Background.-Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAG DA cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown. Methods.-The present study combines Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal. Results.-DREADD-induced activation of locus coeruleus (LC) projections to the vPAG or vPAG DA neurons promoted arousal. Similarly, agonist stimulation of vPAG α1-adrenergic receptors (α1ARs) increased latency to fall asleep, while α1AR blockade had the opposite effect. α1AR stimulation drove vPAG DA activity in a glutamate-dependent, action potential-independent

Psychostimulants and opioids increase dopamine (DA) neurotransmission, activating D1 and D2 G protein-coupled receptors. β-arrestin2 (βarr2) desensitizes and internalizes these receptors and initiates G protein-independent signaling. Previous work revealed that mice with a global or cell-specific knockout of βarr2 have altered responses to certain drugs; however, the effects of βarr2 on the excitability of medium spiny neurons (MSNs), and its role in mediating the rewarding effects of drugs of abuse are unknown. D1-Cre and D2-Cre transgenic mice were crossed with floxed βarr2 mice to eliminate βarr2 specifically in cells containing either D1 (D1 βarr2-KO) or D2 (D2 βarr2-KO) receptors. We used slice electrophysiology to characterize the role of βarr2 in modulating D1 and D2 nucleus accumbens MSN intrinsic excitability in response to DA and tested the locomotor-activating and rewarding effects of cocaine and morphine in these mice. Eliminating βarr2 attenuated the ability of DA to inhibit D2-MSNs and altered the DA-induced maximum firing rate in D1-MSNs. While D1 βarr2-KO mice had mostly normal drug responses, D2 βarr2-KO mice showed dose-dependent reductions in acute locomotor responses to cocaine and morphine, attenuated locomotor sensitization to cocaine, and blunted cocaine reward measured with conditioned place preference. Both D2 βarr2-KO and D1 βarr2-KO mice displayed an enhanced conditioned place preference for the highest dose of morphine. These results indicate that D1-and D2-derived βarr2 functionally contribute to DA-induced changes in MSN intrinsic excitability and behavioral responses to psychostimulants and opioids dose-dependently.

The neuropeptide galanin is reported to attenuate opioid withdrawal symptoms, potentially by reducing neuronal hyperactivity in the noradrenergic locus coeruleus (LC) via galanin receptor 1 (GalR1). We evaluated this mechanism by using RNAscope in situ hybridization to characterize GalR1 mRNA distribution in the dorsal pons and to compare galanin and GalR1 mRNA expression in tyrosine hydroxylase‐positive (TH+) LC cells at baseline and following chronic morphine or precipitated withdrawal. We then used genetically altered mouse lines and pharmacology to test whether noradrenergic galanin (NE‐Gal) modulates withdrawal symptoms. RNAscope revealed that, while GalR1 signal was evident in the dorsal pons, 80.7% of the signal was attributable to TH− neurons outside the LC. Galanin and TH mRNA were abundant in LC cells at baseline and were further increased by withdrawal, whereas low basal GalR1 mRNA expression was unaltered by chronic morphine or withdrawal. Naloxone‐precipitated withdrawal symptoms in mice lacking NE‐Gal (GalcKO‐Dbh) were largely similar to WT littermates, indicating that loss of NE‐Gal does not exacerbate withdrawal. Complementary experiments using NE‐Gal overexpressor mice (NE‐Gal OX) and systemic administration of the galanin receptor agonist galnon revealed that increasing galanin signaling also failed to alter behavioral withdrawal, while suppressing noradrenergic transmission with the alpha‐2 adrenergic receptor agonist clonidine attenuated multiple symptoms. These results indicate that galanin does not acutely attenuate precipitated opioid withdrawal via an LC‐specific mechanism, which has important implications for the general role of galanin in regulation of somatic and affective opioid responses and LC activity.

Psychostimulants and opioids increase dopamine (DA) neurotransmission, activating D1and D2 G protein-coupled receptors. β-arrestin2 (βarr2) desensitizes and internalizes these receptors and initiates G protein-independent signaling. Previous work revealed that mice with a global or cell-specific knockout of βarr2 have altered responses to certain drugs; however, the effects of βarr2 on the excitability of medium spiny neurons (MSNs) and its role in mediating the rewarding effects of drugs of abuse are unknown. D1-Cre and D2-Cre transgenic mice were crossed with floxed βarr2 mice to eliminate βarr2 specifically in cells containing either D1 (D1 barr2-KO ) or D2 (D2 barr2-KO ) receptors. We used slice electrophysiology to characterize the role of βarr2 in modulating D1 and D2 nucleus accumbens MSN intrinsic excitability in response to DA and tested the locomotor-activating and rewarding effects of cocaine and morphine in these mice. We found that eliminating barr2 attenuated the ability of DA to inhibit D2-MSNs but had little effect on the DA response of D1-MSNs. While D1 barr2-KO mice had mostly normal drug responses, D2 barr2-KO mice showed dose-dependent reductions in acute locomotor responses to cocaine and morphine, attenuated locomotor sensitization to cocaine, and blunted cocaine reward measured with conditioned place preference. Both D2 barr2-KO and D1 barr2-KO mice displayed an enhanced conditioned place preference for the highest dose of morphine. These results indicate that D2-derived barr2 functionally contributes to the ability of DA to inhibit D2-MSNs and multiple behavioral responses to psychostimulants and opioids, while loss of barr2 in D1 neurons has little impact on D1-MSN excitability or drug-induced behaviors.

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