The work presented here was funded through NIH HLB U01 HL133360 awarded to JS and RV, NIDA R21 DA036372 awarded to JS and EVB, T32 AA-007463 awarded to Jan Hoek in support of SJO&#39;S, and National Institute of Alcoholism and Alcohol Abuse: R01 AA018873.

<ol>
	<li>. Substance Use and Mental Health Indicators in the United States: Results from the 2019 National Survey on Drug Use and Health Available from: <a target="_blank" href="https://www.samhsa.gov/data/">https://www.samhsa.gov/data/</a> (2020)</li><li>Prevalence of Serious Mental Illness (SMI). NIH Available from: <a target="_blank" href="https://www.nimh.nih.gov/health/statistics/mental-illness.shtml">https://www.nimh.nih.gov/health/statistics/mental-illness.shtml</a> (2020)</li><li>Mattick, R. P., Kimber, J., Breen, C., Davoli, M., Mattick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buprenorphine+maintenance+versus+placebo+or+methadone+maintenance+for+opioid+dependence.">Buprenorphine maintenance versus placebo or methadone maintenance for opioid dependence.</a> Cochrane Database of Systematic Reviews. , (2008).</li><li>Mattick, R. P., Breen, C., Kimber, J., Davoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methadone+maintenance+therapy+versus+no+opioid+replacement+therapy+for+opioid+dependence.">Methadone maintenance therapy versus no opioid replacement therapy for opioid dependence.</a> The Cochrane Database of Systematic Reviews. 2009 (3), (2009).</li><li>Miller, P. M., Book, S. W., Stewart, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+treatment+of+alcohol+dependence:+A+systematic+review.">Medical treatment of alcohol dependence: A systematic review.</a> International Journal of Psychiatry in Medicine. 42 (3), 227-266 (2012).</li><li>Holmes, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lancet+Psychiatry+Commission+on+psychological+treatments+research+in+tomorrow's+science.">The Lancet Psychiatry Commission on psychological treatments research in tomorrow's science.</a> The Lancet. Psychiatry. 5 (3), 237-286 (2018).</li><li>Ford, C. L., Young, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+opportunities+for+circuit-based+social+neuroscience:+advancing+21st+century+psychiatry.">Translational opportunities for circuit-based social neuroscience: advancing 21st century psychiatry.</a> Current Opinion in Neurobiology. 68, 1-8 (2021).</li><li>Holmes, E. A., Craske, M. G., Graybiel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+treatments:+A+call+for+mental-health+science.">Psychological treatments: A call for mental-health science.</a> Nature. 511 (7509), 287-289 (2014).</li><li>Miranda, A., Taca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromodulation+with+percutaneous+electrical+nerve+field+stimulation+is+associated+with+reduction+in+signs+and+symptoms+of+opioid+withdrawal:+a+multisite,+retrospective+assessment.">Neuromodulation with percutaneous electrical nerve field stimulation is associated with reduction in signs and symptoms of opioid withdrawal: a multisite, retrospective assessment.</a> The American Journal of Drug and Alcohol Abuse. 44 (1), 56-63 (2018).</li><li>Metz, V. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ibudilast+on+the+subjective,+reinforcing,+and+analgesic+effects+of+oxycodone+in+recently+detoxified+adults+with+opioid+dependence.">Effects of ibudilast on the subjective, reinforcing, and analgesic effects of oxycodone in recently detoxified adults with opioid dependence.</a> Neuropsychopharmacology. 42 (9), 1825-1832 (2017).</li><li>Heinzerling, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=placebo-controlled+trial+of+targeting+neuroinflammation+with+ibudilast+to+treat+methamphetamine+use+disorder.">placebo-controlled trial of targeting neuroinflammation with ibudilast to treat methamphetamine use disorder.</a> Journal of Neuroimmune Pharmacology. 15 (2), 238-248 (2020).</li><li>Bogenschutz, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psilocybin-assisted+treatment+for+alcohol+dependence:+A+proof-of-concept+study.">Psilocybin-assisted treatment for alcohol dependence: A proof-of-concept study.</a> Journal of Psychopharmacology. 29 (3), 289-299 (2015).</li><li>O'Sullivan, S. J., Schwaber, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+in+alcohol+and+opioid+withdrawal+syndromes+suggest+common+negative+reinforcement+mechanisms+involving+the+interoceptive+antireward+pathway.">Similarities in alcohol and opioid withdrawal syndromes suggest common negative reinforcement mechanisms involving the interoceptive antireward pathway.</a> Neuroscience and Biobehavioral Reviews. 125, 355-364 (2021).</li><li>O'Sullivan, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+systems+neuroscience:+A+growing+frontier+in+mental+illness.">Single-cell systems neuroscience: A growing frontier in mental illness.</a> Biocell. 46 (1), 7-11 (2022).</li><li>O'Sullivan, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+glia+and+neuron+gene+expression+in+the+central+amygdala+in+opioid+withdrawal+suggests+inflammation+with+correlated+gut+dysbiosis.">Single-cell glia and neuron gene expression in the central amygdala in opioid withdrawal suggests inflammation with correlated gut dysbiosis.</a> Frontiers in Neuroscience. 13, 665 (2019).</li><li>O'Sullivan, S. J., McIntosh-Clarke, D., Park, J., Vadigepalli, R., Schwaber, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+scale+neuronal+and+glial+gene+expression+and+putative+cell+phenotypes+and+networks+in+the+nucleus+tractus+solitarius+in+an+alcohol+withdrawal+time+series.">Single cell scale neuronal and glial gene expression and putative cell phenotypes and networks in the nucleus tractus solitarius in an alcohol withdrawal time series.</a> Frontiers in Systems Neuroscience. 15, 739790 (2021).</li><li>O'Sullivan, S. J., Reyes, B. A. S., Vadigepalli, R., Van Bockstaele, E. J., Schwaber, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+laser+capture+microdissection+and+microfluidic+qpcr+to+analyze+transcriptional+profiles+of+single+cells:+A+systems+biology+approach+to+opioid+dependence.">Combining laser capture microdissection and microfluidic qpcr to analyze transcriptional profiles of single cells: A systems biology approach to opioid dependence.</a> Journal of Visualized Experiments. (157), e60612 (2020).</li><li>Achanta, S., Vadigepalli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+high-throughput+qRT-PCR+protocol.">Single cell high-throughput qRT-PCR protocol.</a> Protocols.io. , (2020).</li><li>O'Sullivan, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interoceptive+antireward+pathway+and+gut+dysbiosis+in+addiction.">The interoceptive antireward pathway and gut dysbiosis in addiction.</a> Journal of Psychiatry, Depression &amp; Anxiety. 7 (40), 1-5 (2021).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptional+analysis+reveals+novel+neuronal+phenotypes+and+interaction+networks+involved+in+the+central+circadian+clock.">Single-cell transcriptional analysis reveals novel neuronal phenotypes and interaction networks involved in the central circadian clock.</a> Frontiers in Neuroscience. 10, 481 (2016).</li><li>Staehle, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+patterns+of+gene+expression+in+the+dorsal+vagal+complex+and+the+central+nucleus+of+the+amygdala+-+Non-rhythm-generating+brain+regions.">Diurnal patterns of gene expression in the dorsal vagal complex and the central nucleus of the amygdala - Non-rhythm-generating brain regions.</a> Frontiers in Neuroscience. 14, 375 (2020).</li><li>R&#233;us, G. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammation+and+microglial+activation+in+the+pathophysiology+of+psychiatric+disorders.">The role of inflammation and microglial activation in the pathophysiology of psychiatric disorders.</a> Neuroscience. 300, 141-154 (2015).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+astrocytes+in+major+neuropsychiatric+disorders.">Role of astrocytes in major neuropsychiatric disorders.</a> Neurochemical Research. 46 (10), 2715-2730 (2021).</li><li>Park, J., Ogunnaike, B., Schwaber, J., Vadigepalli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+functional+gene+regulatory+network+phenotypes+underlying+single+cell+transcriptional+variability.">Identifying functional gene regulatory network phenotypes underlying single cell transcriptional variability.</a> Progress in Biophysics and Molecular Biology. 117 (1), 87-98 (2015).</li><li>Lieber, C. S., DeCarli, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+alcohol+feeding+and+liver+injury+in+the+baboon.">An experimental model of alcohol feeding and liver injury in the baboon.</a> Journal of Medical Primatology. 3 (3), 153-163 (1974).</li><li>Lieber, C. S., Decarli, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+ethanol+toxicity.">Animal models of chronic ethanol toxicity.</a> Methods in Enzymology. 233, 585-594 (1994).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inputs+drive+cell+phenotype+variability.">Inputs drive cell phenotype variability.</a> Genome Research. 24 (6), 930-941 (2014).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates: Hard Cover Edition. , (1982).</li></ol>

The authors declare that they have no competing financial interests.

Alcohol use disorder remains a challenging disease to treat. Our group has approached this disorder by investigating antireward processes with a systems neuroscience perspective. We measured gene expression changes in single NTS neurons and microglia in an alcohol withdrawal time series16. The NTS was chosen for its prominent role in the autonomic dysregulation that occurs in alcohol withdrawal syndrome. We combine LCM with single-cell microfluidic RT-qPCR allowing for robust numbers of samples an...

Addiction remains a growing challenge in the developed world1,2. Despite major scientific and clinical advances, rates of addiction continue to increase while the efficacy of established treatments remains stable at best3,4,5. However, advances in biotechnology and scientific approaches have led to novel methods and hypotheses to further investigate the pathophysiology of substance dependence6,7,8. Indeed, recent developments suggest that novel concepts and treatment paradigms may lead to breakthroughs with social, economic, and political consequences9,10,11,12.
We investigated antireward in the withdrawal of alcohol and opioid dependence13,14,15,16. Methods are central to this paradigm17,18. Laser capture microdissection (LCM) can select single cells with high anatomic specificity. This functionality is integral to the neuroinflammation antireward hypothesis as both glia and neurons can be collected and analyzed from the same neuronal subnucleus in the same animal13,14,15,16,19. A relevant portion of the transcriptome of selected cells can then be measured with high-throughput microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR) providing high-dimensional datasets for computational analysis yielding insights into functional networks20,21.
Measuring a subset of the transcriptome in neurons and glia in a specific brain nucleus generates a dataset that is robust in both sample number and genes measured and is sensitive and specific. These tools are optimal for a system&#39;s neuroscience approach to psychiatric disease because glia, mainly astrocytes and microglia, have demonstrated a central role in neurological and psychiatric disease over the past decade22,23. Our approach can measure the expressive response of glia and neurons concomitantly across numerous receptors and ligands involved in local paracrine signaling. Indeed, signaling can be inferred from these datasets using various quantitative methods such as fuzzy logic24. Further, the identification of cellular subphenotypes in neurons or glia and their function can provide insight into how brain cells in specific nuclei organize, respond to, and dysregulate at the single-cell level. The dynamics of this functional system can also be modeled with time series experiments16. Lastly, animal models can be perturbed anatomically or pharmacologically to lend a mechanistic condition to this system&#39;s approach.
Representative experiment: 
Below, we provide an example of the application of these methods. This study investigated rat neuronal and microglia gene expression in the solitary nucleus (NTS) in response to alcohol dependence and subsequent withdrawal16. Rat cohorts comprised 1) Control, 2) Ethanol-dependent (EtOH), 3) 8 h withdrawal (Wd), 4) 32 h Wd, and 5) 176 h Wd (Figure 1A). Following rapid decapitation, brainstems were separated from the forebrain and cryosectioned, and slices were stained for tyrosine hydroxylase-positive (TH +) neurons and microglia (Figure 1B). LCM was used to collect both TH+ and TH- neurons and microglia. All the cells were from the NTS and analyzed as samples of 10-cell pools. Four 96 x 96 microfluidic RT-qPCR dynamic arrays were run on the RT-qPCR platform measuring 65 genes (Figure 1B-C). Data were normalized using a -&#916;&#916;Ct method and analyzed using R, and single-cell selection was validated with molecular markers (Figure 1D-E). Technical validation was further verified by technical replicates analyzed within a single batch and across batches (Figure 2 and Figure 3). TH+ and TH- neurons organized into different sub phenotypes with similar inflammatory gene clusters but differing &#947;-aminobutyric acid (GABA) receptor (R) clusters (Figure 4 and Figure 5). Sub phenotypes that had elevated expression of inflammatory gene clusters were over-represented at 32 h Wd while GABA-receptor (GABAR) expression remained low in protracted alcohol withdrawal (176 h Wd). This work contributes to the antireward hypothesis of alcohol and opioid dependence which conjectures that interceptive feedback from the viscera in withdrawal contributes to the dysregulation of visceral-emotional neuronal nuclei (i.e., NTS and amygdala) resulting in more severe autonomic and emotional sequelae, which contribute to substance dependence (Figure 6).

