Publications by authors named "Nathaniel Heintz"

102 Publications

Serotonin receptor 4 in the hippocampus modulates mood and anxiety.

Mol Psychiatry 2021 Jan 13. Epub 2021 Jan 13.

Laboratory of Molecular Biology, The Rockefeller University, New York, NY, 10065, USA.

Serotonin receptor 4 (5-HTR) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HTR is widely expressed throughout the central nervous system (CNS) and periphery, making it difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HTR knockout mouse line to dissect the function of 5-HTR in specific brain regions and cell types. We show that the loss of functional 5-HTR specifically from excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HTR was necessary to maintain the proper excitability of dentate gyrus (DG) granule cells and cell type-specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HTR. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HTR loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HTR signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits.
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http://dx.doi.org/10.1038/s41380-020-00994-yDOI Listing
January 2021

Parallel ascending spinal pathways for affective touch and pain.

Nature 2020 11 28;587(7833):258-263. Epub 2020 Oct 28.

Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.

The anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain. Projection neurons of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery are channelled through these spinal projection neurons en route to the brain. However, the organizational logic of the anterolateral pathway remains poorly understood. Here we show that two populations of projection neurons that express the structurally related G-protein-coupled receptors (GPCRs) TACR1 and GPR83 form parallel ascending circuit modules that cooperate to convey thermal, tactile and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons. Within this nucleus, axons of spinoparabrachial (SPB) neurons that express Tacr1 or Gpr83 innervate distinct sets of subnuclei, and strong optogenetic stimulation of the axon terminals induces distinct escape behaviours and autonomic responses. Moreover, SPB neurons that  express Gpr83 are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Notably, the valence associated with activation of SPB neurons that express Gpr83 can be either positive or negative, depending on stimulus intensity. These findings reveal anatomically, physiologically and functionally distinct subdivisions of the SPB tract that underlie affective aspects of touch and pain.
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http://dx.doi.org/10.1038/s41586-020-2860-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666110PMC
November 2020

Amygdala inhibitory neurons as loci for translation in emotional memories.

Nature 2020 10 7;586(7829):407-411. Epub 2020 Oct 7.

Center for Neural Science, New York University, New York, NY, USA.

To survive in a dynamic environment, animals need to identify and appropriately respond to stimuli that signal danger. Survival also depends on suppressing the threat-response during a stimulus that predicts the absence of threat (safety). An understanding of the biological substrates of emotional memories during a task in which animals learn to flexibly execute defensive responses to a threat-predictive cue and a safety cue is critical for developing treatments for memory disorders such as post-traumatic stress disorder. The centrolateral amygdala is an important node in the neuronal circuit that mediates defensive responses, and a key brain area for processing and storing threat memories. Here we applied intersectional chemogenetic strategies to inhibitory neurons in the centrolateral amygdala of mice to block cell-type-specific translation programs that are sensitive to depletion of eukaryotic initiation factor 4E (eIF4E) and phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). We show that de novo translation in somatostatin-expressing inhibitory neurons in the centrolateral amygdala is necessary for the long-term storage of conditioned-threat responses, whereas de novo translation in protein kinase Cδ-expressing inhibitory neurons in the centrolateral amygdala is necessary for the inhibition of a conditioned response to a safety cue. Our results provide insight into the role of de novo protein synthesis in distinct inhibitory neuron populations in the centrolateral amygdala during the consolidation of long-term memories.
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http://dx.doi.org/10.1038/s41586-020-2793-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572709PMC
October 2020

Working with Bacterial Artificial Chromosomes (BACs) and Other High-Capacity Vectors.

Cold Spring Harb Protoc 2020 10 1;2020(10). Epub 2020 Oct 1.

Genetic targeting of specific cell types is fundamentally important for modern molecular-genetic studies. The development of simple methods to engineer high-capacity vectors-in particular, bacterial artificial chromosomes (BACs)-for the preparation of transgenic lines that accurately express a gene of interest has resulted in commonplace usage of transgenic techniques in a wide variety of experimental systems. Here we provide a brief description of each of the four major types of large-capacity vectors, with a focus on the use of BAC vectors.
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http://dx.doi.org/10.1101/pdb.top097998DOI Listing
October 2020

Growth of and Preparation of DNA.

Cold Spring Harb Protoc 2020 10 1;2020(10). Epub 2020 Oct 1.

This protocol describes methods for isolation of total DNA from a strain of carrying a recombinant yeast artificial chromosome (YAC). This method is appropriate for preparing DNA that will be subjected to regular agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, polymerase chain reaction (PCR), or other methods that do not require intact high-molecular-weight DNA. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. Drop dialysis should be used to exchange buffers. The expected yield from a 10-mL culture is 2-4 µg of yeast DNA.
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http://dx.doi.org/10.1101/pdb.prot098145DOI Listing
October 2020

Small-Scale Preparations of Yeast DNA.

Cold Spring Harb Protoc 2020 10 1;2020(10). Epub 2020 Oct 1.

In this protocol, yeast DNA is prepared by digestion of the cell wall and lysis of the resulting spheroplasts with SDS. This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in polymerase chain reaction (PCR). Note that yeast colonies can also be used directly in PCR, without purifying yeast DNA.
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http://dx.doi.org/10.1101/pdb.prot098152DOI Listing
October 2020

One-Step Bacterial Artificial Chromosome (BAC) Modification: Transfer of the Reporter Vector into BAC/RecA Cells and Selection of Co-Integrates.

Cold Spring Harb Protoc 2020 07 1;2020(7):098137. Epub 2020 Jul 1.

In one-step bacterial artificial chromosome (BAC) modification, recombination of the reporter vector with the BAC-leading to the modified BAC-is facilitated by the presence of RecA. Recombinants are selected for by growth in the presence of chloramphenicol, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the π protein to replicate. The co-integrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BAC-formation of the co-integrate-is confirmed in separate amplification reactions.
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http://dx.doi.org/10.1101/pdb.prot098137DOI Listing
July 2020

One-Step Bacterial Artificial Chromosome (BAC) Modification: Transformation of the BAC Host with the RecA Vector.

Cold Spring Harb Protoc 2020 07 1;2020(7):098129. Epub 2020 Jul 1.

This protocol outlines the steps for introducing the RecA plasmid into bacterial artificial chromosome (BAC) host cells, and their preparation for subsequent transformation with the reporter plasmid for one-step BAC modification. BAC host cells are rendered chemically competent and transformed with the RecA plasmid, and transformants are selected for tetracycline resistance to ensure the presence of the RecA marker.
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http://dx.doi.org/10.1101/pdb.prot098129DOI Listing
July 2020

One-Step Bacterial Artificial Chromosome (BAC) Modification: Cloning of the A Homology Arm into Reporter-Shuttle Vector.

Cold Spring Harb Protoc 2020 07 1;2020(7):098111. Epub 2020 Jul 1.

In this protocol, the homology arm sequence for one-step bacterial artificial chromosome (BAC) modification is introduced by ligation into the shuttle vector carrying the reporter sequence to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector. To provide further assurance that the homology box has been successfully integrated into the plasmid, the enzyme digestion pattern of the modified plasmid is compared with that of the unmodified plasmid.
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http://dx.doi.org/10.1101/pdb.prot098111DOI Listing
July 2020

One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of the A Homology Arm (A-Box).

Cold Spring Harb Protoc 2020 07 1;2020(7):098103. Epub 2020 Jul 1.

The one-step approach to bacterial artificial chromosome (BAC) modification requires that only one homology arm be cloned into the shuttle vector (in the example presented here, we use the "A-box"). The homology arm, which in this case lies upstream of the ATG start codon, is amplified by polymerase chain reaction (PCR) using purified BAC DNA as template. The resulting amplification product is then digested with the appropriate restriction endonuclease to render it suitable for cloning into the shuttle vector.
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http://dx.doi.org/10.1101/pdb.prot098103DOI Listing
July 2020

One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of Plasmids.

Cold Spring Harb Protoc 2020 07 1;2020(7):098095. Epub 2020 Jul 1.

