Central Amygdala Circuits Mediate Hyperalgesia in Alcohol-Dependent Rats

Abstract

Alcohol withdrawal symptoms contribute to excessive alcohol drinking and relapse in alcohol-dependent individuals. Among these symptoms, alcohol withdrawal promotes hyperalgesia, but the neurological underpinnings of this phenomenon are not known. Chronic alcohol exposure alters cell signaling in the central nucleus of the amygdala (CeA), and the CeA is implicated in mediating alcohol dependence-related behaviors. The CeA projects to the periaqueductal gray (PAG), a region critical for descending pain modulation, and may have a role in alcohol withdrawal hyperalgesia. Here, we tested the roles of (1) CeA projections to PAG, (2) CeA melanocortin signaling, and (3) PAG μ-opioid receptor signaling in mediating thermal nociception and alcohol withdrawal hyperalgesia in male Wistar rats. Our results demonstrate that alcohol dependence reduces GABAergic signaling from CeA terminals onto PAG neurons and alters the CeA melanocortin system, that CeA-PAG projections and CeA melanocortin signaling mediate alcohol withdrawal hyperalgesia, and that μ-opioid receptors in PAG filter CeA effects on thermal nociception.SIGNIFICANCE STATEMENT Hyperalgesia is commonly seen in individuals with alcohol use disorder during periods of withdrawal, but the neurological underpinnings behind this phenomenon are not completely understood. Here, we tested whether alcohol dependence exerts its influence on pain modulation via effects on the limbic system. Using behavioral, optogenetic, electrophysiological, and molecular biological approaches, we demonstrate that central nucleus of the amygdala (CeA) projections to periaqueductal gray mediate thermal hyperalgesia in alcohol-dependent and alcohol-naive rats. Using pharmacological approaches, we show that melanocortin receptor-4 signaling in CeA alters alcohol withdrawal hyperalgesia, but this effect is not mediated directly at synaptic inputs onto periaqueductal gray-projecting CeA neurons. Overall, our findings support a role for limbic influence over the descending pain pathway and identify a potential therapeutic target for treating hyperalgesia in individuals with alcohol use disorder .

Keywords: descending inhibition; electrophysiology; mu opioid receptors; optogenetics; pain.

Figure 1.

CeA-PAG projections mediate thermal nociception in rats. A, Virus expression. Left, Representative 20× image of hSyn-mCherry⁺ neurons in CeA, and map of virus placements. Scale bar, 100 μm. Right, representative 20× image of hSyn-mCherry⁺ terminals in PAG, and map of cannulae placements. Scale bar, 50 μm. BLA, Basolateral amygdala; Aq, aqueduct. B, Thermal hyperalgesia is induced by yellow light inactivation of CeA neurons (inset) transfected with eNpHR3.0 (n = 7 or 8/group). *p < 0.05 versus inactive virus. ^#p < 0.05 versus yellow light off. C, Thermal hyperalgesia is induced by yellow light inactivation of CeA terminals in PAG (inset) transfected with eNpHR3.0 (n = 7 or 8/group). *p < 0.05 versus inactive virus. ^#p < 0.05 versus yellow light off. D, Alcohol dependence-induced thermal hyperalgesia was reversed by blue light activation of CeA terminals in PAG (inset) transfected with ChR2 (n = 7–11/group). *p < 0.05 versus inactive virus. ^#p < 0.05 versus blue light off. B–D, Data are mean ± SEM. Hindpaw withdrawal latencies (s) in Hargreaves Test in response to light stimulation. Yellow bars represent yellow light trials. Blue bars represent blue light trials.

Figure 2.

CeA-PAG circuit activity alterations following alcohol dependence. A, Retrograde labeling of PAG neurons. Left, 4× image of retrobeads (red) injected in PAG, with map of retrobead injections in naive (black) and alcohol-dependent (red) rats. Aq, Aqueduct. Scale bar, 500 μm. Right, 40× image of retrobead-containing (PAG-projecting) neurons in CeA. Nuclei are stained with DAPI and appear blue. White arrow indicates position of a representative retrobead⁺ neuron. Scale bar, 50 μm. B, Map indicating approximate recording positions of PAG-projecting CeA neurons from naive (black) and alcohol-dependent (red) rats; recordings were performed in the medial subdivision of the CeA. C, Average resting membrane potential of PAG-projecting CeA neurons does not significantly differ between naive and dependent rats. D, Average rheobase values do not significantly differ between populations. E, Representative sIPSC traces recorded in PAG-projecting CeA neurons of naive (black) and alcohol-dependent (red) rats. There were no significant differences in baseline sIPSC frequency (F) but a tendency toward an increase in sIPSC amplitude (p = 0.067) (G) of PAG-projecting CeA neurons from naive or alcohol-dependent rats. H, Representative sEPSC traces recorded in PAG-projecting CeA neurons of naive (black) and alcohol-dependent (red) rats. There were no significant differences in baseline sEPSC frequency (I) or amplitude (J) of PAG-projecting CeA neurons from naive or alcohol-dependent rats. C, D, F, G, I, J, Data are mean ± SEM.

