Name: investigating drivers of antireward in addiction behavior with anatomically specific single-cell gene expression methods
Uploaded: 2022-08-04T13:30:00
Description: Watch this Scientific Journal Video about Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods at JoVE.com

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.

This study was carried out in accordance with the recommendations of Animal Care and Use Committee (IACUC) of Thomas Jefferson University. The protocol was approved by Thomas Jefferson University IACUC.
1. Animal model
<ol>
	<li>House male Sprague Dawley (&#62;120 g, Harlan, Indianapolis, IN, USA) rat triplets individually with free access to ethanol-chow (2 rats) or control-chow mixture (1 rat). 
	NOTE: This representative experiment employed the Lieber-DeCarli protoco...

<ol>
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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#946; 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>&#160;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 &#38; 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 ...

The protocol is highly sensitive and allows high throughput capture of gene expression profile at single cell resolution. The technique provides both anatomical spatial and molecular specificity at single cell resolution. After harvesting the brain, selecting the single cells, and preparing the mRNA, inject control line fluid into the qPCR chip for priming.Insert the qPCR chip into the microfluidic mixing device. Select the Prime script and run the program. After the completion of the program, remove the primed qPCR chip and pipette six microliters of the reaction from the PCR sample plate into the corresponding sample well in the primed qPCR chip.Now, pipette six microliters of the reaction from the PCR assay plate into the corresponding assay well in the primed qPCR chip. Then insert the chip into the microfluidic mixing device. Select the Load Mix script and run the program.Turn on the microfluidic RT-qPCR platform and warm up the bulb. Remove the qPCR chip from the microfluidic mixing device and peel the protective sticker from the bottom of the chip. Open the microfluidic RT-qPCR platform and load the qPCR chip into the platform.Launch the data collection software by clicking on Start a New Run. Verify the chip barcode and chip type. Then click on next.Select the chip run file and browse the file location for the data collection storage. Then click on Application Type and select Gene Expression. Select ROX for a passive reference, single probe, and EVAGreen for probe type.Click to select the thermal cycling program and select Biomark HD GE:Fast 96x96 PCR+Melt v2. pcl file. Download the data analysis software.Launch the software to analyze the microfluidic RT-qPCR experiment. Click Chip Run and open the ChipRun. bml file that was created by the experimenter.A window displaying the experimental details including the passive reference, probe, and PCR thermal program will pop up. Under the Chip Explorer tab, click Sample Plate Setup and create a new sample plate template. Copy and paste the sample labels into the software spreadsheet as per the experimental design.Enter the sample name and the RNA concentration used for the standard samples. Now, map the sample set up by clicking map from the task menu and selecting SBS96-Left.dsp. Next, select Detail Views to update these changes in the file and click Analyze.Select Detector Plate Setup and create a new assay plate. Select the appropriate container type and format. Paste the assay names as per the experimental design and click on Analyze.In the analysis settings pane, click the User and set the fit to auto. Now, manually review each reaction in the 96x96 chip. Visualize the amplification and melt curves to determine if each reaction followed the expected qPCR pattern.If the amplification or melt curve does not match with what is expected, fail that reaction. Following QC, export the data by selecting file, clicking export, and saving the dataset as a csv file. As the exported csv file has both a pass fail matrix and a matrix with raw CT values, use the pass fail matrix to replace any failed cells in the dataset with NA.Download the recent version of open Source R software and then download the R Studio application.For median centering, use the R software to calculate the median CT value calculated from all the CT values for an individual sample and then subtract all individual CT values from this median value to get a minus delta CT value. For housekeeping gene normalization, calculate the average expression of the housekeeping genes for each sample and use this value to subtract the individual CT values. To generate a minus delta delta CT value, run the code in the R software.Upload the normalized dataset into R and analyze the data using the scale function, generate the scaled data, and then use the R heat map function or separate software to generate a heat map. To organize the data set for other functionality, calculate the Pearson correlations between each gene followed by the melt function. Export and upload this data into a gene correlation network software.Neurons showed elevated expression of NeuN and microglial samples showed significant expression of the microglial markers Cd34 and Cx3xr1. The Th+neuronal samples demonstrated significantly elevated expression of Th compared to Th-neuronal and microglia samples. The Th-neurons displayed significant expression of Gcg, suggesting that Th-neuronal samples are enriched with neurons that use Glp1 as a neurotransmitter.Linear discriminate analysis showed that these three cell types had differing gene expression profiles across all 65 genes. Cellular sub phenotypes of Glp1 enriched neuron samples through an alcohol withdrawal time series were represented using heat maps. The sub phenotype A, which highly expresses the inflammatory gene cluster one increases in ratio at the eight hour withdrawal time point and reaches a maximum at 32 hour withdrawal.The inflammatory sub phenotype is normalized to control by 176 hour withdrawal. Sub phenotype B, which highly expresses GABA receptor gene cluster two demonstrates an overall suppression in the expression of this gene cluster by the 176 hour withdrawal condition. The gene expression of individual samples was combined into averages so that the expression of gene clusters and the protein location of that gene transcript could be visualized throughout the time series.This technique can be applied to any biological system or tissue to understand the cellular response to a disease or a perturbation. That is to decipher the molecular response at single cell resolution relating to the spatial and the anatomical architecture of the tissue.

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Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...