Chemogenetic inhibition of locus coeruleus to rostromedial tegmental nucleus noradrenergic pathway increases light cycle ethanol drinking in male and female mice and blunts ethanol-induced CTA.

We recently showed that chemogenetic activation of the locus coeruleus (LC) to the rostromedial tegmental nucleus (RMTg) noradrenergic (NE) pathway significantly blunted binge-like ethanol drinking and induced aversive-like behaviors in mice. The aim of the present study is to determine if silencing this TH + LC → RMTg noradrenergic pathway promotes increased levels of binge-like ethanol intake and reduced ethanol-induced conditioned taste aversion (CTA). To this end, both male and female TH-ires-cre mice on a C57BL/6 J background were cannulated in the RMTg and injected in the LC with rAVV viruses that encode cre-dependent Gi-expressing designer receptor exclusively activated by designer drugs (DREADDs), or its control, to directly control the activity of NE neurons. Inhibition of the LC to RMTg pathway had no effect on the binge-ethanol drinking in a "drinking-in-the-dark" (DID) paradigm. However, when using this paradigm during the light cycle, silencing of this circuit significantly increased ethanol intake without altering sucrose drinking. Moreover, we found that inhibition of this circuit significantly attenuated an ethanol-induced CTA. In addition, when compared to control animals, pairing RMTg-directed Clozapine N-oxide (CNO) with an i.p. injection of 1.5 g/kg ethanol reduced c-Fos activation in the LC, and increased c-Fos expression in the ventral tegmental area (VTA) in Gi-expressing mice. Our data show that inhibition of the TH + LC to the RMTg pathway significantly increased ethanol drinking as well as attenuated ethanol-induced CTA, supporting the involvement of the LC to RMTg noradrenergic circuit as an important protective mechanism against excessive ethanol consumption.

Activation of locus coeruleus to rostromedial tegmental nucleus (RMTg) noradrenergic pathway blunts binge-like ethanol drinking and induces aversive responses in mice.

There is strong evidence that ethanol entails aversive effects that can act as a deterrent to overconsumption. We have found that in doses that support the development of a conditioned taste aversion ethanol increases the activity of tyrosine hydroxylase (TH) positive neurons in the locus coeruleus (LC), a primary source of norepinephrine (NE). Using cre-inducible AAV8-ChR2 viruses in TH-ires-cre mice we found that the LC provides NE projections that innervate the rostromedial tegmental nucleus (RMTg), a brain region that has been implicated in the aversive properties of drugs. Because the neurocircuitry underlying the aversive effects of ethanol is poorly understood, we characterized the role of the LC to RMTg circuit in modulating aversive unconditioned responses and binge-like ethanol intake. Here, both male and female TH-ires-cre mice were cannulated in the RMTg and injected in the LC with rAVV viruses that encode for a Gq-expressing designer receptor exclusively activated by designer drugs (DREADDs) virus, or its control virus, to directly control the activity of NE neurons. A Latin Square paradigm was used to analyze both 20% ethanol and 3% sucrose consumption using the "drinking-in-the-dark" (DID) paradigm. Chemogenetic activation of the LC to RMTg pathway significantly blunted the binge-ethanol drinking, with no effect on the sucrose consumption, increased the emission of mid-frequency vocalizations and induced malaise-like behaviors in mice. The present findings indicate an important involvement of the LC to RMTg pathway in reducing ethanol consumption, and characterize unconditioned aversive reactions induced by activation of this noradrenergic pathway.

Distinct and Overlapping Patterns of Acute Ethanol-Induced C-Fos Activation in Two Inbred Replicate Lines of Mice Selected for Drinking to High Blood Ethanol Concentrations.

The inbred high drinking in the dark (iHDID1 and iHDID2) strains are two replicate lines bred from the parent HS/Npt (HS) line for achieving binge levels of blood ethanol concentration (≥80 mg/dL BEC) in a four-hour period. In this work, we sought to evaluate differences in baseline and ethanol-induced c-Fos activation between the HS, iHDID1, and iHDID2 genetic lines in brain regions known to process the aversive properties of ethanol. METHODS: Male and female HS, iHDID1, and iHDID2 mice underwent an IP saline 2 3 g/kg ethanol injection. Brain sections were then stained for c-Fos expression in the basolateral/central amygdala (BLA/CeA), bed nucleus of the stria terminals (BNST), A2, locus coeruleus (LC), parabrachial nucleus (PBN), lateral/medial habenula (LHb/MHb), paraventricular nucleus of the thalamus (PVT), periaqueductal gray (PAG), Edinger-Westphal nuclei (EW), and rostromedial tegmental nucleus (RMTg). RESULTS: The iHDID1 and iHDID2 lines showed similar and distinct patterns of regional c-Fos; however, in no region did the two both significantly differ from the HS line together. CONCLUSIONS: These data lend further support to altered baseline or ethanol-induced activation in brain regions associated with processing the aversive properties of ethanol in the iHDID1 and iHDID2 genetic lines.

Allopregnanolone Decreases Evoked Dopamine Release Differently in Rats by Sex and Estrous Stage.

Mesolimbic dopamine transmission is dysregulated in multiple psychiatric disorders, including addiction. Previous studies found that the endogenous GABAergic steroid (3α,5α)-3-hydroxy-5-pregnan-20-one (allopregnanolone) modulates dopamine levels in the nucleus accumbens and prefrontal cortex. As allopregnanolone is a potent positive allosteric modulator of GABAA receptors, and GABAA receptors can regulate dopamine release, we hypothesized that allopregnanolone would reduce phasic fluctuations in mesolimbic dopamine release that are important in learning and reward processing. We used fast-scan cyclic voltammetry in anesthetized female and male rats to measure dopamine release in the nucleus accumbens evoked by electrical stimulation of the ventral tegmental area, before and after administration of allopregnanolone. Allopregnanolone (7.5-25 mg/kg, IP) reduced evoked dopamine release in both male and female rats, compared to ß-cyclodextrin vehicle. In males, all doses of allopregnanolone decreased dopamine transmission, with stronger effects at 15 and 25 mg/kg allopregnanolone. In females, 15 and 25 mg/kg allopregnanolone reduced dopamine release, while 7.5 mg/kg allopregnanolone was no different from vehicle. Since allopregnanolone is derived from progesterone, we hypothesized that high endogenous progesterone levels would result in lower sensitivity to allopregnanolone. Consistent with this, females in proestrus (high progesterone levels) were less responsive to allopregnanolone than females in other estrous cycle stages. Furthermore, 30 mg/kg progesterone reduced evoked dopamine release in males, similar to allopregnanolone. Our findings confirm that allopregnanolone reduces evoked dopamine release in both male and female rats. Moreover, sex and the estrous cycle modulated this effect of allopregnanolone. These results extend our knowledge about the pharmacological effects of neurosteroids on dopamine transmission, which may contribute to their therapeutic effects.

Description of a new species of Calliostoma (Gastropoda, Calliostomatidae) from Southeastern Brazil.

Calliostoma tupinamba isa new species from Southeastern Brazil, ranging from southern Rio de Janeiro to northern São Paulo, and found only on coastal islands, on rocks and sessile invertebrates at 3 to 5 meters of depth. Shell and soft part morphology is described here in detail. Calliostoma tupinamba is mainly characterized by a depressed trochoid shell; eight slightly convex whorls; a sharply suprasutural carina starting on the third whorl and forming a peripheral rounded keel; and a whitish, funnel-shaped and deep umbilicus, measuring about 5%-10% of maximum shell width. Calliostoma tupinamba resembles Calliostoma bullisi Clench & Turner, 1960 in shape, but differs from it in being taller and wider, having a smaller umbilicus and lacking a strong and large innermost spiral cord at its base. Finally, an identification key of Brazilian Calliostoma species is presented.