Probes for INS

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.

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG) /dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

Significance Statement Clinicians have long had the goal of separating analgesia from anxiety when using deep brain electrical stimulation of the periaqueductal gray (PAG) for difficult to treat pain. Here we show that selective activation of dopamine neurons within the PAG produces analgesia without other behavioral effects, while stimulating glutamate neurons mediates stress-induced anxiety and analgesia. Our results suggest that dopamine agonists may represent a novel class of analgesic drugs and elucidate target neurons that could mediate their effect.

The ventral pallidum (VP) is a critical component of the basal ganglia neurocircuitry regulating learning and decision making; however, its precise role in controlling associative learning of environmental stimuli conditioned to appetitive or aversive outcomes is still unclear. Here, we investigated the expression of preproenkephalin, a polypeptide hormone previously shown to be expressed in nucleus accumbens neurons controlling aversive learning, within GABAergic and glutamatergic VP neurons. Next, we explored the behavioral consequences of chemicogenetic inhibition or excitation of preproenkephalin-expressing VP neurons on associative learning of reward- or aversion-paired stimuli in autoshaping and inhibitory avoidance tasks, respectively. We reveal for the first time that preproenkephalin is expressed predominantly in GABAergic rather than glutamatergic VP neurons, and that excitation of these preproenkephalin-expressing VP neurons was sufficient to impair inhibitory avoidance learning. These findings indicate the necessity for inhibition of preproenkephalin-expressing VP neurons for avoidance learning, and suggest these neurons as a potential therapeutic target for psychiatric disorders associated with maladaptive aversive learning.