In the one-step approach to bacterial artificial chromosome (BAC) modification, two plasmids are introduced into the BAC host cells. The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the π protein to replicate, must be grown in PIR1- or PIR2-competent Our preference for these vectors is PIR1, because these cells are able to maintain about 250 copies of the donor vector. This small-sized vector is stable in PIR1. The RecA plasmid pSV1.RecA has a temperature-sensitive origin of replication and can be grown in most competent bacteria at 30°C; here we use DH5α competent cells. This protocol describes preparation of the vector DNAs. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).
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http://dx.doi.org/10.1101/pdb.prot098095DOI Listing
July 2020

Selective Neuronal Vulnerability in Alzheimer's Disease: A Network-Based Analysis.

Neuron 2020 09 29;107(5):821-835.e12. Epub 2020 Jun 29.

Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10065, USA.

A major obstacle to treating Alzheimer's disease (AD) is our lack of understanding of the molecular mechanisms underlying selective neuronal vulnerability, a key characteristic of the disease. Here, we present a framework integrating high-quality neuron-type-specific molecular profiles across the lifetime of the healthy mouse, which we generated using bacTRAP, with postmortem human functional genomics and quantitative genetics data. We demonstrate human-mouse conservation of cellular taxonomy at the molecular level for neurons vulnerable and resistant in AD, identify specific genes and pathways associated with AD neuropathology, and pinpoint a specific functional gene module underlying selective vulnerability, enriched in processes associated with axonal remodeling, and affected by amyloid accumulation and aging. We have made all cell-type-specific profiles and functional networks available at http://alz.princeton.edu. Overall, our study provides a molecular framework for understanding the complex interplay between Aβ, aging, and neurodegeneration within the most vulnerable neurons in AD.
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http://dx.doi.org/10.1016/j.neuron.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580783PMC
September 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Verification of Co-Integrates and Selection of Resolved BAC Clones.

Cold Spring Harb Protoc 2020 04 1;2020(4):098087. Epub 2020 Apr 1.

Successful modification of the bacterial artificial chromosome (BAC) after two-step BAC engineering is confirmed in two separate polymerase chain reactions (PCRs). The first reaction (5' co-integrate PCR) uses a forward 5' co-integrate primer (a sequence located upstream of the 5' end of the A-box) and a reverse 3' primer on the vector (175PA+50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3' co-integrate PCR) uses a forward 5' primer on the gene (RecA1300S) and a reverse 3' co-integrate primer (a sequence located downstream from the 3' end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including and genes become UV light sensitive.
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http://dx.doi.org/10.1101/pdb.prot098087DOI Listing
April 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Electroporation of Competent BAC Host Cells with the Recombinant Shuttle Vector.

Cold Spring Harb Protoc 2020 04 1;2020(4):098079. Epub 2020 Apr 1.

Bacterial artificial chromosome (BAC) clones are rendered electrocompetent and transformed with the recombinant shuttle vector, pLD53SCAB/AB-box. Cointegrates are selected by growth on chloramphenicol and ampicillin to ensure recombination of the shuttle vector into the BAC.
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http://dx.doi.org/10.1101/pdb.prot098079DOI Listing
April 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation and Verification of the Recombinant Shuttle Vector.

Cold Spring Harb Protoc 2020 04 1;2020(4):098061. Epub 2020 Apr 1.

Plasmid DNA is prepared from the recombinant shuttle vector pLD53.SCAB/A-B created by cloning of the A and B homology arms for two-step bacterial artificial chromosome (BAC) engineering. To confirm that the A-box and B-box arms have been successfully incorporated into pLD53.SCAB, the pattern of enzyme digestion of the modified plasmid is compared with that of the unmodified pLD53.SCAB. Once the shuttle vector is shown to carry the proper sequences, it is ready for transfer into the BAC host.
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http://dx.doi.org/10.1101/pdb.prot098061DOI Listing
April 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Cloning of the A and B Homology Arms into the Shuttle Vector.

Cold Spring Harb Protoc 2020 04 1;2020(4):098053. Epub 2020 Apr 1.

This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector.
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http://dx.doi.org/10.1101/pdb.prot098053DOI Listing
April 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box).

Cold Spring Harb Protoc 2020 04 1;2020(4):098046. Epub 2020 Apr 1.

The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector.
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http://dx.doi.org/10.1101/pdb.prot098046DOI Listing
April 2020

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of Shuttle Vector DNA.

Cold Spring Harb Protoc 2020 04 1;2020(4):098038. Epub 2020 Apr 1.

In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The shuttle vector pLD53.SCAB (or pLD53.SCAEB) carries the gene and the R6Kγ origin, which requires the π protein to replicate. PIR2 cells, expressing π, are typically used for the amplification of the vector and maintain about 15 copies/cell of the donor vector, which is relatively stable in this host.
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http://dx.doi.org/10.1101/pdb.prot098038DOI Listing
April 2020

The habenular G-protein-coupled receptor 151 regulates synaptic plasticity and nicotine intake.

Proc Natl Acad Sci U S A 2020 03 25;117(10):5502-5509. Epub 2020 Feb 25.

Laboratory of Molecular Biology, The Rockefeller University, New York, NY 10065;

The habenula, an ancient small brain area in the epithalamus, densely expresses nicotinic acetylcholine receptors and is critical for nicotine intake and aversion. As such, identification of strategies to manipulate habenular activity may yield approaches to treat nicotine addiction. Here we show that GPR151, an orphan G-protein-coupled receptor (GPCR) highly enriched in the habenula of humans and rodents, is expressed at presynaptic membranes and synaptic vesicles and associates with synaptic components controlling vesicle release and ion transport. Deletion of inhibits evoked neurotransmission but enhances spontaneous miniature synaptic currents and eliminates short-term plasticity induced by nicotine. We find that GPR151 couples to the G-alpha inhibitory protein Gα to reduce cyclic adenosine monophosphate (cAMP) levels in mice and in GPR151-expressing cell lines that are amenable to ligand screens. knockout (KO) mice show diminished behavioral responses to nicotine and self-administer greater quantities of the drug, phenotypes rescued by viral reexpression of in the habenula. These data identify GPR151 as a critical modulator of habenular function that controls nicotine addiction vulnerability.
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http://dx.doi.org/10.1073/pnas.1916132117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071848PMC
March 2020

Cell-type-specific drug-inducible protein synthesis inhibition demonstrates that memory consolidation requires rapid neuronal translation.

Nat Neurosci 2020 02 20;23(2):281-292. Epub 2020 Jan 20.

Center for Neural Science, New York University, New York, NY, USA.

New protein synthesis is known to be required for the consolidation of memories, yet existing methods of blocking translation lack spatiotemporal precision and cell-type specificity, preventing investigation of cell-specific contributions of protein synthesis. Here we developed a combined knock-in mouse and chemogenetic approach for cell-type-specific drug-inducible protein synthesis inhibition that enables rapid and reversible phosphorylation of eukaryotic initiation factor 2α, leading to inhibition of general translation by 50% in vivo. We use cell-type-specific drug-inducible protein synthesis inhibition to show that targeted protein synthesis inhibition pan-neuronally and in excitatory neurons in the lateral amygdala (LA) impaired long-term memory. This could be recovered with artificial chemogenetic activation of LA neurons, although at the cost of stimulus generalization. Conversely, genetically reducing phosphorylation of eukaryotic initiation factor 2α in excitatory neurons in the LA enhanced memory strength but reduced memory fidelity and behavioral flexibility. Our findings provide evidence for a cell-specific translation program during consolidation of threat memories.
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http://dx.doi.org/10.1038/s41593-019-0568-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147976PMC
February 2020

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions.

Elife 2019 12 23;8. Epub 2019 Dec 23.

Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

Methyl-CpG-binding-Protein 2 (MeCP2) is an abundant nuclear protein highly enriched in neurons. Here we report live-cell single-molecule imaging studies of the kinetic features of mouse MeCP2 at high spatial-temporal resolution. MeCP2 displays dynamic features that are distinct from both highly mobile transcription factors and immobile histones. Stable binding of MeCP2 in living neurons requires its methyl-binding domain and is sensitive to DNA modification levels. Diffusion of unbound MeCP2 is strongly constrained by weak, transient interactions mediated primarily by its AT-hook domains, and varies with the level of chromatin compaction and cell type. These findings extend previous studies of the role of the MeCP2 MBD in high affinity DNA binding to living neurons, and identify a new role for its AT-hooks domains as critical determinants of its kinetic behavior. They suggest that limited nuclear diffusion of MeCP2 in live neurons contributes to its local impact on chromatin structure and gene expression.
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http://dx.doi.org/10.7554/eLife.51449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957317PMC
December 2019

Examination of Bacterial Artificial Chromosome (BAC) DNA Quality and Quantity by Pulsed-Field Gel Electrophoresis.