Figure 3.

CeA inputs to vlPAG are weaker in alcohol-dependent rats. A, Top, Map indicating approximate positions of PAG neurons recorded from naive (black) and alcohol-dependent (red) rats. ChR2-containing CeA terminals were stimulated in PAG-containing brain slices (inset). Closed circles represent neurons in which an optically evoked postsynaptic current was observed. Open circles represent neurons in which optical stimulation evoked no events. Bottom, Sample recordings of optically evoked postsynaptic current of PAG neuron from naive (black) and alcohol-dependent (red) rats. B, No significant differences exist in the proportion of neurons in which evoked events were observed between groups. C, Average amplitude of evoked postsynaptic current was significantly lower in PAG neurons from alcohol-dependent rats compared with naive controls. *p < 0.05. D, Representative trace showing optically evoked current of PAG neuron in the absence (black) and presence (orange) of PTX. E, Superfusion of PTX significantly decreased the relative amplitude of evoked postsynaptic currents of PAG neurons. C, E, Data are mean ± SEM.

Figure 4.

Alcohol dependence reduces MC4R expression in CeA. A, 10× image of αMSH expression in CeA of naive (left) and alcohol-dependent (right) rat. Approximate positions of CeA and BLA indicated. Scale bar, 200 μm. CeM, Medial subdivision of CeA; CeL, lateral subdivision of CeA. B, Average expression of αMSH in CeA, BNST, and PVN of alcohol-dependent (red) and naive (white) rats. C, CeA MC4R expression is significantly decreased in CeA of alcohol-dependent rats (red) compared with naive controls (white). *p < 0.05. Inset, Representative image of MC4R and tubulin levels from naive (N) and alcohol-dependent (D) rats. B, C, Data are mean ± SEM.

Figure 5.

MC4R in CeA and MOR in vlPAG modulate thermal nociception. A, Hindpaw withdrawal latencies of naive (white) and alcohol-dependent (red) rats following intra-CeA infusion of HS014. *p < 0.05 versus naive. ^#p < 0.05, effect of HS014. B, Hindpaw withdrawal latency of naive rats is significantly reduced following intra-CeA infusion of αMSH. *p < 0.05 versus control. C, Hindpaw withdrawal latencies of naive rats following intra-PAG infusion of DAMGO and intra-CeA infusion of 0.3 μg αMSH. Intra-PAG administration of DAMGO before αMSH prevents thermal hyperalgesia. *p < 0.05 versus 0 μg DAMGO. A–C, Top, Cannulae placement. D, IPSPs are evoked in putative PAG pyramidal neurons by nearby electrical stimulation (left). Superthreshold current injection evoked action potentials (APs) under baseline conditions (middle), but APs are blocked by concurrent stimulation of local inhibition (right). Bath application of DAMGO reduces IPSPs in the same neuron shown in top. In the presence of DAMGO, stimulation of local inhibition fails to block AP generation while depolarizing neurons (middle, right). E, DAMGO reduces IPSP amplitude in 4 of 6 neurons recorded. F, DAMGO reduces integrated IPSP area in 6 of 6 neurons. *p < 0.05. G, Under control conditions, inhibition blocks AP generation. *p < 0.05. H, In the presence of DAMGO, inhibition fails to significantly block AP generation; 1 of 6 neurons was still blocked by inhibition. A–C, Data are mean ± SEM.

Figure 6.

Adaptations in CeA-PAG circuitry contribute to hyperalgesia following alcohol dependence. Left, Schematic depicting CeA-PAG circuitry under baseline conditions. CeA GABAergic neurons project to PAG, ultimately resulting in disinhibition of MOR-containing PAG neurons, initiating the descending pain inhibition pathway. Right, Following repeated cycles of alcohol exposure and withdrawal, MC4R expression is decreased in the CeA. There is a decrease in the strength of CeA projections to PAG, resulting in increased inhibition of PAG output neurons to RVM and/or forebrain regions (e.g., TH⁺ projections to BNST) and/or midbrain regions (e.g., VTA/SN). Ultimately, these adaptations contribute to dependence-induced hyperalgesia. Illustration generated by modifying images purchased in the Illustration Toolkit-Neuroscience from Motifolio.