Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods
Summary
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.
Abstract
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.
Introduction
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'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'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 -ΔΔCt 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 γ-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).
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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'S, and National Institute of Alcoholism and Alcohol Abuse: R01 AA018873.
Materials
| 20X DNA Binding Dye | Fluidigm | 100-7609 | NA |
| 2x GE Assay Loading Reagent | Fluidigm | 85000802-R | NA |
| 96.96 Dynamic Array IFC for Gene Expression (referred to as qPCR chip in text) | Fluidigm | BMK-M-96.96 | NA |
| Anti-Cd11β Antibody | Genway Biotech | CCEC48 | Microglia Stain |
| Anti-NeuN Antibody, clone A60 | EMD Millipore | MAB377 | Neuronal Stain |
| Anti-tyrosine hydroxylase antibody | abcam | ab112 | Stain for TH+ neurons |
| ArcturusXT Laser Capture Microdissection System | Arcturus | NA | NA |
| Biomark HD | Fluidigm | NA | RT-qPCR platform |
| Bovine Serum Antigen | Sigma-Aldrich | B4287 | |
| CapSure Macro LCM Caps | ThermoFisher Scientific | LCM0211 | NA |
| CellDirect One-Step qRT-PCR Kit | ThermoFisher Scientific | 11753500 | Lysis buffer solution components |
| CellsDirect Resuspension & Lysis Buffer Kit | ThermoFisher Scientific | 11739010 | Invitrogen |
| DAPI | ThermoFisher Scientific | 62248 | Nucleus Stain |
| DNA Suspension Buffer | TEKnova | T0221 | |
| Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Donkey anti-Rabbit IgG (H+L) ReadyProbe Secondary Antibody, Alexa Fluor 488 | ThermoFisher Scientific | R37118 | Seconadry Antibody |
| Exonuclease I | New Englnad BioLabs, Inc. | M0293S | NA |
| ExtracSure Sample Extraction Device | ThermoFisher Scientific | LCM0208 | NA |
| FisherbrandTM Superfrost Plus Microscope Slides | ThermoFisher Scientific | 22-037-246 | Plain glass slides |
| GeneAmp Thin-Walled Reaction Tube | ThermoFisher Scientific | N8010611 | |
| Goat anti-Mouse IgG (H+L), Superclona Recombinant Secondary Antibody, Alexa Fluor 555 | ThermoFisher Scientific | A28180 | Seconadry Antibody |
| IFC Controller | Fluidigm | NA | NA |
| RNaseOut | ThermoFisher Scientific | 10777019 | |
| SsoFast EvaGreen Supermix with Low Rox | Bio-Rad | PN 172-5211 | NA |
| SuperScript VILO cDNA Synthesis Kit | ThermoFisher Scientific | 11754250 | Contains VILO and SuperScript |
| T4 Gene 32 Protein | New Englnad BioLabs, Inc. | M0300S | NA |
| TaqMan PreAmp Master Mix | ThermoFisher Scientific | 4391128 | NA |
| TE Buffer | TEKnova | T0225 | NA |
| TempPlate Semi-Skirted 96-Well PCR Plate, 0.2 mL | USA Scientific | 1402-9700 | NA |
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