Cold Spring Harb Protoc 2019 11 1;2019(11). Epub 2019 Nov 1.

Pulsed-field gel electrophoresis (PFGE) is the preferred method to measure the size of an insert in a high-capacity vector. After digestion with a restriction enzyme (e.g., PI-SceI or NotI), the DNA fragments are separated by PFGE through a 1% agarose gel.
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http://dx.doi.org/10.1101/pdb.prot098020DOI Listing
November 2019

Large-Scale Preparation and Linearization of Bacterial Artificial Chromosome (BAC) DNA.

Cold Spring Harb Protoc 2019 11 1;2019(11). Epub 2019 Nov 1.

A critical step for creating bacterial artificial chromosome (BAC) transgenic organisms is the production of high-quality BAC DNA. The method described here provides a source of DNA for further manipulation and modification, but can also be used for preparing modified BAC DNA, resulting from the one-step or two-step strategy, for use in other applications such as BAC transgenesis. An analysis of precautions to take when working with large DNA molecules is also provided.
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http://dx.doi.org/10.1101/pdb.prot098012DOI Listing
November 2019

Small-Scale Isolation of Bacterial Artificial Chromosome (BAC) DNA and Verification by Polymerase Chain Reaction (PCR).

Cold Spring Harb Protoc 2019 11 1;2019(11). Epub 2019 Nov 1.

In this protocol, small amounts of selected bacterial artificial chromosome (BAC) DNAs are prepared from 5-mL cultures of the host transformed with the BAC clone. DNA is isolated by an adaptation of the alkaline lysis method. The yield is 0.1-0.4 µg, and the BAC DNA is suitable for analysis by polymerase chain reaction (PCR). Considerations for clone selection are also addressed.
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http://dx.doi.org/10.1101/pdb.prot098004DOI Listing
November 2019

Species and cell-type properties of classically defined human and rodent neurons and glia.

Elife 2018 10 15;7. Epub 2018 Oct 15.

Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, United States.

Determination of the molecular properties of genetically targeted cell types has led to fundamental insights into mouse brain function and dysfunction. Here, we report an efficient strategy for precise exploration of gene expression and epigenetic events in specific cell types in a range of species, including postmortem human brain. We demonstrate that classically defined, homologous neuronal and glial cell types differ between rodent and human by the expression of hundreds of orthologous, cell specific genes. Confirmation that these genes are differentially active was obtained using epigenetic mapping and immunofluorescence localization. Studies of sixteen human postmortem brains revealed gender specific transcriptional differences, cell-specific molecular responses to aging, and the induction of a shared, robust response to an unknown external event evident in three donor samples. Our data establish a comprehensive approach for analysis of molecular events associated with specific circuits and cell types in a wide variety of human conditions.
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http://dx.doi.org/10.7554/eLife.37551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188473PMC
October 2018

Molecular and Functional Sex Differences of Noradrenergic Neurons in the Mouse Locus Coeruleus.

Cell Rep 2018 05;23(8):2225-2235

Department of Genetics and Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Preclinical work has long focused on male animals, though biological sex clearly influences risk for certain diseases, including many psychiatric disorders. Such disorders are often treated by drugs targeting the CNS norepinephrine system. Despite roles for noradrenergic neurons in behavior and neuropsychiatric disease models, their molecular characterization has lagged. We profiled mouse noradrenergic neurons in vivo, defining over 3,000 high-confidence transcripts expressed therein, including druggable receptors. We uncovered remarkable sex differences in gene expression, including elevated expression of the EP3 receptor in females-which we leverage to illustrate the behavioral and pharmacologic relevance of these findings-and of Slc6a15 and Lin28b, both major depressive disorder (MDD)-associated genes. Broadly, we present a means of transcriptionally profiling locus coeruleus under baseline and experimental conditions. Our findings underscore the need for preclinical work to include both sexes and suggest that sex differences in noradrenergic neurons may underlie behavioral differences relevant to disease.
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http://dx.doi.org/10.1016/j.celrep.2018.04.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070358PMC
May 2018

Cell-Type-Specific Contributions of Medial Prefrontal Neurons to Flexible Behaviors.

