Probes for CRE

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pro-nociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here we demonstrate the different contributions of genetically-defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Significance Statement The PAG is a midbrain region critical for the modulation of pain. However, the roles played by the distinct cell types within the PAG in nociceptive processing are poorly understood. This work addresses the divergent roles of glutamatergic and GABAergic PAG neuronal subpopulations in nociceptive processing. We demonstrate that activation of glutamatergic neurons or inhibition of GABAergic neurons suppresses nociception. Whereas inhibition of glutamatergic neuronal activity or activation of GABAergic neuronal activity potentiates nociception. This report identifies distinct roles for these neuronal populations in modulating nociceptive processing.

The circadian transcription factor neuronal PAS domain 2 (NPAS2) is linked to psychiatric disorders associated with altered reward sensitivity. The expression of Npas2 is preferentially enriched in the mammalian forebrain, including the nucleus accumbens (NAc), a major neural substrate of motivated and reward behavior. Previously, we demonstrated that down-regulation of NPAS2 in the NAc reduces the conditioned behavioral response to cocaine in mice. We also showed that Npas2 is preferentially enriched in dopamine receptor 1 containing medium spiny neurons (D1R-MSNs) of the striatum. To extend these studies, we investigated the impact of NPAS2 disruption on accumbal excitatory synaptic transmission and strength, along with the behavioral sensitivity to cocaine reward in a cell-type specific manner. Viral-mediated knockdown of Npas2 in the NAc of male and female C57BL/6J mice increased the excitatory drive onto MSNs. Using Drd1a-tdTomato mice in combination with viral knockdown, we determined these synaptic adaptations were specific to D1R-MSNs relative to non-D1R-MSNs. Interestingly, NAc-specific knockdown of Npas2 blocked cocaine-induced enhancement of synaptic strength and glutamatergic transmission specifically onto D1R-MSNs. Lastly, we designed, validated, and employed a novel Cre-inducible short-hairpin RNA virus for MSN-subtype specific knockdown of Npas2 Cell-type specific Npas2 knockdown in D1R-MSNs, but not D2R-MSNs, in the NAc reduced cocaine conditioned place preference. Together, our results demonstrate that NPAS2 regulates excitatory synapses of D1R-MSNs in the NAc and cocaine reward-related behavior.SIGNIFICANCE STATEMENTDrug addiction is a widespread public health concern often comorbid with other psychiatric disorders. Disruptions of the circadian clock can predispose or exacerbate substance abuse in vulnerable individuals. We demonstrate a role for the core circadian protein, NPAS2, in mediating glutamatergic neurotransmission at medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a region critical for reward processing. We find that NPAS2 negatively regulates functional excitatory synaptic plasticity in the NAc and is necessary for cocaine-induced plastic changes in MSNs expressing the dopamine 1 receptor (D1R). We further demonstrate disruption of NPAS2 in D1R-MSNs produces augmented cocaine preference. These findings highlight the significance of cell-type specificity in mechanisms underlying reward regulation by NPAS2 and extend our knowledge of its function.

Abstract

KEY POINTS:

Alpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide, and injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, but did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is likely activity dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine dependent behaviors.

ABSTRACT:

The mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviors including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward, and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA), and our lab previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. The cellular mechanisms underlying the effects of intra-VTA α-MSH on feeding and food reward are unknown, however. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing EYFP in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is likely activity dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward.