Background & Aims
Although initially formulated to treat pain, opioids are highly addictive and have triggered a devastating public health crisis, affecting over 16 million people worldwide. Tapering opioids is difficult for patients due to the excruciating set of physiological and psychological symptoms. Furthermore, opioid withdrawal induces widespread changes in reward circuitry, including within the nucleus accumbens (NAc), through maladaptive synaptic plasticity and transcriptional changes. Prior work demonstrated that transcription of the neuropeptide precursor VGF (non-acronymic) increased in the NAc when opioid exposure was periodically interrupted by withdrawal. The VGF-derived peptide TLQP-62 is a known mediator of plasticity, but its action on NAc physiology and withdrawal-evoked behaviors has never been examined. Our objective is to characterize VGF expression in the NAc and determine the effect of its derived peptides on synaptic transmission and opioid withdrawal-evoked behaviors.
Methods
To characterize expression in the NAc, we used fluorescent RNA in situ hybridization to determine the colocalization of VGF with markers of NAc neurons. HALO analysis software quantified the distribution of VGF-expressing cells across NAc subregions and cell types. Initial experiments used the calcium indicator Fluo-4 and live two-photon microscopy to visualize neuronal responses to glutamate exposure before and after TLQP-62 application. To disrupt VGF and assess changes to opioid withdrawal-evoked behaviors, we used a Cre-dependent retrograde virus injected into the NAc of floxed VGF mice. We then treated mice with an interrupted pattern of morphine exposure using continuously-releasing osmotic pumps and naloxone injections to precipitate withdrawal. Open field locomotion determined the necessity of VGF in the NAc for psychomotor sensitization, von Frey tests demonstrated its contribution to mechanical hypersensitivity, and pairwise social behaviors showed the effect on sociability.
Results
Preliminary data showed that VGF transcripts are expressed fairly ubiquitously across the NAc, but may be enriched in the medial shell of the NAc and somatostatin-expressing interneurons. Calcium imaging revealed that both dopamine receptor 1-expressing (D1) and non-expressing cells have potentiated responses to glutamate following exposure to the VGF-derived peptide TLQP-62. Furthermore, knocking out VGF in the NAc eliminated the behavioral changes typically evoked by opioid exposure and withdrawal in mice. More specifically, the psychomotor sensitization normally produced by repeated interrupted opioid exposure is absent, and instead, the mice undergoing precipitated withdrawal show psychomotor tolerance.
Conclusions
Our expression characterization and calcium imaging studies suggest that VGF is enriched in somatostatin interneurons and released onto medium spiny neurons, the major population in the NAc, to augment excitatory neurotransmission. Disrupting VGF expression in the NAc attenuated psychomotor sensitization, which reflects withdrawal-induced plasticity, following interrupted morphine exposure. Prior studies showed that stimulation and inhibition of somatostatin cells in the NAc increase and decrease drug-induced locomotor activity, respectively. Together, this suggests that VGF is released by somatostatin interneurons onto medium spiny neurons and evokes psychomotor sensitization following opioid withdrawal. Future directions will distill the effect of TLQP-62 using whole-cell patch-clamp electrophysiology to record excitatory postsynaptic currents. We will also evaluate how VGF disruption affects other opioid withdrawal-evoked behaviors, including hypersensitivity and sociability.
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