilitates memory-guided escape navigation

Forty-eight hours after NAc Shelter labeling with Cal-Light, we assessed memory-guided escape in the absence of shelter by reactivating NAc Shelter ensemble during escape (Fig. 6a ). Without reactivation, mice fled to random locations in the arena lacking shelter upon threat delivery (Fig. 6b ), showing that their escape was not necessarily guided by memory from previous shelter experience during labeling that occurred 48 h ago. Reactivating NAc Shelter during threat presentation, however, directed their escape to the previous shelter location (Fig. 6b–f ). We further explored whether co-activating engaged neural circuits including NAc Shelter and dPAG could replicate memory-guided escape behavior. Similarly to the effect of threat presentation, while the activation of dPAG alone led mice to random escape routes in the arena lacking shelter (Fig. 6g ), co-activation of dPAG and NAc Shelter directed their escape to the previous shelter location (Fig. 6g–l and Supplementary Video 6 ). Escape vigor measured by movement speed and initial acceleration was not different between dPAG activation alone and co-activation of dPAG and NAc Shelter (Extended Data Fig. 9a,b ). The goal-directedness of the escape, measured as the cosine of the angle between heading direction and direction to the previous shelter location, was higher during co-activation of dPAG and NAc Shelter than during dPAG activation alone (Fig. 6i–l ).

a , Schematic for reactivating shelter-associated NAc ensembles (NAc Shelter ). Left: labeling session with blue light to the NAc at shelter entry. 48 h were given for TRE-ChrimsonR-mEmerald expressions in NAc Shelter . Right: probe trials with co-delivery of either threat stimulus or optogenetic activation of dPAG, and amber light (589 nm) to NAc to reactivate labeled neurons (NAc Shelter ::ChrimsonR) in the absence of shelter. b – f , Escape behavior in NAc Shelter ::ChrimsonR reactivation test ( n  = 6 mice) during probe trials with threat stimuli alone (top) or with concurrent NAc Shelter reactivation (bottom). b , Distance to previous shelter location aligned to flight onset, for each escape trial. c , Normalized flight traces to previous shelter location. d , Polar histogram of head direction to previous shelter location. e , Normalized distance to previous shelter location aligned to threat onset. Gray indicates threat alone; red indicates reactivation of NAc Shelter alone; orange indicates both. f , Head-shelter direction for threat delivery alone (gray) and with concurrent NAc Shelter reactivation (orange; threat: 80.7 ± 6.30 from 26 trials; threat + NAc Shelter : 58.9 ± 4.39 from 28 trials, n  = 6 mice, t 52  = 2.87, P  = 5.87 × 10 −3 , two-sided Student’s t -test). g , Example escape traces triggered by activation of dPAG alone (left) or with concurrent NAc Shelter reactivation (right) during probe trials. h – l , Escape behavior in NAc Shelter ::ChrimsonR reactivation test with dPAG activation ( n  = 5 mice) during probe trials, with dPAG activation, which occurred either alone (top) or with concurrent NAc Shelter reactivation (bottom). h , Distance to previous shelter location aligned to flight onset, for each escape trial. i , Normalized flight traces to previous shelter location. j , Polar histogram of head direction to previous shelter location. k , Normalized distance to previous shelter location aligned to dPAG activation onset. Gray indicates dPAG activation alone; red indicates reactivation of NAc Shelter alone; orange indicates both. l , Head-shelter direction for dPAG activation alone (gray) and with concurrent NAc Shelter reactivation (orange; dPAG: 94.7 ± 5.83 from 48 trials; dPAG + NAc Shelter : 49.1 ± 3.82 from 24 trials, n  = 5 mice, t 70  = 5.24, P  = 1.64 × 10 −6 , two-sided Student’s t -test). Box plots show the mean (square), the median (horizontal line) and percentile ranges (25th–75th, box; 10th–90th, whiskers). Shaded area indicates the s.e.m. ** P  < 0.01, *** P  < 0.001. For detailed statistical information, see Supplementary Table 1 .

To determine whether goal-directed escape is guided by NAc Shelter and not by arbitrary neuronal sets, we conducted random labeling control (NAc Random ) and dark control (sham labeling with no blue light; NAc Dark ) experiments. In these control conditions, neither the co-activation of NAc Random and dPAG nor the co-activation of NAc Dark and dPAG elicited goal-directed escapes to the previous shelter location (Extended Data Fig. 9c–e ), indicating that the behavioral causality of shelter ensemble reactivation was not triggered by simply activating a set of random neuronal populations. Notably, activating NAc Shelter alone, without threat presentation or dPAG activation, did not change locomotion patterns (Fig. 6e,k ), suggesting its function as a memory module, whose manifestation depends on an escape drive to guide goal-directed behavior.

Cell-type-specific compositions of NAc Shelter ensemble

To determine cell-type-specific contributions to shelter memory, we identified the transcriptome compositions of the NAc Shelter ensemble via in situ single-molecule mRNA fluorescence in situ hybridization (FISH), focusing on three cell types: Drd1 medium spiny neurons (MSNs), Drd2 MSNs and cholinergic (ChAT) interneurons. We generated a Cal-Light solo construct by incorporating two Cal-Light constructs into one vector and removing a viral infection fluorescence marker, allowing us to link neuronal activities in the ensemble to their cell types identified by FISH (Fig. 7a ). To increase the temporal precision of functional labeling, we implemented one additional CreER T2 system. In combination with the Cal-Light solo and TRE-CreER T2 as a reporter, we were able to label a selective population undergoing coincident neuronal activity and blue light exposure in a task-induced manner (Fig. 7a,b ). To suppress task-irrelevant TRE-CreER T2 expression, animals were fed with doxycycline (DOXY)-containing food for 2 weeks before the blue light labeling session. Normal food was resumed 2 days before the labeling session, and tamoxifen was administered to induce task-relevant CreER T2 expressions 1 day after the labeling session (Fig. 7c ). Goal-directed behavioral causality during escape behavior was reproduced using this construct (Fig. 7d,e ).

a , Illustration of Cal-Light solo system, consisting of AAV 1 -hSyn-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA-P2A-M13-TEV-C, AAV 1 -TRE-CreER T2 and AAV 5 -Syn-Flex-ChrimsonR-tdTomato; a mixture of the above three viruses was transfected into NAc neurons. b , Experiment schematic: a mixture of Cal-Light solo constructs and ChR2-EYFP was injected into the NAc and dPAG, respectively, with optic fibers implanted. c , Experiment timeline using Cal-Light solo system. After virus injection, DOXY food was given to mice to suppress CreER T2 expression. DOXY food was then stopped for 2 days before the labeling session of NAc Shelter , in which blue light was delivered to the NAc upon shelter entry, followed by tamoxifen injection to promote Cre-dependent gene expression (ChrimsonR-tdTomato) and reintroduction of DOXY food to prevent potential leaky reporter gene expression in nonrelevant neurons. Two days after tamoxifen injection, a probe session was performed to induce memory-guided escape behavior in a shelter-removed arena by co-activation of dPAG and NAc Shelter . d , Confocal image of NAc medial shell expressing Cal-Light solo-positive neurons (indicated by red arrows) and DAPI (blue). Scale bar, 100 µm. e , Example escape traces for dPAG activation alone (left) and co-activation with NAc Shelter neurons (right). Color indicates speed. f – h , Multiplex fluorescence detection of Drd1 (yellow), Drd2 (magenta), ChAT (red) and tdTomato (green; tdT, neuronal activity reporter; n  = 4 mice). Scale bar, 100 µm. aca, anterior commissure ( f ). Representative images of NAc neurons depicting expression of transcripts for Drd1 , Drd2 , ChAT and tdTomato expression in NAc medial shell ( g ). Example cells classified as ChAT + (left), Drd1 + (middle) and Drd2 + (right) ( h ). i , Top: 3D distribution of Drd1 +, Drd2 + and ChAT + transcripts. Each sphere represents a cell, with size indicating tdTomato transcript intensity and color denoting classified cell types (total, 31,751 cells); Drd1 + (yellow), Drd2 + (magenta), ChAT + (red) and ‘not specified’ (gray). Bottom: percentages of classified cell types, with Double+ indicating cells classified into two types. j , Doughnut charts showing the distribution of tdTomato+ cells in total NAc neurons (top) and cell types among active tdT+ cells (bottom). k , Distributions of tdTomato mRNA (activity reporter) in Drd1+, Drd2+, ChAT+ and ‘not specified’ cell types ( n  = 4 mice, F 3,30469  = 1421.2, P  = 0, one-way ANOVA with Bonferroni post hoc test). Box plots show the median (horizontal line) and percentile ranges (25th–75th, box; 10th–90th, whiskers). l , Doughnut charts showing the percentages of tdT+ cells that coexpress ChAT mRNA (ChAT+/tdT+), Drd1 mRNA (Drd1+/tdT+), Drd2 mRNA (Drd2+/tdT+) or other (other/tdT+) in the NAc. For detailed statistical information, see Supplementary Table 1 .

The mRNAs of Drd1 , Drd2 , ChAT subtypes and tdTomato (activity marker) in NAc neurons were confirmed by FISH using a multiplex RNAscope assay (Fig. 7f–h ). Machine-learning-based categorization identified subtypes of ~30,000 cells in the NAc medial shell, analyzing cells with clear tdTomato+ (activity marker) signals (Fig. 7i ). All three Drd1 +, Drd2 + and ChAT + signals were detected in the NAc Shelter ensemble labeled by Cal-Light solo, with the Drd1 + subset being the most abundant (Fig. 7j ). The ChAT + subset showed the highest tdTomato expression in its mRNA (Fig. 7k ) and the highest percentage of tdTomato + cells (Fig. 7l ). Drd1 + and Drd2 + subsets ranked second and third in these expressions, respectively. These results suggest that ChAT interneurons, along with Drd1 + and Drd2 + neurons, contribute to forming a location-associated ensemble. The single-cell RNAscope analysis suggests that shelter-memory formation is mediated by coordinated activity among multiple neuronal cell types, not by a single type alone.

Animals rapidly encode spatial memory even after a brief experience of shelter location, which subsequently guides effective escape under threat 3 . Despite the prevalence of such purposeful spatial navigation across animal species, an understanding of the neural mechanisms underlying how the brain sets a spatial goal and directs navigation toward it is limited. Here we showed that the convergence of spatial-contextual vHPC inputs and reward VTA DA signals onto the NAc contributes to the formation of a shelter-related representation. We further uncovered the causal role of a distinct NAc ensemble that arises in response to shelter experience, specifically in guiding memory-based escape behavior. Our findings support a crucial function of DA in facilitating the rapid formation of NAc cell assemblies and in translating spatial memory into actionable behaviors through these assemblies, especially when driven by specific motivational needs.

While the role of VTA DA signals in reward processing and reinforcement learning has long been identified under appetitive settings 33 , here we explored the role of VTA DA signals in encoding safety during escape behavior and supporting the formation of spatial memory under aversive settings. We observed that the activity of VTA DA neurons transiently decreased upon exposure to threat stimuli. Such decrease may be mediated by VTA GABA neurons, which have been shown to be activated and in turn inhibit neighboring VTA DA neurons when a looming stimulus was presented 34 . VTA GABA neurons reduce the activation of VTA DA neurons and DA release in the NAc 35 . Although the firing rates of VTA GABA neurons are known to change during movement 36 , it is unlikely that movement itself directly mediates the activation of VTA GABA neurons, as increased VTA DA activity was observed during rest inside the shelter and at the termination of threat stimulus. Instead, the source of VTA GABA neuron activation may come from the detection of a threat or an aversive state, as supported by evidence that VTA GABA neurons are robustly activated by aversive or stressful stimuli such as foot shock, air-puff or looming stimulus 37 , 38 and may mediate defensive behavior 34 . We observed that the activity of VTA DA neurons increased upon shelter entry and termination of threat, suggesting that VTA DA neurons encode a safety signal as mice experience a state of relief at these events. Similarly, DA in the NAc showed an initial dip followed by a rapid increase in DA before and peaking upon shelter entry. Indeed, it has been shown in an active avoidance learning task that successful escape from an impending threat is accompanied by a long-lasting increase in ventromedial striatal DA levels 39 . In addition, increase in DA release in the NAc has been reported during periods of safety after escape from an aversive stimuli 40 . These results are consistent with our observation of peak DA signals upon shelter entry and support the theory that the neural correlates of safety are a form of reward 26 encoded by DA.

It is noteworthy that DA dynamics in this threat-induced approach toward safety contrasts with the well-known ramping of DA signals during approach toward food reward, which is thought to provide a sustained motivational drive toward a goal 18 , 41 . While conventional ramping under appetitive settings displays a steady increase in DA over a prolonged time frame (5–10 s), DA release during threat-induced approach increased rapidly (1–2 s) and was preceded by a noticeable dip. These distinct DA dynamics displayed in aversive and appetitive settings may result from valence coding in VTA DA neurons 37 . Importantly, the bidirectional modulation of DA activity by experiences of shelter and threat suggests its role in encoding not only simple motivational states but also an internal state of safety reflecting the onset of a threat and position with respect to shelter. The dynamic dip and peak DA patterns shown in this study may trigger the activation of Drd2 receptors during DA dip and Drd1 receptors of MSNs upon DA peak, which can together mediate bidirectional synaptic plasticity in NAc neurons for the suppression and facilitation of actions 30 , 42 , 43 . We cannot rule out the possibility of distinct DA dynamics between the VTA and NAc, which may be due to control of extracellular DA release in the NAc by inputs from the lateral hypothalamus, whose activity has been shown to increase DA in the NAc during an escape task 44 . Alternatively, it may be attributed to local DA control mechanisms in the NAc that are independent of VTA DA neuron firing 18 , such as through ChAT interneurons whose synchronized activity has been shown to directly generate DA signals in the striatum 45 .

Our data showed that phasic optogenetic stimulation of VTA DA neurons in the absence of shelter elicited escape behavior to the stimulation location upon threat delivery. It is well known that direct phasic stimulation of VTA DA neurons establishes conditioned place preference of one location over another 46 , consistent with our findings. Here, we further showed the involvement of phasic VTA DA stimulation in forming a spatial memory of a rewarding place. The spatial precision of the location-locked optogenetic stimulation used in this study suggests that the role of phasic VTA DA neurons may be more specific than establishing a conditioned preference between two chambers as previously shown in conditioned place preference paradigms. Pairing phasic VTA DA signals with a specific location within an environment may support the formation of spatial memory of the location, a result that complements a theoretical model accounting for the sufficiency of phasic VTA DA activity to drive learning via the effects of DA on inference 47 . Next, phasic VTA DA stimulation has been shown to influence motivated behavior under aversive settings. For instance, a past study showed that phasic VTA DA stimulation or inhibition during the onset of a warning signal enhances or diminishes avoidance of aversive stimuli, respectively, and particularly stimulation promotes the selection of active over passive defensive behaviors 48 . Here, we showed that spatial memory induced by phasic VTA DA stimulation can be retrieved by salient motivational states such as threat to guide navigation under aversive settings. Taken together, heightened phasic VTA DA signals during periods of shelter experience may act as safety reward signals to induce the learning of spatial memory of shelter location to be subsequently retrieved under threat.

We found that DA inputs to the NAc medial shell during shelter experience are required to form location-specific responses in NAc subpopulations. Unconditioned rewarding stimuli such as unfamiliar appetitive tastes or addictive drugs cause preferential DA release in the NAc medial shell, but not in other regions of the ventral or dorsal striatum 28 , 49 , 50 , 51 , 52 , 53 . Conversely, unconditioned aversive tastes reduce DA release in the NAc shell but not in the NAc core 53 . Thus, NAc shell neurons encode both directions of hedonic valence associated with unconditioned stimuli. In addition, DA responsiveness in the NAc shell has been shown to habituate after a single episode of food experience 49 , suggesting that NAc shell DA transmission is dependent on the novelty of stimuli. In contrast, DA release in the NAc core has been shown to be responsive to Pavlovian food-conditioned stimuli and reward-predictive cues 51 , 54 , 55 , independent of the hedonic valence of delivered stimuli, and lack habituation after single episodes of stimulus experience 49 . Several studies have suggested that the role of NAc core DA transmission may be in encoding motivational value evoked by conditioned stimuli, such as the recruiting of active or passive forms of behavioral strategies via increased or decreased DA release, respectively 40 , 56 .

Unconditioned rewarding stimuli selectively modify excitatory synapses on DA neurons projecting to the NAc medial shell 57 . Such synaptic plasticity may be involved in shaping a NAc ensemble representing a memory of the rewarding stimuli, such as a shelter location providing safety as observed in this study. Moreover, optogenetically induced DA release in the NAc shell during exposure to unconditioned stimuli has been shown to bias a preference for the stimulus option when presented as a choice 58 . Consistently, we showed that shelter memory (a ‘preference’ for the location in context of threat) can be formed via phasic optogenetic stimulation of VTA DA neurons. Thus, elevated DA signals from the VTA to the NAc shell induced by safety may drive the formation of spatial memory of the shelter.

Combining the role of DA signals in encoding internal affective states and reward value, we propose that NAc DA signals encode a combined form of state–value functions rather than either motivation or reward per se. Such a state–value function of DA can be used to update the value of each location based on physiological states. It can effectively control the suppression of irrelevant responses and facilitate actions toward the current goal. Decreasing NAc activity during shelter approach is consistent with electrophysiological data showing sustained inhibition in the firing rate of a subset of NAc neurons, whose onset precedes goal-directed behavior. Such inhibition is important for suppressing competing behaviors 59 .

Cellular dynamics of NAc neurons revealed that distinct ensembles for coding shelter and non-shelter locations emerge with shelter experience. Such a separation is consistent with an electrophysiological study that showed the presence of two principal classes of NAc neurons involved in consummatory behavior: a class of neurons whose firing rate decreased before reinforcer consumption and another class whose firing rate increased during consumption based on the value of reinforcers 60 . In addition to DA-mediated bidirectional synaptic plasticity in NAc neurons, distinct firing patterns of goal-related excitation and inhibition in vHPC neurons to the NAc may contribute to the conversion of spatial–episodic hippocampal information into goal-related spatial coding in NAc neurons 61 , although we cannot completely rule out the possibility of other HPC target brain areas such as the hypothalamus, which was shown to be important for controlling context-dependent escape behavior 62 . As the functional interface between limbic and motor systems, the NAc projects to downstream basal ganglia circuits 63 and the VTA 64 for the control of actions and modulation of reward processing, respectively. Notably, the NAc medial shell predominantly innervates the ventromedial ventral pallidum (VP), whose GABAergic and glutamatergic neurons regulate reward-seeking and threat-avoidance behaviors, respectively 65 . Thus, we speculate that the emerged distinct populations of GABAergic afferents in the NAc to the VP differentially affect the subpopulation of GABAergic and glutamatergic neurons in the VP to control actions for goal-directed escape 66 . Furthermore, distinct subpopulations of Drd1 MSNs in the NAc, which receive efficacious excitatory afferents from the vHPC, directly project to the VTA 67 , 68 . Given that the convergence of vHPC Glu inputs and VTA DA inputs to the NAc is required for goal-memory formation and heterogeneous spatiotemporal DA control of NAc neurons, this pathway-specific Drd1 MSN output to the VTA could regulate mesolimbic DA release to further facilitate DA-mediated synaptic plasticity that may underlie specific goal-memory formations.

Given that the NAc ensemble associated with shelter consisted of coordinated activity of ChAT interneurons, Drd1 MSNs and Drd2 MSNs, future studies investigating cell-type-specific interactions could further clarify mechanisms that enable rapid memory encoding and retrieval. A tripartite model has been suggested, according to which ChAT interneurons, Drd1 MSNs and Drd2 MSNs function cooperatively in the NAc to enable predictive learning of appropriate responses to pleasurable or aversive stimuli 69 . It is thought that ChAT interneurons detect salient motivational stimuli 70 and mediate distinct Drd1-dependent and Drd2-dependent striatal plasticity needed for learning 45 , 71 . Because our data depicting the functional contributions of these cell types to memory ensembles recapitulate this circuit model, further investigation of the dynamics of this circuit will be fruitful for our evolving understanding of the cellular–molecular mechanisms of learning and memory.

Our study showed that the NAc adaptively encoded shelter experience at the level of individual neurons and formed distinct functional subpopulations, and targeted optogenetic manipulation of these ensembles unveiled their causal role in guiding escape behavior, but not triggering escape behavior itself. This feature is distinct from other behavioral causality tests via reactivation of memory-associated neurons 72 . The NAc ensemble was differentiated in several aspects from a memory engram, which is formally defined by (1) the persistence of chemical and/or physical changes induced by experience in a population of neurons and by (2) its sufficiency to retrieve memory and the associated memory-driven behavior upon reactivation 73 . Shelter memory could be formed by brief shelter experience rather than by repeated associations between threat stimuli and shelter experience, indicating that escape behavior was guided by expectation of safety available at the shelter rather than conditioned responses. The effects of this rapid learning were dependent on CaMKII expression, suggesting that shelter memory is mediated by DA-dependent synaptic plasticity 30 during shelter experience. Instead of persistent storage of memory by engram cells, the shelter memory shown here may reflect flexible and transient aspects of working memory to be selectively recruited under salient motivational states. Importantly, after acclimation with shelter, the memory-guided escape behavior was reliably triggered by the novel encounter of threats or the activation of dPAG, suggesting that the shelter memory is retrieved by salient internal states such as threats. This property differs from the reinstatement of memory-driven behavior by the reactivation of engram cells. While artificial activation of engram cells was sufficient to reinstate memory-driven behavior in previous studies 72 , 74 , 75 , the artificial activation of the NAc ensemble alone did not trigger escape behavior in our study. This contingent function of the NAc ensemble suggests its role as a goal representation that guides but does not initiate goal-directed escape behavior. Taken together, goal-directed escape behavior may be controlled by the concurrent activity of a motivational drive and an expectation of the desired outcome. Future studies will need to investigate whether different types of reward, such as food and water, give rise to dynamic goal representations in the NAc to facilitate the selection of appropriate goal-directed behavior under corresponding motivational states such as hunger and thirst, respectively.

We propose a cognitive module that bridges memory and action, drawing inspiration from Tolman’s Cathexis theory 76 that describes the dependence of goal-directed action on two distinct processes: the strength of connections (‘cathexis’) between representations of potential goals and motivational states, and the instrumental association between action and outcome. A modular memory stores only a representation of the outcome, whose value as a goal depends solely on the strength of its connection with a distinct system that provides motivational drive based on innate or learned consummatory behaviors associated with the outcome. Consequently, it requires inputs from other brain regions to establish it as a goal within a specific context before the agent can initiate appropriate actions to achieve the desired outcome. Our observations suggest that the dPAG acts as a threat system determining the agent’s internal motivational state, while the NAc contains a modular memory of the shelter location, ready to be activated as a goal by motivational input. VTA DA signals likely encode the agent’s relief responses during shelter experiences, reinforcing the connection between the threat system and shelter representation. This reinforcement increases the reward value of the shelter within the motivational context of threat. Taken together, the NAc adaptively provides a goal-related memory module during motivated navigation, raising the possibility that a complex repertoire of cognitive actions is accomplished through a flexible combination of separate cognitive modules, rather than by creating an exhaustive set of predetermined action sequences.

Adult male and female C57BL/6 (stock no. 664), DAT-Cre mice (stock no. 6660), and Camk2a fl/fl knockout (KO) mice (stock no. 6575) from Jackson Laboratory were used for experiments (4–12 weeks old). All mice were maintained on a 12-h light–12-h dark cycle at an ambient temperature of 22–24 °C and a relative humidity level of 35–60%. All experimental procedures were carried out in accordance with protocols approved by Johns Hopkins University Animal Care and Use Committee, the Max Planck Florida Institute for Neuroscience Institutional Animal Care and Use Committee and National Institutes of Health (NIH) guidelines.

Stereotaxic surgery for viral injections and optical fiber implants

Surgeries were performed aseptically on 4–6-week-old mice using a stereotaxic instrument (Kopf Instruments). Mice were anesthetized with an intraperitoneal injection of an anesthetic cocktail containing ketamine (87.5 mg per kg body weight) and xylazine (12.5 mg per kg body weight; Sigma-Aldrich). The scalp was shaved and treated with hair removal lotion (Nair, Church & Dwight). Ophthalmic ointment (Puralube Vet Ophthalmic Ointment) was applied to prevent the eyes from drying. Mice were then placed in the stereotaxic device, and the surgical site was scrubbed with 10% betadine solution (Purdue product LP). The body temperature (37–38 °C) was maintained with a thermostatically controlled heating pad (Harvard Apparatus). A small incision was made on the scalp, followed by a craniotomy (0.5 mm in diameter) above the injection site; Coordinates were as follows: NAc for virus injection alone, anteroposterior (AP): +1.3 mm, mediolateral (ML): ±0.5 mm, dorsoventral (DV): −3.9 mm from the brain surface; NAc for 7° angled virus injection and fiber implant, AP: +1.3 mm, ML: ±1.0 mm, DV: −4 mm from the brain surface; VTA for virus injection and fiber implant, AP: −3.25 mm, ML: ±0.5 mm, DV: −4 mm from the brain surface; vHPC for virus injection and fiber implant, AP: −3.7 mm, ML: ±3 mm, DV: −4 mm from the brain surface; dPAG for virus injection and fiber implant, AP: −4.5 mm, ML: ±0.25 mm, DV: −2.2 mm from bregma for virus injection and −1.3 mm from the brain surface for fiber implant. Viral constructs were injected with a beveled glass micropipette (10–20-μm tip diameter, Braubrand) backfilled with mineral oil at a flow rate of 100–150 nl min −1 using a syringe pump (World Precision Instruments). After injection, the micropipette was left in place for 5 min to prevent backflow. Following virus injection, either an optic fiber cannula (200-μm core diameter; Thorlabs) for optogenetic manipulation or a large-core optic fiber cannula (400-μm core diameter; Doric Lenses) for fiber photometry was implanted above the injection site and secured with dental cement (C&B Metabond, Parkell). Analgesia (Buprenorphine SR, 0.6 mg per kg body weight) was given subcutaneously, and mice were monitored until fully mobile.

Behavior experiments

Standard escape behavior.

Experiments were conducted in a black open-top chamber (86 cm wide, 69.5 cm deep, 56.5 cm high) with four distinctive visual cues on surrounding walls. The cues varied in shape and color, and had dimensions ranging from 15 cm to 28 cm. The LCD monitor was placed on top of the chamber. A white turntable circular arena of 53.4 cm in diameter was placed 39 cm below the center of the monitor in the chamber. A small shelter (9 cm wide, 5 cm deep, 11 cm high) consisting of three corrugated board walls and an arch-shaped top with an entrance facing the arena center was placed in the arena. The location of the shelter was determined randomly, peripherally for each mouse. A small piece of home-cage bedding was placed inside the shelter to help with acclimation. Mice were placed in the arena and given 7 min to explore and visit the shelter for acclimation. If they did not visit the shelter during this period, an additional 5 min of acclimation was allowed. Sessions were excluded if mice did not find shelter during the acclimation period or stayed in it for 20 min. One session each for a Camk2a- knockout mouse and a vHPC-NAc mouse under the CNO condition were excluded from data analysis based on these criteria. After acclimation, four to six rounds of auditory or visual stimuli were presented in a pseudorandom manner when mice were outside the shelter to minimize desensitization to the threat stimulus due to repeated exposures. When mice were outside the shelter, these threat stimuli were delivered to mice with a minimum 1-min interval to test whether the stimulus triggers escape behavior (the number of stimuli presented per animal: 3.83 ± 0.27 for the auditory, 2.5 ± 0.23 for the visual, mean ± s.e.m). Auditory stimulus (15 kHz or 17 kHz pure tone, 10 s, ~70–80 dB) was delivered through portable speakers from the top of the chamber. The visual stimulus consisted of an expanding black disk, known as a ‘looming disk,’ on a gray background. The visual stimulus expanded from a diameter of 2° of visual angle to 20° within 250 ms, and the expanded circle remained for an additional 250 ms 24 . The visual stimulus was repeated ten times with a 500-ms interstimulus interval (ISI) (duration of 10 s). Consistent with a previous study 3 , both sensory modalities of stimuli triggered escape behavior. To remove the influence of odor from a previous session on behavior, the arena and shelter were wiped and cleaned with 70% ethanol between individual sessions. All the experiments were video-recorded by a camera (GoPro HERO3 Silver Edition or Ethovision camera) placed above the arena. Behavioral variables of mice, such as position, speed, acceleration and head direction, were assessed using DeepLabCut 77 combined with custom scripts in MATLAB (MathWorks) or Ethovision XT 14 (Noldus). Shelter-reaching percentage was defined as the percentage of correct escape during threat presentation to the shelter location or previous shelter location when the shelter was removed.

Escape behavior task without shelter acclimation

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, but the shelter was removed during the acclimation period and reintroduced afterwards. Only 1–2 rounds of auditory or visual stimuli were presented to mice for testing escape behavior to prevent mice from acclimating to the shelter during the test period.

Escape behavior task with a change in shelter location after acclimation

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, except that the shelter location was changed after the acclimation period. Next, four rounds of visual stimuli were presented to mice for testing escape behavior. For each visual stimulus, the location of the shelter was changed to a new location during the ISl.

Escape behavior task in the absence of shelter after shelter acclimation

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, except that the shelter was removed after the acclimation period. Next, four to six rounds of auditory or visual stimuli were presented to mice in a pseudorandom order for testing whether they escaped to the previous shelter location.

Escape behavior task with rotated visual cues after shelter acclimation

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, except that all visual cues on the surrounding walls were rotated 180° and the shelter was removed after the acclimation period. During this rearrangement, mice were isolated in a black cylinder in the center of the arena. Upon release, four to six rounds of auditory or visual stimuli were presented in a pseudorandom order to test whether mice escaped to the previous shelter location based on the new cue arrangement.

Escape behavior task in the dark following shelter acclimation

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, except that the shelter was removed, and escape behavior was tested in darkness after the acclimation period using only auditory threats. Two infrared illuminators were activated inside the chamber while room light and the monitor were turned off. During this transition to darkness, mice were isolated into a black cylinder. Upon release, three rounds of auditory stimuli were given to assess escape behavior to the previous shelter location.

Escape behavior task with shelter acclimation in the dark

Mice were placed in the arena in the same manner as ‘Standard escape behavior’, except that they were acclimated to the shelter in darkness and the shelter was removed after the acclimation. Two infrared illuminators were activated inside the chamber while room light and the monitor were turned off during the acclimation period. After acclimation, mice were isolated in a black cylinder, the shelter was removed, the room light and the monitor were turned on, then the animals were released from the cylinder. Upon release, four to six rounds of auditory or visual stimuli were presented in a pseudorandom order to test whether mice escaped to the previous shelter location.

Identification of behavior bouts

We classified each behavior frame (sampling rate, 100 Hz) as rest or movement. Rest was defined by movement speed under 0.02 m s −1 and acceleration below 0.2 m s −1 . Movement occurred over speeds of 0.03 m s −1 , excluding rest frames. Flight onset required speeds above 0.1 m s −1 for at least 0.3 s. Shelter stay was noted when the mouse was within 8 cm of the shelter center for over 0.5 s. These criteria were established by visual comparison with videos and corresponding locomotion traces. The goal angle was defined as the angle between the mouse’s heading and the direction to the shelter.

Fiber photometry recording

For monitoring the activity of VTA DA neurons, we used a viral approach to express the genetically encoded calcium indicator GCaMP6f 78 . Under anesthesia with an intraperitoneal injection of an anesthetic cocktail containing ketamine (87.5 mg per kg body weight) and xylazine (12.5 mg per kg body weight) (Sigma-Aldrich), AAV 1 -Syn-Flex-GCamk6f-WPRE-SV40 (Addgene, 100833; 400 nl) was slowly injected (100–150 nl min −1 ) through a beveled glass micropipette (tip size 10–20-μm diameter, Braubrand) backfilled with mineral oil into the VTA (AP: −3.25 mm, ML: +0.5 mm, DV: −4.0 mm from the brain surface) of DAT-Cre mice. After virus injection, a 400-μm diameter optical fiber with a high numerical aperture (NA; 0.66) attached to a metal ferrule (Doric Lenses) was implanted 200 μm above the injection site and secured with dental cement (C&B Metabond). Three weeks later, fluorescence data were acquired during an escape behavior task.

For monitoring the activity of NAc neurons in response to optogenetic stimulation of vHPC Glu neurons, we injected AAV 1 -hSyn-GCaMP6f (400 nl, Addgene) into the NAc and AAV 2 -CaMKIIa-bReaChES-TS-eYFP (400 nl, UNC Virus Vector Core) into the vHPC. Following virus injection, we implanted a large-core (400-μm diameter), high-NA (0.66) optical fiber attached to a metal ferrule (Doric Lenses) into the NAc for photometry recording and a fiber-optic cannula (fiber: 200 μm core, 0.37 NA, BFL37-200, Thorlabs; ceramic ferrule: CFLC 230, Thorlabs) into the vHPC for optogenetic stimulation ~100–150 µm above the virus injection site. For photometry recording upon optogenetic stimulation, we delivered a series of lights to mice in their home cage. Light at a wavelength of 589 nm (5-ms pulse duration, 8 Hz, 15 mW power at tip) was delivered to the vHPC for a duration of 1 s, 3 s or 5 s.

For all GCaMP6 imaging, a continuous sinusoidally modulated light-emitting diode (LED) of 470 nm (M470F3, Thorlabs, at 10–30 μW light intensity for imaging) was used. For dual-color imaging of dopamine and calcium transients in NAc neurons, we injected a mixture (1:1 ratio, total 750 nl) of AAV 9 -CAG-dLight1.1 (Vigene Biosciences, 1.42 × 10 13 genome copies (GC) per ml) and AAV 1 -Syn-NES-jRGECO1a-WPRE-SV40 (Addgene, 100854) into the NAc. Following virus injection, a large-core (400-μm diameter), high-NA (0.66) optical fiber, attached to a metal ferrule (Doric Lenses), was implanted 200 μm above the virus injection site. Two continuous sinusoidally modulated LEDs of 470 nm (M470F3, Thorlabs) and 565 nm (M565F3, Thorlabs, at 10–30 μW light intensity for imaging) were used. For optogenetic activation of VTA DA neurons, AAV 5 -Syn-Flex-rc[ChrimsonR-tdTomato] (Addgene, 62723) was unilaterally injected into the VTA and a fiber-optic cannula was implanted 150–300 μm above the injection site. Brief pulses of a 589-nm laser (5-ms pulse duration, 15 mW power at the tip) at various frequencies (5–50 Hz) were delivered to the VTA. Using a fluorescence minicube (FMC5, Doric Lenses), each LED light source was coupled to an optical fiber patch cord (400-μm diameter, NA 0.48, Doric Lenses). Each fluorescence signal was collected through the same fiber and Fluorescence MiniCube (FMC5, Doric Lenses; excitation range: 460–490 nm, 555–570 nm; emission range: 500–540 nm, 580–680 nm). The signals were delivered to a femtowatt silicon photoreceiver (New Focus 2151, Newport, Low mode).

Outputs from the photoreceivers were acquired and digitized by the photometry data acquisition system (RZ5P, Tucker-Davis Technologies) at a sampling rate of 100 Hz, and event timings were acquired by the same system via transistor–transistor logic (TTL) inputs. Photometry and event data were recorded using Synapse Essentials software (v95, Tucker-Davis Technologies).

Fiber photometry data analysis

The fluorescence signal was normalized in each mouse by calculating Δ F/F 0 . The time-varying baseline of fluorescence trace ( F 0 ) was calculated by the following procedure. The preliminary baseline of fluorescence trace ( F Prelim ) was estimated by taking a moving median of raw fluorescence trace ( F Raw ) with 10-min time windows. Then, preliminary ΔF Prelim was calculated by subtracting F Base from F Raw . The standard deviation of ΔF Prelim was estimated as a noise level. The baseline of fluorescence data ( F 0 ) was calculated by subtracting the noise level from F Prelim . Δ F/F 0 was then calculated as ( F Raw  −  F 0 )/ F 0 . In the dual-color fiber photometry recording, the conjunctive signal was calculated as the product of moment-to-moment Δ F/F 0 signals obtained from each channel. Data were analyzed using custom-written scripts in MATLAB.

Viral neuronal tracing

We used virus tracing to map and characterize NAc cell populations. For anterograde tracing, we bilaterally injected AAV 1 -CAG-Flex-GFP (400 nl) in the VTA (AP: −3.25 mm, ML: ±0.4 mm, DV: −4.0 mm from the dural surface) to label DA neurons and a mixture (1:1 ratio, 400 nl total) of AAV 1 -CamKII-0.4-Cre-SV40 and AAV 1 -CAG-Flex-tdTomato-WPRE-bGH in the vHPC (AP: −3.7 mm, ML: ±3.0 mm, DV: −4.0 mm from the dural surface) to label glutamatergic neurons in DAT-Cre mice. For retrograde tracing, we injected AAV rg -Flex-tdTomato (Addgene, 28306, 400 nl) in the NAc (AP: +1.3 mm, ML: +0.5 mm, DV: −3.9 mm) and AAV 1 -CamKII-0.4-Cre-SV40 (400 nl) into the vHPC (AP: −3.7 mm, ML: ±3.0 mm, DV: −4.0 mm from the dural surface) to trace vHPC neurons projecting to the NAc, and we injected AAV rg -Flex-tdTomato (400 nl) in the NAc in DAT-Cre mice for tracing VTA neurons projecting to the NAc.

Intracranial injections for manipulation with pharmacological drugs, 6-OHDA and region-specific Camk2a conditional knockout

Pharmacological manipulation was performed via intracranial injection of drugs using a stereotaxic setup (Kopf Instruments) via glass micropipettes with a beveled edge (30–40 μm, tip size). SCH23390 (Drd1 antagonist, 0.1 mg per kg body weight, Tocris, 925), haloperidol (Drd2 antagonist, 1 mg per kg body weight, Tocris, 931) or the neurotoxin 6-OHDA (9 µg in 400 nl, Tocris, 2547) was bilaterally injected into the NAc medial shell. The drug solutions were prepared with sterile PBS (pH 7.4). To prevent oxidation, ascorbic acid (0.25%, Sigma-Aldrich, A92902) was added to the 6-OHDA solution. Control mice received an equivalent vehicle solution. After injection, mice recovered on a heating pad until fully mobile.

For mice injected with SCH23390 or haloperidol, standard escape behavior tests were conducted 30 min after injection. For those injected with 6-OHDA, the tests were performed 7–10 days later. Experiments of Camk2a conditional knockout involved bilateral intracranial injection of either AAV9-CaMKII-HI.eGFP-Cre-WPRE-SV40 (105551, Addgene, 750 nl) or AAV9- CaMKII0.4.-eGFP-WPRE-rBG for control (105541, Addgene, 750 nl) into the NAc medial shell of the NAc in a stereotaxic setup (Kopf Instruments). After injection, mice recovered on a heating pad until fully mobile. Standard escape behavior tests were then conducted 3 weeks after the virus injection.

Tissue fixation, immunohistochemistry and acquisition of confocal and light-sheet microscope images

Animals were deeply anesthetized with isoflurane or a mixture of ketamine and xylazine, and then perfused transcardially with PBS (pH 7.4) followed by 4% paraformaldehyde (PFA) dissolved in PBS. The brains were removed and postfixed in 4% PFA overnight at 4 °C. For confocal microscopy, the brains were embedded into a 10% melted gelatin solution for 50 min at 50 °C. The melted gelatin solution was refreshed, and the embedded brains were solidified at 4 °C for ~30 min. The solidified gel was trimmed to a small cube around the brain and the cube was stored in 4% PFA overnight. The gelatin cube brain was sectioned (100-μm thick) using a vibratome (VT1000, Leica Biosystems). The coordinates of sections were labeled using landmarks and neuroanatomical nomenclature in accordance with the mouse brain atlas 79 . For DAPI staining to visualize the nucleus of the fixed cell, brain slices were mounted on the slide glass with a mounting solution containing DAPI (DAPI Fluoromount-G, SouthernBiotech). The images of brain sections were acquired using an upright laser scanning confocal microscope (Zeiss LSM880) with a ×20 or ×40 objective. TH staining was accomplished as follows: (1) coronally section the brain (40-μm thick) using a vibratome (Leica Biosystems); (2) rinse 40-μm-thick slices in PBS (pH 7.4) three times; (3) block slices in 10% normal goat serum (Thermo Fisher, 50062Z) with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 1 h at room temperature (RT); (4) incubate slices in rabbit polyclonal anti-TH primary antibody (1:700 dilution in PBS; Sigma-Aldrich, T8700) for 24 h at 4 °C; (5) rinse slices in PBS three times; (6) incubate slices in Cy3-conjugated goat anti-rabbit IgG (1:100 dilution in PBS, Jackson ImmunoResearch Laboratories, 111–165–003) and DAPI (1:1,000 dilution) for 2 h at RT on a rotary shaker; (7) rinse slices in PBS three times and mount with an Aqua-Poly/Mount solution (Polysciences). TH-positive fibers were imaged using a confocal microscope (Zeiss LSM800) and quantified by measuring the optical density of TH immunofluorescence using MATLAB. Confocal images were analyzed using FIJI software (ImageJ, NIH). For light-sheet microscope images, brains were perfused as described above and were coronally sectioned (2-mm thick) using a vibratome (Leica Biosystems). The brain slices were cleared by using the Passive Clarity protocol 80 . Images of the NAc medial shell were collected with a Zeiss Light-sheet microscope at ×25 magnification. Light-sheet microscope image processing and three-dimensional rendering were performed using Arivis Vision4D (Zeiss).

Chemogenetic manipulations

For chemogenetic silencing of glutamatergic neurons in the vHPC projecting to the NAc, we bilaterally injected retrograde Cre-dependent AAV rg -hSyn-DIO-hM4Di(Gi)-mCherry (400 nl, Addgene, 44362) 81 into NAc medial shell and AAV 1 -CamKII-0.4-Cre-SV40 (400 nl, Addgene, 51503) into the vHPC (AP: −3.7 mm, ML: ±3.0 mm, DV: −4.0 mm from the dural surface). For chemogenetic silencing of NAc neurons, we bilaterally injected AAV 5 -hSyn-hM4Di(Gi)-mCherry (400 nl, a gift from B. Roth, Addgene, 50475) into the NAc medial shell. CNO (Tocris, 4936/10) was freshly prepared from a stock solution of 0.5 mg ml −1 in 100% dimethyl sulfoxide and diluted to a final concentration of 1.5 mg per kg body weight with PBS for intraperitoneal administration 45 min before behavioral tests.

Optogenetic manipulations

Phasic and tonic optogenetic activation of vta activity.

We bilaterally injected AAV 1 -EF1-dFlox-hChR2-mCherry-WPRE-hGHpA (400 nl, Addgene, 20297) into the VTA in DAT-Cre mice and implanted a fiber-optic cannula (fiber: 200-μm core, 0.37 NA, BFL37-200, Thorlabs; ceramic ferrule: CFLC 230, Thorlabs) 200 μm above the injection site. After 3 weeks, we tested whether phasic or tonic activation of DA neurons sufficiently induces goal memory. Mice were placed in the arena and given a 3-min acclimation period without shelter. Blue light generated by a 473-nm laser (MBL-FN-473, Changchun New Industries Optoelectronic Technology; 15 mW power at the fiber tip) was delivered through the optic fibers at the phasic mode (5 ms, 20 pulses at 25 Hz for 5 s, 60 times) or the tonic mode (5 ms, 20 pulses at 4 Hz for 5 s, 60 times) when mice were located at a target location that was predetermined by the experimenter. TTL pulses were generated by Pulse Pal (Sanworks). After the induction with light, visual or auditory stimuli were presented in a pseudorandom order to examine flight behavior to the target location.

Optogenetic inactivation of NAc neurons

We bilaterally injected AAV 5 -hSyn-eNpHR3.0-EYFP (400 nl, Addgene, 26972) into the NAc and implanted fiber-optic cannulae (BFL37-200) 100–150 µm above the injection site. To inactivate NAc activity, yellow light generated by a 589-nm laser (MBL-FN-589, Changchun New Industries Optoelectronic Technology; 10 mW power at the fiber tip) was delivered through the optic fibers continuously during a 7-min acclimation period. After the acclimation period, the shelter was removed, and visual or auditory threat stimuli were presented in a pseudorandom order.

In vivo Ca 2+ imaging in freely behaving mice

Briefly, for Ca 2+ imaging in mice, AAV 1 -Syn-GCaMP6s-WPRE-SV40 (Addgene, 100843) virus was injected, and a GRIN lens (0.6-mm diameter, ~7.3-mm length, ProView Lens Probe, Inscopix) was implanted into the NAc medial shell, followed by a microscope baseplate (Inscopix) attachment 3–4 weeks later. Mice were anesthetized with 1.5% to 2.0% isoflurane mixed with oxygen for surgical procedures and placed into a stereotaxic frame (Kopf Instruments). Surgical procedures maintained aseptic conditions and body temperature was maintained as in stereotaxic surgeries for virus injection and optical fiber implants described above. An incision was made in the scalp, and the skin and muscle were retracted or removed. The fascia on the skull was removed, and the skull surface was cleaned. A craniotomy (0.6–1 mm in diameter) was made above the implant site. Once the bone was removed, a durotomy was performed. The exposed brain tissue was irrigated with artificial cerebrospinal fluid (ACSF). Cortical superficial layers (1–2 mm) above the NAc were gently aspirated. GCaMP6s virus was injected into the NAc using a beveled glass micropipette (tip size, 10–20-μm diameter, Braubrand) backfilled with mineral oil at 0.1 μl min −1 , regulated by a syringe pump (World Precision Instruments). For Ca 2+ imaging with 6-OHDA lesions, we bilaterally injected a mixture of GCaMP6s (300 nl) and 6-OHDA (9 µg in 300 nl) into the NAc medial shell. For Ca 2+ imaging with chemogenetic silencing of vHPC Glu -NAc neurons, we bilaterally injected a mixture of GCaMP6s (300 nl) and retrograde Cre-dependent AAV rg -hSyn-DIO-hM4Di(Gi)-mCherry (300 nl, Addgene, 44362) into NAc medial shell and AAV 1 -CamKII-0.4-Cre-SV40 (400 nl, Addgene, 51503) into the vHPC. Following virus injections, GRIN lens implantation was performed either on the same day or 1–2 weeks apart. Using a GRIN lens gripper (Inscopix), the GRIN lens was carefully inserted 100–300 μm above the target brain region. Silicone sealant (Kwik-Sil, World Precision Instruments) was applied around the lens to cover exposed brain tissue, and dental cement (C&B Metabond) was used to securely attach the lens to the skull. The lens top was sealed with silicone sealant for 1–2 weeks before assessing imaging quality. A microscope baseplate (Inscopix) was mounted using dental cement at the optimal focal distance (C&B Metabond). After surgery, mice were monitored until fully mobile and individually housed under a reverse light–dark cycle. Epifluorescence signals were initially checked 3–4 weeks after injection.

Data acquisition

Before behavior experiments, mice with a baseplate implant were acclimated using a plastic ‘dummy’ microscope (nVoke dummy, Inscopix) for about 30 min. On experiment days, mice were gently restrained, connected to a miniature microscope (nVoke2, Inscopix), and given 10 min of habituation to the attached microscope in their home cage. Subsequently, microendoscopic Ca 2+ imaging was conducted during standard escape behavior tasks using Inscopix Data Acquisition Software (Inscopix). Imaging data were collected at 10 Hz in grayscale (32 bit, TIFF format) with a 455-nm blue light LED at 20–80% power. The attached microscope did not noticeably impair locomotion or escape behaviors.

Imaging data processing

Imaging and behavioral data were synchronized and preprocessed using Inscopix Data Processing Software (version 1.2.1, Inscopix) and custom MATLAB scripts. Imaging data were preprocessed with a spatial band-pass filter cutoff (low: 0.005, high: 0.5) and motion correction based on the mean image. Then Δ F/F 0 was calculated as (F Raw  −  F 0 ) / F 0 , where F Raw and F 0 were raw fluorescence signals and the baseline fluorescence level, respectively. Individual regions of interest (ROIs) were semiautomatically drawn using principal component analysis–independent component analysis or a custom algorithm with the Cell Magic Wand plugin (ImageJ) based on fluorescence intensity, cell size and cell shape. We visually inspected raw frames of movies, average of movies, standard deviation of movies and selected neurons that showed a fluorescence transient at least once during the session. All selected ROIs were manually inspected and corrected. All pixels within each ROI in Δ F/F 0 data were averaged to create a fluorescence time series, Δ F/F 0 , for each ROI.

Ca 2+ imaging data analysis

We analyzed Ca 2+ imaging data together with behavioral data using custom MATLAB scripts. Ca 2+ transient event rates were calculated based on a nonnegative deconvolution algorithm using an auto-regressive model with an order of one and a nonnegative least square problem with three frame window shifts 82 .

Calcium-gated and light-gated labeling of the activated neuronal population

Experiments using cal-light constructs and surgery.

For labeling active neuronal population within a specific time window, a mixture (1:1:2 ratio, total 750 nl) of AAV 1 -Cal-Light (plasmid: AAV-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA, Addgene, 92392; virus production from Vigene Biosciences), AAV 1 -M13-TEV-C-P2A-TdTomato (plasmid from Addgene, 92391; virus production from Vigene Biosciences) and a reporter virus (AAV DJ -TRE-ChrimsonR-mEmerald, virus production from B. Lim’s laboratory at UCSD; AAV DJ -TRE-eNpHR-YFP, virus production from B. Lim’s laboratory at UCSD; or AAV 1 -TRE-EGFP, virus production from Vigene Biosciences) was bilaterally injected into the NAc (AP: +1.3 mm, ML: ±1.05 mm, DV: −4.05 mm from the dural surface with 7° angle) and fiber-optic cannulae (fiber: 200 μm core, 0.37 NA, BFL37-200, Thorlabs; ceramic ferrule: CFLC 230, Thorlabs) were implanted with a 7° angle 200 μm above the injection site. For optogenetic activation of dPAG neurons, we bilaterally injected AAV 5 -hSyn-hChR2(H134R)-EYFP (200 nl, Addgene, 26973) into the dPAG (AP: −4.5 mm, ML: ±0.25 mm, DV: −1.7 mm from brain surface), implanted a fiber-optic cannula (fiber: 200 μm core, 0.37 NA, BFL37-200, Thorlabs; ceramic ferrule: CFLC 230, Thorlabs) and located the fiber tips at 200 μm above the injection site.

Cal-Light induction

Mice were allowed to recover from the surgery and to express the virus for 18–25 days before undergoing behavioral experiments. Neuronal subpopulations associated with shelter experience were labeled by delivering blue light laser (473 nm, 3 s, 100 times, 15 mW power at the fiber tip, MBL-FN-473, Changchun New Industries Optoelectronic Technology) to the NAc synchronously with shelter entry. Control experiments included random labeling, where blue light was delivered at random locations in the arena when mice were outside the shelter regardless of the mouse’s position or behavior (for example, at rest or moving), and a dark control, with all procedures identical except no blue light was delivered at shelter entry. Despite the larger area outside the shelter, we ensured that the intensity and overall duration of blue light illumination matched between the shelter and random labeling conditions to balance the impact of light-gated gene expression. The minimum interval between each light pulse was 30 s. During the session, mice were monitored by an overhead video camera, with their locations tracked with Ethovision (Noldus). Light delivery timing was managed by a custom code written in MATLAB or Ethovision (Noldus). Following the labeling session, approximately 48 h were given for the induction of reporter gene expression in target neurons, which were activated during blue light delivery.

Cal-Light probe test

Two days after the labeling session, we conducted probe tests to assess the causal effects of (1) inactivation and (2) reactivation of the Cal-Light-labeled NAc subpopulation, using AAV DJ -TRE-eNpHR-YFP and AAV DJ -TRE-ChrimsonR-mEmerald reporters, respectively. Threat stimuli or optogenetic activation of dPAG neurons expressing AAV 5 -hSyn-hChR2(H134R)-EYFP were used to trigger escape behaviors. For dPAG optogenetic activation, blue light intensity (473 nm, 10-ms pulses at 10 Hz for 3 s) was adjusted in the home cage to trigger escape responses similar to those triggered by threat stimuli, with intensities tested from 0.1–0.4 mW/m 2 until consistent light-evoked escape behavior was observed.

For inactivation experiments, we tested four groups: two blue light shelter labeling groups with TRE-NpHR-EYFP reporter (one undergoing probe tests with threat stimuli and the other with dPAG activation), one with blue light random location labeling with TRE-NpHR-EYFP reporter, and one with blue light shelter labeling with TRE-EGFP reporter. In probe tests, mice acclimated for 7 min in the arena with the shelter in the same position as during the Cal-Light induction. In the threat-alone condition, after acclimation, auditory threat stimuli (10 s) with a minimum 1-min ISI were presented to elicit escape responses. In the dPAG-alone control condition, the dPAG was activated by blue light of the previously measured intensity that sufficiently evoked escape behavior (473 nm, 10-ms pulses at 10 Hz for 3 s) to assess escape behavior. For the NAc alone control condition, the labeled NAc population expressing TRE-NpHR-EYFP was optogenetically inactivated alone by a 589-nm laser (continuous for the duration of threat stimulus or dPAG activation, 10–15 mW). For concurrent NAc inactivation and either dPAG activation or threat delivery conditions, the labeled NAc population expressing TRE-NpHR-EYFP was optogenetically inactivated by the 589-nm laser (continuous for the duration of threat stimulus or dPAG activation, 10–15 mW) together with the threat stimulus or with the optogenetic activation of dPAG neurons (473 nm, 10-ms pulses at 10 Hz for 3 s). Each condition was tested four to eight times per mouse in a pseudorandom sequence.

For reactivation experiments, we tested four groups with TRE-ChrimsonR-mEmerald reporter with varying labeling conditions mentioned above: two blue light shelter labeling groups (one undergoing probe tests with threat stimuli and the other with dPAG activation), one with blue light random location labeling and one as a dark control (no blue light at shelter entry). In probe tests, mice acclimated for 7 min in the arena lacking shelter. In the threat-alone condition, after acclimation, auditory threat stimuli with at minimum 1-min ISI were presented to trigger escape responses. In the dPAG-alone control condition, the dPAG was activated by blue light of the previously measured intensity that sufficiently evoked escape behavior (473 nm, 10-ms pulses at 10 Hz for 3 s) to assess escape behavior. For the NAc alone control condition, the labeled NAc population expressing TRE-ChrimsonR-mEmerald was optogenetically activated alone by 589-nm laser (15-ms pulses at 8 Hz for 3 s, ~10–15 mW power at tip end). For concurrent NAc reactivation and either dPAG activation or threat delivery conditions, the labeled NAc population expressing TRE-ChrimsonR-mEmerald was optogenetically activated by a 589-nm laser (15-ms pulses at 8 Hz for 3 s, ~10–15 mW power at tip end) together with the threat stimulus or with the optogenetic activation of dPAG neurons (473 nm, 10-ms pulses at 10 Hz for 3 s). Each condition was tested four to eight times per mouse in a pseudorandom order. TTL pulses controlling laser output were managed by Pulse Pal (Sanworks) and Ethovision XT 14 (Noldus), except where noted. Behaviors were video-recorded using a camera (GoPro HERO3 Silver Edition or Ethovision camera) placed above the arena. Behavioral data, including position, speed, acceleration and head direction, were assessed with DeepLabCut 77 combined with custom MATLAB scripts or Ethovision XT 14 (Noldus).

Cal-Light solo induction and probe experiment

To reduce the induction time required for labeling active neuronal populations by blue light and to achieve stable labeling, we developed a solo version of Cal-Light (AAV1-hSyn-TM-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA-P2A-M13_TEV-C, plasmid construction by Z.G. in the laboratory of R.M.C., virus production from Vigene Biosciences, 1.25 × 10 13 GC per ml) and AAV1-TRE-CreER T2 (1.43 × 10 13 GC per ml, virus production from Vigene Biosciences). Briefly, the Cal-Light solo requires concurrent neural activity and blue light exposure to induce reporter gene expressions via nuclear translocation of the tetracycline transactivator (tTA), similarly to the original Cal-Light construct. This construct, when combined with a TRE-CreER T2 system, enables stable, irreversible Cre-dependent reporter gene expression after brief light exposure. A mixture (1:1:1 ratio, 750 nl per side) of Cal-Light solo, AAV1-TRE-CreER T2 (provided by R.M.C.) and AAV5-Syn-Flex-rc[ChrimsonR-tdTomato] (Addgene, 62723) was bilaterally injected into the NAc, with optic fibers implanted 150–300 μm above the injection site. Mice received ad libitum DOXY-containing food (200 mg per kg body weight, Bio-Serv) a week before the induction to inhibit tTA binding to its target tetracycline-responsive element (TRE) site that induces the expression of the gene of interest, thereby preventing driving of the CreER T2 expression. Two days before the labeling session, mice were switched to a normal diet without DOXY. During labeling, blue light to the NAc selectively labeled active NAc neurons with CreER T2 , enabling reporter gene (Flex-ChrimsonR) expression. After labeling, the DOXY diet resumed to prevent further task-irrelevant gene activation. We used a TRE-CreER T2 system based on the tamoxifen-dependent Cre recombinase (CreER T2 ). Tamoxifen (20 mg ml −1 in corn oil with 10% ethanol; Sigma, T5648) was prepared, incubated at 37 °C for 4–6 h and vortexed every 30 min for solubilization. One day after the labeling, tamoxifen was administered to mice via intraperitoneal injection (100 mg per kg body weight) to activate the CreER T2 for further reporter gene expression. Two days later, probe tests were conducted as described for the Cal-Light probe experiment.

Cal-Light-labeled population analysis

The Cal-Light-labeled population was categorized into four types based on red and green fluorescent signals from post hoc confocal imaging analysis. Individual ROIs for cells were semiautomatically drawn using a custom algorithm with the Cell Magic Wand plugin (ImageJ) based on fluorescence intensity, cell size, and cell shape. Average red and green fluorescent signals were calculated for each ROI and normalized by dividing by the mean background value outside the ROIs. Then, the red and green signals were log-normalized and logarithmized for categorization, respectively. ROIs were allocated into four different quadrants divided by thresholds in two fluorescent colors ( x axis: red tdTomato signal, red threshold value: −1.5; y axis: green TRE reporter signal, green threshold value: 1). ROIs with red signals above or below the red threshold value were categorized as tdT+ or tdT−, respectively. ROIs with green signals above or below the green threshold value were categorized as a reporter fluorescent protein positive or negative, such as mEmerald+ or mEmerald−, respectively. Percentages of each population among all selected ROIs and densities in the NAc were calculated.

Single-molecule FISH

For tissue processing, mice were euthanized, and brains were quickly harvested and briefly washed in ice-cold 1× PBS before overnight fixation in 4% PFA. After fixation, brains were incubated in 30% sucrose solution until they sank, then transferred and incubated in a 50:50 30% sucrose solution optimal cutting temperature embedding compound before being frozen. Brains were coronally sectioned at 14 μm using a cryostat (Leica Biosystems) and stored in antifreeze solution at −20 °C. Sections were mounted on Superfrost Plus slides (Fisher Scientific) and dried at RT for 30 min before immunohistochemistry.

For RNAscope Hiplex assay (Advanced Cell Diagnostics), fresh frozen sections were prepared as described above, and slide-mounted sections were stored at −80 °C. All other procedures for sample preparation and Hiplex assay were performed by following the manufacturer’s instructions. Probes targeting Drd1 (461901-T5), Drd2 (406501-T6), ChAT (408731-T2) and tdTomato (317041-T1) were hybridized and visualized through two rounds of fluorescent labeling and imaging, which allows for simultaneous detection of four different RNA targets in the same tissue. Images were taken using ×20 and ×40 objectives on a confocal microscope (Zeiss 800) and analyzed in custom MATLAB scripts. In brief, ROIs of cells were semiautomatically drawn using a custom algorithm based on DAPI fluorescence intensity, cell size and cell shape by visually inspecting images. For each fluorescence image channel, all pixels within each ROI were averaged to calculate mean fluorescence values. The analysis utilized a classification and regression tree (CART) model for classifying three cell types ( Drd1+ , Drd2+ and ChAT+ ) 83 . The proportion of each type of molecular marker in individual cells is used by CART to predict the underlying cell type. Initially, the CART model was trained on a manually classified dataset. Predictions from this model based on discriminant analysis classification were used to classify the remaining data.

Preparation of acute brain slices for patch-clamp electrophysiology

Mice were anesthetized with isoflurane and quickly decapitated. The brain was rapidly removed from the skull and placed into a cutting solution containing 205 mM sucrose, 2.5 mM KCl, 20 mM glucose, 4 mM MgCl 2 , 0.5 mM CaCl 2 , 1.25 mM NaH 2 PO 4 , 4 mM MgSO 4 and 26 mM NaHCO 3 . Coronal brain slices (300 μm thickness) were made using a vibratome (VT1000, Leica Microsystems). The slices were then incubated at 32 °C for 30 min in a holding chamber filled with ACSF containing 124 mM NaCl, 3 mM KCl, 1.3 mM MgSO 4 , 2.5 mM CaCl 2 , 10 mM glucose, 1.25 mM NaH 2 PO 4 and 26 mM NaHCO 3 , followed by a recovery period at RT for 1 h before recording. For electrophysiology, slices were transferred to a recording chamber (Scientifica) and continuously perfused with ACSF using a peristaltic pump. Recordings were performed at RT. All solutions were saturated with carbogen (95% O 2 , 5% CO 2 ) for at least 30 min.

Patch-clamp electrophysiology

Ex vivo patch-clamp electrophysiological recordings were performed at RT on visually identified neurons within 50 μm from the surface of an acute NAc brain slice in ACSF by using a MultiClamp 700B amplifier controlled by Clampex 10.2 and the Digidata 1440A data acquisition system (Molecular Devices). Cells expressing tdTomato and ChrimsonR-mEmerald, indicating Cal-Light transfection and reporter gene expression, were identified under 565 nm and 470 nm LED (pE-100, CoolLED), respectively. ChrimsonR was activated by 5-ms pulses of 589-nm laser light (Changchun New Industries Optoelectronic Technology; 15 mW power at the fiber tip), controlled by Pulse Pal (Sanworks). Recording pipettes were fabricated from a capillary glass tubing (G150F-3, Warner Instruments), pulled via a micropipette puller (P-1000, Sutter Instrument) to achieve 4–8 MΩ resistance. The pipettes were filled with a potassium-based internal solution (124 mM potassium gluconate, 10 mM HEPES, 16 mM KCl, 10 mM NaCl, 1 mM MgCl 2 , 4 mM NA2-ATP, 0.4 mM NA-GTP, 10 mM Na 2 phosphocreatine and ~300 mOsm, ~pH 7.25). Whole-cell current-clamp and voltage-clamp recordings were performed on the target cells to confirm opsin expression. In the whole-cell current-clamp mode, membrane potential was maintained at −70 mV, and 589-nm light was delivered through an optical fiber (200 μm core, 0.37 NA, BFL37-200, Thorlabs) resting on the brain slice at a distance of approximately 5 mm. In the voltage-clamp mode, opsin expression was confirmed by observing inward currents in response to light pulses (1 Hz, 589 nm) delivered during recording.

Cellular activity analysis

Categorization of location preference.

Individual neurons were categorized based on their location dependence of Ca 2+ fluorescence signals: shelter preferring, outside preferring and nonspecific. Cells for which responses were notably higher in the shelter than outside were categorized as shelter-preferring cells. Cells with responses notably higher outside than inside were categorized as outside-preferring cells. Cells that showed a nonsignificant difference in responses between the shelter and outside were categorized as nonspecific. The SPI was defined as the log of the ratio between Ca 2+ event rates inside and outside the shelter.

SI statistics

To quantify the specificity of neuronal activity over the arena in terms of the information content, we computed the SI based on the Shannon entropy. The positions of animals were mapped onto the bins of the arena, and time spent in each bin was calculated. Event-rate maps for a single ROI were computed by partitioning the arena into 5 cm × 5 cm bins and dividing the number of events that occurred in each bin by the total time spent in that bin. Bins in which mice did not explore during the session were excluded from the analysis. The maps were smoothed with a Gaussian kernel of width 3 × 3 bins and σ  = 1 to create the final map for display. Spatial coding of neuronal activity was examined through the estimation of spatial SI 84 , which is equal to

where \({r}_{i}\) is the event rate of individual cell in the i th bin in the event-rate map, and \({p}_{i}\) is the probability of mice being in the i th bin equal to time spent in the i th bin divided by total session time. \(r={\varSigma }_{i}{p}_{i}{r}_{i}\) is the overall average event rate of the cell across bins.

Sparsity statistics

Sparsity of neuronal activity was measured to compare the firing pattern of experiment data with random firing (Extended Data Fig. 5 ). Sparsity 84 is equal to

in the range between 0 and 1. To estimate average Ca 2+ responses of cells with respect to the distance to shelter, mouse distances to shelter were binned by a distance of 2 cm, and the corresponding normalized Δ F/F value for each bin was averaged. The preferred distance to shelter of each cell was determined as the distance to shelter at which the cell showed maximal average Ca 2+ responses. For both statistics, we corrected for the sampling bias problem by using shuffled distributions of Ca 2+ responses. We assigned randomly chosen time bins to each cell and calculated the resulting statistics. We simulated the shuffling for 1,000 trials and then compared the statistics from the empirical data to the resulting distribution of the shuffled data.

Spatial sparseness

We quantified the spatial sparseness of individual neuronal activities as the kurtosis of spatial distributions of skewness maps. The significance of the spatial sparseness was tested with a one-sample t -test by comparing the sparseness value estimated from empirical data with the sparseness value estimated from the randomly shuffled dataset (100 simulations per cell).

No statistical method was used to predetermine sample size. Statistical analysis was performed using MATLAB and OriginPro (OriginLab Corporation). Data collection and analysis were not performed blind to the conditions of the experiments. Data are reported as the mean ± s.e.m unless otherwise noted. Data distribution was assumed to be normal, but this was not formally tested. Statistical significance was considered when P  < 0.05. Asterisks indicate significant differences (* P  < 0.05, ** P  < 0.01 and *** P  < 0.001). All statistical tests are described in Supplementary Table 1 .

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All other data generated during the current study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The code for semiautomated ROI selection can be found at https://github.com/fitzlab/CellMagicWand/ . Custom codes used for analyses in this study are available from a GitHub repository ( https://github.com/KanghoonJ/Jung_NatNeuro_2024 ) and the corresponding authors upon reasonable request. Further information and requests for analysis code should be directed to and will be fulfilled by H.-B.K.

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Acknowledgements

We thank members of the H.-B.K. laboratory for helpful discussions. We thank M. G. Hussain Shuler and R. Smith for constructive comments on the manuscript. We thank M. E. Shin and R. Yasuda for Camk2a fl/fl knockout mice, B. Lim for the production of AAV DJ -TRE-ChrimsonR-mEmerald and L. Tian for dLight1.1 plasmid. We thank the Johns Hopkins Multiphoton Imaging Core for assistance with microscopy. GCaMP6 virus was available from the Genetically Encoded Neuronal Indicator and Effector (GENIE) Project and the Janelia Farm Research Campus, especially V. Jayaraman, R. A. Kerr, D. S. Kim, L. L. Looger and K. Svoboda. This work was supported by Johns Hopkins School of Medicine (to H.-B.K.), Max Planck Florida Institute for Neuroscience (to H.-B.K.), NARSAD Young Investigator Grant (to Z.G.) and NIH grants R01MH107460 (to H.-B.K.), 5U19NS104649 (to R.M.C.), K99 NS119788 (to Z.G.), DK108230 (to S.B.) and DP1MH119428 (to H.-B.K.).

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Present address: Department of Neuroscience, The University of Texas at Dallas, Richardson, Texas, USA

Authors and Affiliations

Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, USA

Kanghoon Jung, Sarah Krüssel, Sooyeon Yoo, Benjamin Burke, Nicholas Schappaugh, Youngjin Choi, Seth Blackshaw & Hyung-Bae Kwon

Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA

Kanghoon Jung, Sarah Krüssel, Myungmo An, Nicholas Schappaugh & Hyung-Bae Kwon

Allen Institute for Neural Dynamics, Seattle, WA, USA

Kanghoon Jung

Allen Institute, Seattle, WA, USA

Kanghoon Jung & Rui M. Costa

Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, USA

Zirong Gu & Rui M. Costa

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Contributions

K.J. and H.-B.K. conceived and designed the study. K.J., S.K., B.B. and M.A. performed behavioral experiments. K.J., B.B., M.A. and N.S. performed viral injections for experiments. K.J. performed electrophysiology recording, fiber photometry recording and microendoscopic calcium imaging. K.J. wrote behavior and calcium imaging data analysis programs and analyzed all data. B.B., M.A., N.S. and Y.C. helped in histology. Z.G. and R.M.C. developed a modified version of Cal-Light viruses for the initial set of experiments. S.Y. and S.B. helped with the RNAscope experiment. R.M.C. provided critical advice on the paper. K.J. wrote the paper, and K.J., Y.C. and H.-B.K. edited the paper. All authors discussed and commented on the paper.

Corresponding authors

Correspondence to Kanghoon Jung or Hyung-Bae Kwon .

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Nature Neuroscience thanks Afif Aqrabawi, Kay Tye, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended data fig. 1 behavioral analyses on spatial memory for goal-directed escapes..

(a) Top left, video frame depicting shelter-directed escape after threat stimulus (green arrows indicate shelter direction). Top right: speed, distance to shelter, and goal-angle (angle between head direction and shelter direction) during escape (shaded area indicates threat duration). Bottom left: goal angle schematic. Bottom right: video frames showing real-time goal-angle changes during escape. (b) Top, averaged cumulative time in shelter (dotted lines in the inset indicate 7-minute acclimation). Bottom, cumulative shelter visits for each mouse (both time and visits increased over time). (c) Bout duration distributions of inside and outside the shelter. (d) Top, representative trajectory (XY plane) plotted over time (Z-axis) with blue escape flight traces under threat. Bottom, probability distribution of animal position. (e) Flight trajectories under auditory and visual threats, showing shelter escape is independent of stimulus modality. The shelter location was randomly selected among four cardinal directions. For display purposes, the shelter location and the trajectories were rotated relative to the shelter location on the west side of the area. Scale bar, 10 cm. (f) Peak flight speed comparison between auditory and visual threats (Auditory vs. Visual, 46 auditory threat trials, 30 visual threat trials, from 12 mice, t 74  = 1.238, P  = 0.220, two-sided Student’s t-test). (g) Percentage of movement types (darting, hesitating, and freezing) under auditory and visual threats. (h) Normalized flight trajectories under auditory and visual threats. (i) Shelter reaching percentages for auditory and visual threats (n = 46 auditory threats, n = 30 visual threats, from 12 mice, t 74  = −0.173, P  = 0.862, two-sided Student’s t-test). (j) Polar histogram of goal-angles during exploration (left) and escape flights (right). 0 degrees represents movement towards shelter. Speed is color-coded. (k) Example traces from mice with (left) and without (right) shelter acclimation; different colors indicate each trial. Scale bar, 10 cm. (l) Polar histogram of goal-angles during escape flights from mice with (left) and without (right) shelter acclimation. 0 degrees represents movement towards shelter. Speed is color-coded. (m) Cumulative probability distribution of movement speed during exploration (gray) and flight (blue) under threat (n = 7 mice, D  = 0.26, P  = 0, two-sided Kolmogorov-Smirnov test; Speed = 8.9 ± 0.02 cm/s for exploration, Speed = 16.6 ± 0.20 cm/s for flight under threat, mean ± s.e.m). Box plots (inset) show mean (square), median (horizontal line), and percentile ranges (25–75%, box; 10–90%, whiskers). (n-o) Left, example flight trajectories from mice of shelter-unacclimated condition (n) and of shelter-removed condition (o) . Blue arrows indicate head direction. Gray arrows indicate movement direction. Middle, normalized flight trajectories. Right, average speed, distance to shelter, and goal-angle. (p) Video frames of a mouse escaping to a previous shelter location (blue) upon threat in the presence of shelter in a new location (orange). (q) Comparison of reaching percentages between previous and current shelter locations (n = 6 mice, t 5  = 3.71, P  = 0.014, two-sided paired Student’s t-test). (r) Video frames showing escape to previous shelter location in relation to visual cues. After acclimation, the surrounding visual cues were rotated 180°, influencing the mice to navigate based on the rotated cues. (s) Shelter reaching percentages in standard and cue-rotated conditions (Standard, n = 12; cue-rotated, n = 6, t 16  = 0.44, P  = 0.67; two-sided Student’s t-test). (t) Video frames of escape behavior in the dark: mice that experienced the shelter showed a reduced reaching percentage to the previous shelter location when auditory threats were given in the dark. (u) Video frames of escaping behavior in mice acclimated with the shelter in the dark. After acclimation in the dark, mice showed deficits in escaping to the previous shelter location upon threats in the absence of the shelter. (v) Shelter reaching percentages for standard escapes (n = 12 mice), escape in the dark condition (n = 5 mice), and acclimation in the dark condition (n = 6 mice) (Standard escapes vs. escape in the dark, t 15  = 7.38, P  = 1.15×10 −6 ; Standard escapes vs. acclimation in the dark, t 16  = 6.33, P  = 5×10 −6 ; Escape in the dark vs. acclimation in the dark, t 9  = 0.52, P  = 0.62; two-sided Student’s t-test). *** P  < 0.001; n.s., not significant; Error bar and shaded error bar represent s.e.m. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 2 The activity of VTA DA neurons during shelter experience.

(a) Top, schematic of injection of AAV 1 -Syn-Flex-GCaMP6f into VTA of DAT-Cre mice. Middle, confocal image of VTA neurons. Scale bar, 100 µm. Bottom, schematic of fiber photometry recording from VTA DA neurons during behavior. (b) Example traces of homing and leaving shelter, with corresponding Ca 2+ fluorescence signals of VTA DA neurons (color-coded). (c) Example trace of distance to shelter and Ca 2+ fluorescence signals (Δ F/F ) of VTA DA neurons. Shaded areas indicate duration of shelter stay. Horizontal scale bar, 10 s. (d) Example heatmap of Ca 2+ transient signals of VTA DA neurons from a mouse aligned to shelter entry time. (e) Example heatmaps for distance to shelter (top), speed (middle), and Ca 2+ transient signals of VTA DA neurons (bottom) from a mouse aligned to the first five threat onsets. (f) Normalized Ca 2+ transient signals of VTA DA neurons aligned to threat onset (left), shelter entry (middle), and threat offset (right). (g) Average Ca 2+ transient signals of VTA DA neurons (blue) and distance to shelter (black) aligned to shelter entry time. The Ca 2+ signals rapidly increased when animals entered the shelter (n = 5 mice). (h) Quantification of normalized Ca 2+ transient signals by location (outside/shelter) and threat (on/off) (n = 5 mice; Location, F 1, 80  = 15.71, P  = 1.60×10 −4 ; Threat, F 1, 80  = 2.93, P  = 0.09; two-way ANOVA followed by a Fisher’s least significant difference (LSD) post-hoc test). * P  < 0.05, *** P  < 0.001; Box plots show mean (square), median (horizontal line), and percentile ranges (25–75%, box; 10–90%, whiskers). Error bar or shaded area indicates s.e.m. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 3 Converging synaptic inputs from the vHPC and the VTA to the medial shell of the NAc.

(a) Left, schematic and confocal images showing convergent dopaminergic VTA (VTA DA ) and glutamatergic vHPC (vHPC Glu ) inputs in the medial shell of NAc. VTA DA neurons express AAV 1 -CAG-Flex-EGFP (green) and vHPC excitatory neurons expressing CaMKII-Cre and Flex-TdTomato (red) in DAT-Cre mice. Axons of VTA DA neurons (EGFP) and vHPC Glu neurons (TdTomato) are intermingled in the NAc. Blue: DAPI. Scale bar, 200 µm. Right, 3-D light-sheet microscopy image of input convergence. (b) Left, schematic of retrograde tracing of vHPC Glu neurons projecting to the medial shell of NAc by injecting retrograde AAV rg -CAG-Flex-tdTomato in the NAc and AAV 1 -CaMKII-0.4-Cre-SV40 in the vHPC. Right, confocal image showing Cre-dependent expression of tdTomato in vHPC neurons. Scale bar, 500 μm. Glu, glutamatergic axonal projection; vHPC, ventral hippocampus; Sub, subiculum. (c) vHPC Glu inputs drive NAc activity (n = 3 mice). Left, fiber photometry recording in NAc neurons using AAV 1 -hSyn-GCaMP6f and optogenetic activation of vHPC Glu neurons expressing red-shifted excitatory opsin, AAV 2 -CaMKIIa-bReaChES-TS-EYFP (589 nm, 8 Hz laser pulses). Right, confocal images of GCaMP6f expression in NAc neurons (top) and bReaChES-EYFP in vHPC Glu neurons (bottom), with photometry optic fiber placement indicated (dotted line). Scale bars, 500 μm. (d) Top, heatmaps of the activity of NAc neurons expressing GCaMP6f in response to vHPC Glu optogenetic activation (5 trials per condition; stimulation: 589 nm light, 8 Hz; 1 s, 3 s, 5 s durations). Note that the optogenetic activation of vHPC Glu neurons reliably triggered NAc activity (5 of 5 trials), inducing the fluorescence increase persisted throughout the stimulation duration. Bottom, average Δ F/F response. Orange region: stimulation duration. (e) Left, schematic of retrograde tracing of VTA DA projecting to NAc medial shell by AAV rg -CAG-Flex-tdTomato injection in the NAc of DAT-Cre mice. Right, coronal and sagittal brain sections displaying tdTomato in VTA DA neurons. Scale bars are 500 μm (coronal) and 1 mm (sagittal). SNc, substantia nigra pars compacta. (f) Schematic of fiber photometry recording of dLight 1.1 in NAc medial shell in DAT-Cre mice with optogenetic stimulation of VTA DA neurons using either AAV 5 -Syn-Flex-ChrimsonR-tdTomato or AAV1-EF1-dFlox-hChR2-mCherry-WPRE-hGHn. (g) Heatmaps and average Δ F/F response of dLight1.1 signals during optogenetic stimulation (left, 589-nm laser for ChrimsonR; right, 473-nm laser for ChR2; top, 20 Hz, 1 s duration; middle, 5 Hz, 3 s duration; bottom, 5 Hz, 10 s duration; 10 trials per condition). Brief pulses (5 ms) of laser to VTA DA neurons consistently increased the signals (10 of 10 at 20 Hz, 1 s; 10 of 10 at 5 Hz, 3 s; 10 of 10 at 5 Hz, 10 s), inducing the fluorescence increase persisted throughout the stimulation duration. High-frequency stimulation (20 Hz) produced larger signal changes compared to 5 Hz. (h) Normalized dLight1.1 fluorescence changes compared to baseline (average of 3 s pre- stimulation) across different stimulation frequencies (color coded). Fluorescence increases with frequency and saturates at around 40 Hz. Orange region: stimulation duration. Shaded area error bar: s.e.m.

Extended Data Fig. 4 Context-dependent DA signals in the NAc medial shell.

(a) Two representative shelter inbound and outbound trajectories of (xy plane) over time (z axis) and normalized Δ F/F of Ca 2+ from jRGECO (left), DA from dLight (middle), and their conjunctive signals (right) (signal intensity shown in heatmap). Shelter location marked by a black circle. (b) Schematic of photometry recording of NAc DA signals during threat (top), and an example flight trace to shelter (bottom). (c) Representative traces of distance to shelter, speed, and normalized NAc DA transients during shelter-directed escape behavior. Event timings were indicated by dashed lines for threat onset, flight onset, shelter-in, and threat end. (d) Average changes in distance to shelter and speed during threat presentation, aligned to key events (threat onset, flight onset, shelter-in, and threat end). Peak escape speed occurred 0.66 s after flight onset. DA signal traces (n = 5 mice, 10 escape trials to shelter) showed a decrease at threat onset, a momentary rise at shelter entry (peak at 214.5 ms, green arrow). A depression in DA signals sustained for the remaining threat presentation, followed by a robust increase of DA signals upon the threat end. The red and green shaded areas indicate threat stimulus (10 s) and shelter-stay, respectively. (e) Changes in normalized DA concentrations for different events (threat onset, flight onset, shelter-in, and threat end, n = 5 mice, F 2, 18  = 3.76, P  = 0.043, for Threat onset; F 2, 18  = 0.17, P  = 0.846, for Flight onset; F 3, 27  = 4.50, P  = 0.011, for Shelter-In; F 4, 36  = 7.537, P  = 0.00016, for Flight onset; one-way repeated measures ANOVA). * P  < 0.05, *** P  < 0.001; Error bars and shaded error bars represent s.e.m. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 5 Shelter-related cellular responses in NAc neurons.

(a) Video frame of a freely exploring mouse with microendoscope for in vivo Ca 2+ imaging of NAc neurons expressing AAV 1 -hSyn-GCaMP6s and field-of-view (FOV) of the NAc neurons. Blue circle indicates shelter location. (b) Arena-activity maps for example NAc neurons. A small circle on the arena indicates the shelter location. Top, shelter-preferring neurons showing peaks at the shelter; Bottom, outside-preferring neurons of which peak locations varied across the arena. (c) Box plot (left; mean ± s.e.m, 0.27 ± 0.008, experimental data; 0.13 ± 0.003, surrogate data; n = 704 neurons from 7 mice, t 703  = 23.98, P  = 1.27×10 −93 , two-sided paired Student’s t-test) and cumulative distribution (right; D  = 0.46, P  = 0, two-sided Kolmogorov-Smirnov test) of spatial information of NAc neurons for empirical and shuffled data. (d) Box plot (left; mean ± s.e.m, 0.69 ± 0.006, experimental data; 0.82 ± 0.003, surrogate data; n = 704 neurons from 7 mice, t 703  = 29.33, P  = 2.04×10 −124 , two-sided paired Student’s t-test) and cumulative distribution (right; D  = 0.43, P  = 0, two-sided Kolmogorov-Smirnov test) of sparsity of NAc neurons for empirical and shuffled data. In (c) and (d) , boxes and whiskers cover the middle 50%, and 10–90% of values. Horizontal lines denote medians. (e) Spatial sparseness of neural activity for empirical and shuffled datasets (n = 704 neurons from 7 mice, t 703  = 20.62, P  = 1.50×10 −74 , two-sided paired Student’s t-test). *** P  < 0.001. (f) Example NAc activity map, color-coded to show each neuron’s preferred distance to shelter, defined as the distance where neural activity peaks during exploration. (g) Pairwise correlation between NAc neuronal pairs relative to cell-to-cell distance (green, empirical data; black, shuffled data). (h) Heatmap showing normalized Δ F/F responses of individual NAc neurons relative to preferred distance to shelter. (i) Probability and cumulative distribution of preferred distance to shelter among NAc neurons (n = 704 neurons from 7 mice). (j) Neuronal responses aligned to shelter entry at four time points within a session. Top, distance to shelter. Middle, individual neurons’ activities. Bottom, average population activities by preference: outside-preferring (magenta), non-specific (gray), and shelter-preferring (green). The changes in population activity of outside-preferring and shelter-preferring neurons become more distinct over time. (k) Heatmaps of maximum-normalized NAc neuron activity aligned to shelter entry at 3, 15, and 40 minutes from the session start, showing the emerging pattern of increased activity post-entry. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 6 Manipulation of vHPC Glu , DA, and αCaMKII signalings in the NAc.

(a) Schematic and timeline of pharmacological manipulations of Drd1 and Drd2 in the NAc medial shell. (b) Shelter reaching percentage during escape behaviors (saline control, mean ± s.e.m: 91.7 ± 8.3%, n = 6 mice; SCH23390, 19.2 ± 7.7%, n = 6 mice; Haloperidol, 60.8 ± 11.2%, n = 5 mice; F 2, 14  = 17.4, P  = 1.61×10 −4 , one-way ANOVA with Bonferroni post-hoc test). (c) Exploration speed across conditions (saline control, mean ± s.e.m: 9.19 ± 0.49 cm/s, n = 6; SCH23390, 7.94 ± 0.75 cm/s, n = 6 mice; Haloperidol, 8.34 ± 0.58 cm/s, n = 5; F 2, 14  = 1.34, P  = 0.294, one-way ANOVA with Bonferroni post-hoc test). (d) Distance traveled per minute across conditions (saline control, mean ± s.e.m: 0.68 ± 0.20 m/min, n = 6; SCH23390, 0.57 ± 0.09 m/min, n = 6 mice; Haloperidol, 1.34 ± 0.32 m/min, n = 5; F 2, 14  = 3.67, P  = 0.052, one-way ANOVA with Bonferroni post-hoc test). (e) Left, schematic and timeline of calcium/calmodulin-dependent protein kinase II α ( αCaMKII ) conditional knockout (KO). Right, shelter reaching percentages in αCaMKII KO mice for shelter-present (left) and shelter-removed (right) conditions after acclimation period in the presence of shelter (n = 5 mice, αCaMKII-WT ; n = 8, αCaMKII KO ; shelter-present, t 11  = 4.48, P  = 9.27×10 −4 ; shelter-removed, t 11  = 3.84, P  = 2.74×10 −3 ; two-sided Student’s t-test). Disruption of αCaMKII signaling impairs instant place learning. (f) Shelter reaching percentage for conditional αCaMKII KO and control mice in the NAc in shelter-present condition (n = 10 mice, αCaMKII -floxed:CaMKII-EGFP; n = 12, αCaMKII -floxed:CaMKII-Cre-EGFP; t 20  = 5.05, P  = 6.11×10 −5 , two-sided Student’s t-test). (g) Exploration speed across conditions. The administration of CNO did not have a significant effect on speed (n = 13 for control; n = 7 for NAc::hM4Di; n = 6 for vHPC-NAc::hM4Di; Two-way ANOVA, F group (2)  =  1.93 , P  = 0.156; F CNO (1)  =  0.68, P  =  0.414 ). (h) Distance traveled during exploration across conditions. The administration of CNO did not significantly affect distance traveled (n = 13 for control; n = 7 for NAc::hM4Di; n = 6 for vHPC-NAc::hM4Di; Two-way ANOVA, F group (2)  =  4.83 , P  = 0.012; F CNO (1)  =  3.89, P  =  0.054 ). Box plots show mean (square), median (horizontal line), and percentile ranges (25–75%, box; 10–90%, whiskers). * P  < 0.05, ** P  < 0.01, *** P  < 0.001; Error bars represent s.e.m. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 7 NAc Shelter inactivation experiment with escape behavior triggered by dPAG activation.

(a) Left, schematic of virus injection for inactivation experiments using the cal-light system (n = 8 mice). A mixture of cal-light constructs with TRE-NpHR-EYFP reporter were bilaterally injected into the NAc medial shell. Optic fibers were bilaterally implanted 200 µm above the virus injection site. AAV 5 -hSyn-hChR2(H134R)-EYFP was bilaterally injected into the dPAG. Optic fibers were implanted 200 µm above the virus injection site. Right, confocal images of NAc neurons expressing cal-light constructs with M13-tdTomato (top, cell marker), NpHR-EYFP reporter (middle), and merged (bottom). Scale bar, 100 µm. (b) Behavioral experiment schematics for inactivating NAc Shelter ensembles. During labeling, mice exploring the arena and shelter received 100 instances of blue light (473 nm, 3 s each) upon entering the shelter to label NAc Shelter . After 48 hours, allowing for TRE-NpHR-EYFP expression, amber light (589 nm) was applied in probe trials to inactivate these neurons, coinciding with optogenetic dPAG activation to trigger escape behaviors. (c) Normalized distance to the previous shelter location aligned to the onset time of optogenetic activation. The shaded area indicates the duration of light delivery (3 s). Gray, dPAG activation alone; red, inactivation of NAc Shelter alone; orange, concurrent dPAG activation and NAc Shelter inactivation). Shaded error bars indicate s.e.m. (d) Polar histograms of head-shelter direction distribution in different trial types. (e) Head-shelter angles across conditions (n = 19 trials for NAc Shelter ::NpHR, n = 39 trials for dPAG, n = 37 trials for dPAG + NAc Shelter ::NpHR; 8 mice; F 2, 90  = 9.11, P  = 2.50×10 −4 , one-way ANOVA with Bonferroni post-hoc test). Box plots show mean (square), median (horizontal line), and percentile ranges (25–75%, box; 10–90%, whiskers). * P  < 0.05; *** P  < 0.001. For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 8 Quantification of NAc Shelter neurons.

(a) Schematic of virus injection for the NAc Shelter reactivation experiment. Cal-light constructs with TRE-ChrimsonR-mEmerald reporter and ChR2-EYFP were bilaterally injected into the NAc and dPAG, respectively, with optic fibers implanted accordingly. (b) Left, a confocal image showing TRE-ChrimsonR-mEmerald expression in NAc medial shell under shelter labeling condition (n = 6 mice). Red: M13-tdTomato (virus-infected cell marker), Green: ChrimsonR-mEmerald (labeled neurons). Right, magnified images of the region (yellow box). (c) Whole-cell patch-clamp electrophysiological recording from tdT + /mEmerald+ neurons labeled for NAc Shelter . Left, DIC image of a tdT + /mEmerald+ cell. Right, excitatory postsynaptic currents (left) and action potential (right) traces elicited by a short pulse of 589 nm light (5 ms, red tick). (d) Top, schematics of three labeling conditions: blue light at shelter entry, at random locations outside the shelter, and no blue light. Bottom, the composition of labeled NAc neuronal populations: cells with no viral infection (tdT-/mEmerald-, black), with only ChrimsonR-mEmerald (tdT-/mEmerald + , green), with only M13-tdTomato (tdT + /mEmerald-, red), with both ChrimsonR-mEmerald and M13-tdTomato (tdT + /mEmerald + , yellow). (e) Density quantification of labeled NAc neuronal ensembles (tdT + /mEmerald+ cells in the NAc medial shell) under three conditions: blue light at shelter (n = 6 mice), at random location outside shelter (n = 5 mice), and no blue light (n = 5 mice) ( F 2, 13  = 27.4, P  = 2.18×10 −5 , one-way ANOVA with Bonferroni post-hoc test). Error bars indicate s.e.m. (f) Probability distributions of intersomatic distances among neurons: all pairs (gray), tdT + /mEmerald+ pairs (yellow), and tdT + /mEmerald- and tdT + /mEmerald+ pairs (orange). For detailed statistics information, see Supplementary Table 1 .

Extended Data Fig. 9 Escape behaviors in control conditions for the NAc Shelter reactivation experiment.

(a) Movement speeds reflecting escape vigor aligned to flight onset in dPAG activation alone (black) and coactivation with labeled NAc population (colored) for shelter labeling (n = 5 mice) (left), random location labeling control (n = 5 mice) (middle), and dark (no blue-light) control (n = 5 mice) (right). The shaded area indicates the initial escape flight period (0-0.5 s from flight onset). Maximum escape speeds showed no significant differences between conditions (shelter labeling condition, n = 5 mice, 63.7 ± 3.76 cm/s for 37 dPAG activation trials, 61.8 ± 3.51 cm/s for 28 dPAG + NAc Shelter activation trials, t 63  = 0.357, P  = 0.722; random labeling condition, n = 5 mice, 35.0 ± 2.26 cm/s for 40 dPAG activation trials, 34.6 ± 2.51 cm/s for 40 dPAG + NAc Random activation trials, t 78  = 0.115, P  = 0.909; dark control, n = 5 mice, 41.3 ± 3.08 cm/s for 42 dPAG activation trials, 33.5 ± 2.43 cm/s for 35 dPAG + NAc Dark activation trials, t 75  = 1.933, P  = 0.057; two-sided Student’s t-test). (b) Average initial acceleration for escape (0-0.5 s from escape onset) for dPAG activation alone (black) and coactivation with labeled NAc population (colored) (shelter labeling condition, n = 5 mice, 57.7 ± 5.9 cm/s 2 for 37 dPAG activation trials, 54.5 ± 5.80 cm/s 2 for 28 dPAG + NAc Shelter activation trials, t 63  = 0.373, P  = 0.711; random labeling condition, n = 5 mice, 45.7 ± 2.2 cm/s 2 for 40 dPAG activation trials, 45.9 ± 2.8 cm/s 2 for 40 dPAG + NAc Random activation trials, t 78  = 0.0308, P  = 0.976; dark control, n = 5 mice, 58.3 ± 3.6 cm/s 2 for 42 dPAG activation trials, 46.0 ± 2.4 cm/s 2 for 35 dPAG + NAc Dark activation trials, t 75  = 1.599, P  = 0.114; two-sided Student’s t-test). Box plots show median (horizontal line), and percentile ranges (25-75%, box; 10-90%, whiskers). (c) Normalized distance to previous shelter location aligned to optogenetic activation onset for dPAG alone (black), and with NAc Random (top) or NAc Dark (bottom) coactivation. The shaded area indicates the 3 s activation duration. (d) Goal-directedness (cosine of head-shelter angle) of escape behavior over time from flight onset. (e) Goal-directedness relative to distance to previous shelter location. Shaded error bars represent s.e.m. n.s., not significant. For detailed statistics information, see Supplementary Table 1 .

Supplementary information

Supplementary information.

Supplementary Table 1.

Reporting Summary

Supplementary video 1.

Video depicting a visual or auditory threat stimulus evoking escape behavior to the shelter.

Supplementary Video 2

Video from a mouse that acclimated to the arena without shelter depicting failure in escape to the shelter upon threat stimuli.

Supplementary Video 3

Video depicting escape behavior upon a threat stimulus to the previous shelter location where the mouse was acclimated rather than the currently visible shelter.

Supplementary Video 4

Video depicting escape behaviors triggered by optogenetic stimulation of dPAG neurons.

Supplementary Video 5

Video depicting a light-sheet microscope image displaying the convergence of VTA DA and vHPC Glu inputs onto the medial shell of the NAc.

Supplementary Video 6

Video depicting escape behaviors to random locations triggered by optogenetic stimulation of dPAG neurons alone and goal-directed escape behaviors to the previous shelter location triggered by optogenetic activation of both dPAG neurons and NAc Shelter ::ChrimsonR neurons.

Source Data Fig. 1

Statistical source data.

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Jung, K., Krüssel, S., Yoo, S. et al. Dopamine-mediated formation of a memory module in the nucleus accumbens for goal-directed navigation. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01770-9

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Received : 05 November 2021

Accepted : 23 August 2024

Published : 27 September 2024

DOI : https://doi.org/10.1038/s41593-024-01770-9

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