<table><tbody><tr><td>20X DNA Binding Dye</td><td>Fluidigm</td><td>100-7609</td><td>NA</td></tr><tr><td>2x GE Assay Loading Reagent</td><td>Fluidigm</td><td>85000802-R</td><td>NA</td></tr><tr><td>96.96 Dynamic Array IFC for Gene Expression (referred to as qPCR chip in text)</td><td>Fluidigm</td><td>BMK-M-96.96</td><td>NA</td></tr><tr><td>Anti-Cd11&amp;beta; Antibody</td><td>Genway Biotech</td><td>CCEC48</td><td>Microglia Stain</td></tr><tr><td>Anti-NeuN Antibody, clone A60</td><td>EMD Millipore</td><td>MAB377</td><td>Neuronal Stain</td></tr><tr><td>Anti-tyrosine hydroxylase antibody</td><td>abcam</td><td>ab112</td><td>Stain for TH+ neurons</td></tr><tr><td>ArcturusXT Laser Capture Microdissection System</td><td>Arcturus</td><td>NA</td><td>NA</td></tr><tr><td>Biomark HD</td><td>Fluidigm</td><td>NA</td><td>RT-qPCR platform</td></tr><tr><td>Bovine Serum Antigen</td><td>Sigma-Aldrich</td><td>B4287</td><td></td></tr><tr><td>CapSure Macro LCM Caps</td><td>ThermoFisher Scientific</td><td>&amp;nbsp;LCM0211</td><td>NA</td></tr><tr><td>CellDirect One-Step qRT-PCR Kit</td><td>ThermoFisher Scientific</td><td>11753500</td><td>Lysis buffer solution components</td></tr><tr><td>CellsDirect Resuspension &amp;amp; Lysis Buffer Kit</td><td>ThermoFisher Scientific</td><td>11739010</td><td>Invitrogen</td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>62248</td><td>Nucleus Stain</td></tr><tr><td>DNA Suspension Buffer</td><td>TEKnova</td><td>T0221</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Alexa Fluor 488</td><td>ThermoFisher Scientific</td><td>R37118</td><td>Seconadry Antibody</td></tr><tr><td>Exonuclease I</td><td>New Englnad BioLabs, Inc.</td><td>M0293S</td><td>NA</td></tr><tr><td>ExtracSure Sample Extraction Device</td><td>ThermoFisher Scientific</td><td>LCM0208</td><td>NA</td></tr><tr><td>FisherbrandTM Superfrost Plus Microscope Slides</td><td>ThermoFisher Scientific</td><td>22-037-246</td><td>Plain glass slides</td></tr><tr><td>GeneAmp Thin-Walled Reaction Tube</td><td>ThermoFisher Scientific</td><td>N8010611</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L), Superclona Recombinant Secondary Antibody, Alexa Fluor 555</td><td>ThermoFisher Scientific</td><td>A28180</td><td>Seconadry Antibody</td></tr><tr><td>IFC Controller</td><td>Fluidigm</td><td>NA</td><td>NA</td></tr><tr><td>RNaseOut</td><td>ThermoFisher Scientific</td><td>10777019</td><td></td></tr><tr><td>SsoFast EvaGreen Supermix with Low Rox</td><td>Bio-Rad</td><td>PN 172-5211</td><td>NA</td></tr><tr><td>SuperScript VILO cDNA Synthesis Kit</td><td>ThermoFisher Scientific</td><td>11754250</td><td>Contains VILO and SuperScript</td></tr><tr><td>T4 Gene 32 Protein</td><td>New Englnad BioLabs, Inc.</td><td>M0300S</td><td>NA</td></tr><tr><td>TaqMan PreAmp Master Mix</td><td>ThermoFisher Scientific</td><td>4391128</td><td>NA</td></tr><tr><td>TE Buffer</td><td>TEKnova</td><td>T0225</td><td>NA</td></tr><tr><td>TempPlate Semi-Skirted 96-Well PCR Plate, 0.2 mL</td><td>USA Scientific</td><td>1402-9700</td><td>NA</td></tr></tbody></table>

investigating drivers of antireward in addiction behavior with anatomically specific single-cell gene expression methods

Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift away from reward and commencement necessitates novel perspectives, paradigms, and hypotheses along with an expansion of the methods applied to investigate addiction. Here, we provide an example: A systems biology approach to investigate antireward that combines laser capture microdissection (LCM) and high-throughput microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). Gene expression network dynamics were measured and a key driver of neurovisceral dysregulation in alcohol and opioid withdrawal, neuroinflammation, was identified. This combination of technologies provides anatomic and phenotypic specificity at single-cell resolution with high-throughput sensitivity and specific gene expression measures yielding both hypothesis-generating datasets and mechanistic possibilities that generate opportunities for novel insights and treatments.

Validation of single-cell collection is performed visually during LCM procedures. Cell nuclei are assessed at the QC station. The cell type can be determined by emission of tagged fluorophore for that cell type and its general morphology. If non-desired cells have been selected on the cap, their genetic material can be destroyed with a UV laser at the QC station. Further validation by molecular analysis is also necessary. In this representative example16, two types of neurons were selected-tyrosin...

Watch this Scientific Journal Video about Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods at JoVE.com

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods

The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction.

Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift ...

investigating-drivers-antireward-addiction-behavior-with-anatomically

Research

JoVE Journal

Neuroscience

2.1K Views.  Thomas Jefferson University. Het protocol is zeer gevoelig en maakt het mogelijk om genexpressieprofiel met hoge doorvoer vast te leggen bij een enkele celresolutie. De techniek biedt zowel anatomische ruimtelijke als moleculaire specificiteit bij eencellige resolutie. Na het oogsten van de hersenen, het selecteren van de afzonderlijke cellen en het voorbereiden van het mRNA, injecteert u controlelijnvloeistof in de qPCR-chip voor priming.Plaats de qPCR-chip in het microfluïdische mengapparaat. Selecteer het Prim...

Video: Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Behavior

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular ...

Genetics

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video