J Neurosci 2018 05 12;38(19):4490-4504. Epub 2018 Apr 12.

Laboratory of Molecular Biology, Rockefeller University, New York, New York 10065

Behavioral flexibility and impulse control are necessary for successful execution of adaptive behavior. They are impaired in patients with damage to the prefrontal cortex (PFC) and in some clinically important conditions, such as obsessive-compulsive disorder. Although the medial prefrontal cortex (mPFC) has been investigated as a critical structure for behavioral flexibility and impulse control, the contribution of the underlying pyramidal neuron cell types in the mPFC remained to be understood. Here we show that interneuron-mediated local inactivation of pyramidal neurons in the mPFC of male and female mice induces both premature responses and choice bias, and establish that these impulsive and compulsive responses are modulated independently. Cell-type-specific photoinhibition of pyramidal deep layer corticostriatal or corticothalamic neurons reduces behavioral flexibility without inducing premature responses. Together, our data confirm the role of corticostriatal neurons in behavioral flexibility and demonstrate that flexible behaviors are also modulated by direct projections from deep layer corticothalamic neurons in the mPFC to midline thalamic nuclei. Behavioral flexibility and impulse control are indispensable for animals to adapt to changes in the environment and often affected in patients with PFC damage and obsessive-compulsive disorder. We used a probabilistic reversal task to dissect the underlying neural circuitry in the mPFC. Through characterization of the three major pyramidal cell types in the mPFC with optogenetic silencing, we demonstrated that corticostriatal and corticothalamic but not corticocortical pyramidal neurons are temporally recruited for behavioral flexibility. Together, our findings confirm the role of corticostriatal projections in cognitive flexibility and identify corticothalamic neurons as equally important for behavioral flexibility.
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http://dx.doi.org/10.1523/JNEUROSCI.3537-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943977PMC
May 2018

Retrograde inhibition by a specific subset of interpeduncular α5 nicotinic neurons regulates nicotine preference.

Proc Natl Acad Sci U S A 2017 12 20;114(49):13012-13017. Epub 2017 Nov 20.

Laboratory of Molecular Biology, The Rockefeller University, New York, NY 10065;

Repeated exposure to drugs of abuse can produce adaptive changes that lead to the establishment of dependence. It has been shown that allelic variation in the α5 nicotinic acetylcholine receptor (nAChR) gene is associated with higher risk of tobacco dependence. In the brain, α5-containing nAChRs are expressed at very high levels in the interpeduncular nucleus (IPN). Here we identified two nonoverlapping α5 cell populations (α5- and α5- ) in mouse IPN that respond differentially to nicotine. Chronic nicotine treatment altered the translational profile of more than 1,000 genes in α5- neurons, including neuronal nitric oxide synthase () and somatostatin (). In contrast, expression of few genes was altered in the α5- population. We show that both nitric oxide and SST suppress optically evoked neurotransmitter release from the terminals of habenular (Hb) neurons in IPN. Moreover, in vivo silencing of neurotransmitter release from the α5- but not from the α5- population eliminates nicotine reward, measured using place preference. This loss of nicotine reward was mimicked by shRNA-mediated knockdown of in the IPN. These findings reveal a proaddiction adaptive response to chronic nicotine in which nitric oxide and SST are released by a specific α5 neuronal population to provide retrograde inhibition of the Hb-IPN circuit and thereby enhance the motivational properties of nicotine.
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http://dx.doi.org/10.1073/pnas.1717506114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724287PMC
December 2017

5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes.

Proc Natl Acad Sci U S A 2017 09 28;114(37):E7812-E7821. Epub 2017 Aug 28.

Laboratory of Molecular Biology, The Rockefeller University, New York, NY 10065;

5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in "functional" demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization.
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http://dx.doi.org/10.1073/pnas.1708044114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604027PMC
September 2017

Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP.

Cell Rep 2017 04;19(3):655-667

Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre.
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http://dx.doi.org/10.1016/j.celrep.2017.03.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476221PMC
April 2017