Nucleus Accumbens Single Nucleus RNA Sequencing Identifies Cell Subtype-Specific Maladaptations after Prolonged Spared Nerve Injury

Abstract

<b>Abstract ID 14714</b> <b>Poster Board 278</b> Chronic pain is a highly prevalent condition that is difficult to treat because of molecular maladaptations that occur at several levels of the nociceptive circuitry. Currently prescribed therapeutics show limited efficacy and cause substantial side effects after prolonged use. Several regions of the mesocorticolimbic circuit, such as the medial prefrontal cortex, nucleus accumbens (NAc), and ventral tegmental area play a key role in the maintenance of sensory hypersensitivity, pain perception, and chronic pain-associated affective comorbidities, such as depression and addiction vulnerability. Here, we performed single nucleus RNA sequencing (snRNA-seq) on NAc tissue from adult male DBA/2J mice three months after spared nerve injury (SNI). Cell clusters were annotated against a publicly-available single cell RNA-seq NAc dataset by Avey et&nbsp;al. (“Single-Cell RNA-Seq Uncovers a Robust Transcriptional Response to Morphine by Glia”). Bioinformatic analysis identified the following cell clusters with &gt;100 differentially-expressed genes (DEGs; p-nominal &lt; 0.05): Astrocyte_1, Astrocyte_2, Endothelial, Microglia, Neuron_1, Neuron_2, Neuron_3, NSC_1, OL_2, OL_3, OL_4, OPC, VLMC. When considering neuronal clusters, we identified Ingenuity Pathway Analysis (IPA)-generated Top 15 Canonical Pathways that were directionally conserved across at least two of the three clusters, including impaired “Synaptogenesis Signaling” and “Synaptic Long-term Potentiation”. Unpublished work from our group also suggests that NAc medium spiny neurons become hyperexcitable at 1.5-2 months post-SNI. A key transcription factor that negatively regulates excitatory synaptic plasticity in adult neurons is MEF2C. At three months post-SNI we observed a decrease in <i>Drd1</i>+/<i>Drd2</i>+/<i>Mef2c</i>+ co-expressing neurons. Taken together, we hypothesized that overexpression of<i> Mef2c</i> in NAc neurons with an AAV8-hSyn-<i>Mef2c</i>-<i>Gfp</i> viral vector would modulate these maladaptations. Indeed, virus-mediated overexpression of <i>Mef2c</i> in NAc MSNs reversed SNI-induced hyperexcitability and reduced miniature excitatory post-synaptic current amplitudes relative to SNI-<i>Gfp</i> animals. IPA analysis of NAc bulk RNA-seq comparing SNI-<i>Mef2c</i> and SNI-<i>Gfp</i> conditions (2 months post-SNI) predicted an upregulation of the “Synaptic Long-term Potentiation” pathway. These <i>Mef2c</i> overexpression-induced counteractions of SNI maladaptations were associated with a reversal of SNI-induced punctate mechanical hypersensitivity and anxiety-like behavior in the marble burying assay. Future directions include assessing circuit-level effects on neurotransmitter levels upon <i>Mef2c</i> overexpression in the NAc by use of voltammetry. Overall, this work emphasizes that prolonged peripheral nerve injury induces transcriptional changes in a majority of cell subtypes in the NAc, and cell subtype-specific therapeutic strategies can alleviate behavioral manifestations of SNI. <b>Support/Funding Information:</b> R01 NS086444 (VZ), R01 NS086444-07S1 (VZ &amp; RAS), ASRA Graduate Student Award (RAS)