Role of neuropeptide neuromedin U in the nucleus accumbens shell in cocaine self-administration in male rats

Abstract

The nucleus accumbens shell (NAcSh) and its afferent and efferent neuronal projections control key aspects of motivation for cocaine. A recently described regulator of γ-aminobutyric acid (GABA) projections from the dorsal raphe nucleus (DRN) to the NAcSh (DRN → NAcSh) is the neuropeptide neuromedin U (NMU). Here, we find that systemic administration of NMU decreases breakpoint for cocaine on a progressive ratio schedule of reinforcement in male rats. Employing a retrograde adeno-associated virus (AAV), we found that RNAi-mediated knockdown of the NMU receptor 2 (NMUR2) in afferent DRN projections to the NAcSh increases the breakpoint for cocaine. Our previous studies demonstrated that NMU regulates GABA release in the NAcSh, and our current investigation found that systemic NMU administration suppresses cocaine-evoked GABA release in the NAcSh and increases phosphorylated c-Fos expression in neurons projecting from the NAcSh to the ventral pallidum (VP). To further probe the impact of NMU/NMUR2 on neuroanatomical pathways regulating motivation for cocaine, we employed multi-viral transsynaptic studies. Using a combination of rabies virus and retrograde AAV helper virus, we mapped the impact of NMU across three distinct brain regions simultaneously and found a direct connection of GABAergic DRN neurons to the NAcSh → VP pathway. Together, these data reveal that NMU/NMUR2 modulates a direct connection within the GABAergic DRN → NAcSh → VP circuit that diminishes breakpoints for cocaine. These findings importantly advance our understanding of the neurochemical underpinnings of pathway-specific regulation of neurocircuitry that may regulate cocaine self-administration, providing a unique therapeutic perspective.

Fig. 1. NMU and NMUR2 controls breakpoints for cocaine.

A NMU decreased test day lever-presses for cocaine (0.75 mg/kg/inf) on a PR schedule (n = 10–12 per group; one individual Vehicle rat at 2536 lever presses not shown). B NMU decreased test day data breakpoints for cocaine. The breakpoint value is identified on the left axis and the corresponding ratio value of lever presses needed to receive one infusion is located on the right axis. Statistical comparisons were conducted on total infusions. C The schematic diagram for administration of retrograde knockdown of NMUR2 in the NAcSh is clarified. D Representative immunohistochemistry images of control hairpin (top) and NMUR2 hairpin (bottom) are illustrated with NMUR2 (blue) and virus GFP (magenta). E–F Daily acquisition of self-administration is illustrated by active lever presses and infusions. G–H Knockdown of NMUR2 increased test day active lever presses and breakpoints for cocaine (n = 10 per group; one individual shNMUR2 rat at 4585 lever presses not shown). Statistical comparisons were conducted on total infusions. Bar graph shows mean ± SEM. *p < 0.05.

Fig. 2. NMU prevented cocaine-induced increase in GABA and preferentially increased phosphorylated c-Fos on NAcSh→VP projection neurons.

A Cocaine and NMU alter GABA concentrations in the NAcSh measured by microdialysis and mass spectrometry. The arrow indicates time of cocaine (10 mg/kg, IP), NMU (0.3 mg/kg, IP) or vehicle administration. ^#p < 0.05 compared to basal and *p < 0.05 between groups. B This illustration depicts microinjection sites of retrograde viral vector tracers into target brain regions (VTA or VP) and presents confocal images at 20X objective in the NAcSh. C Representative NAcSh confocal images of vehicle (left) and NMU-treated (right) illustrate NAcSh → VP cell bodies labeled with GFP (green), NAcSh → VTA cell bodies with mCherry (red), and phosphorylated c-Fos (blue). D NMU increased the average quantity of phosphorylated c-Fos positive cell bodies per rat in the NAcSh (n = 6–7 per group). E NMU preferentially increased phosphorylated c-Fos colocalization in the NAcSh → VP (GFP) pathway but not in the NAcSh → VTA (mCherry) pathway quantified from images of tissue from control or NMU-treated rats (n = 3–4 per group). Bar graph shows mean ± SEM. *p < 0.05.

Fig. 3. The schematic diagram of transsynaptic study to label all neurons that project onto the NAcSh → VP pathway is presented.

A Retrograde AAV6 was administered to the VP and infected neurons to express TVA, rabies glycoprotein, and mCherry. B Neurons in the NAcSh that express TVA were infected with EnvA pseudotyped rabies virus expressing GFP. Only neurons with TVA can be infected by this pseudotyped rabies. C Rabies replicated in neurons with glycoprotein and transsynaptically “jumps” one synapse in the retrograde direction. D Neurons that express GFP (pseudocolored to magenta) directly synapsed onto NAcSh neurons that project to the VP.

Fig. 4. GABAergic and serotonergic DRN → NAcSh neurons differentially innervated NAcSh → VTA and NAcSh → VP pathways.

A schematic diagram illustrates the viral placement with representative data in the DRN showing rabies (magenta) infected neurons that project onto the (A) NAcSh → VTA pathway or (B) NAcSh → VP pathway, colocalizing with GAD67 (yellow) or TPH (cyan). White arrows indicate colocalization events. C Total number of rabies positive neurons in the DRN from each rat in a survey of the DRN (15 coronal slices per rat and n = 3 per group). D Percentage of the rabies-positive neurons colocalizing with GAD67 or TPH. E The illustration summarizes the proposed pathway.