Highlights d Rostromedial tegmental (RMTg) neurons are activated by punishment-related stimuli d Distinct RMTg afferents communicate distinct punishmentrelated signals d These RMTg afferents drive correspondingly distinct aspects of punishment learning d Negative valence encoding in the ventral tegmental area depends on the RMTg

Background Psychiatric disorders such as addiction and mania are marked by persistent reward-seeking despite highly negative or aversive outcomes, but the neural mechanisms underlying this aberrant decision-making are unknown. The recently identified rostromedial tegmental nucleus (RMTg) encodes a wide variety of aversive stimuli and sends robust inhibitory projections to midbrain dopamine neurons, leading to the hypothesis that the RMTg provides a brake to reward signaling in response to aversive costs (1, 2). Methods To test the role of the RMTg in punished reward seeking, adult male Sprague Dawley rats were tested in several cost-benefit decision tasks after excitotoxic lesions of the RMTg, or temporally specific optogenetic inhibition of RMTg efferents in the ventral tegmental area (VTA). Results RMTg lesions drastically impaired the ability of footshock to suppress operant responding for food. Optogenetic inhibition showed that this resistance to punishment was due in part to RMTg activity at the precise moment of shock delivery and mediated by projections to the VTA, consistent with an aversive “teaching signal” role for the RMTg during encoding of the aversive event. We observed a similar resistance to punishment when the RMTg was selectively inhibited immediately prior to the operant lever press, consistent with a second distinct role for the RMTg during action selection. These effects were not attributable to RMTg effects on learning rate, locomotion, shock sensitivity, or perseveration. Conclusions The RMTg has two strong and dissociable roles during both encoding and recall of aversive consequences of behavior.

The rostromedial tegmental nucleus (RMTg), also known as the tail of the ventral tegmental area (tVTA), is a GABAergic structure identified in 2009 that receives strong inputs from the lateral habenula and other sources, sends dense inhibitory projections to midbrain dopamine (DA) neurons, and plays increasingly recognized roles in aversive learning, addiction, and other motivated behaviors. In general, little is known about the genetic identity of these neurons. However, recent work identified the transcription factor FoxP1 as enhanced in the mouse RMTg (Lahti et al. 2016). Hence, in the current study, we used RNA sequencing to identify genes significantly enhanced in the rat RMTg as compared to adjacent VTA, and then examined the detailed distribution of two genes in particular, prepronociceptin (Pnoc) and FoxP1. In rats and mice, both Pnoc and FoxP1 were expressed at high levels in the RMTg and colocalized strongly with previously established RMTg markers. FoxP1 was particularly selective for RMTg neurons, as it was absent in most adjacent brain regions. We used these gene expression patterns to refine the anatomic characterization of RMTg in rats, extend this characterization to mice, and show that optogenetic manipulation of RMTg in mice bidirectionally modulates real-time place preference. Hence, RMTg neurons in both rats and mice exhibit distinct genetic profiles that correlate with their distinct connectivity and function.

Angiotensin II (Ang II) acts at central type 1 (AT 1 ) receptors to increase intake of water and saline. In vitro studies demonstrated rapid desensitization of the AT 1 receptor after Ang II exposure, and behavioural studies in rats suggest that exposure to Ang II decreases the dipsogenic potency of subsequent Ang II. Nevertheless, the effect of repeated Ang II injections on saline intake remains untested, and a reliable protocol for examining this purported behavioural desensitization has not emerged from the literature. To address these issues, we established a reliable approach to study Ang II-induced dipsetic desensitization and used this approach to test the requirement of central AT 1 receptors and the specificity of the effect for water intake. Rats given a treatment regimen of three injections of Ang II (300 ng, intracerebroventricular), each separated by 20 min, drank less water than control rats after a subsequent test injection of Ang II. The effect was relatively short lasting, dependent on the dose and timing of Ang II, and was almost completely blocked by the AT 1 receptor antagonist losartan. In further testing, when rats were given access to both water and 1.5% saline, animals that received an Ang II treatment regimen drank less water than control animals, but saline intake was unaffected. These data support previous suggestions that Ang II-induced water and saline intakes are separable. Given the role of G protein uncoupling in desensitization of the AT 1 receptor, these data are consistent with the emerging hypothesis that AT 1 receptor G protein-dependent intracellular signalling pathways are more relevant for water, but not saline, intake.

Angiotensin II (AngII) plays a key role in maintaining body fluid homeostasis. The physiological and behavioral effects of central AngII include increased blood pressure and fluid intake. In vitro experiments demonstrate that repeated exposure to AngII reduces the efficacy of subsequent AngII, and behavioral studies indicate that prior icv AngII administration reduces the dipsogenic response to AngII administered later. Specifically, rats given a treatment regimen of three icv injections of a large dose of AngII, each separated by 20 min, drink less water in response to a test injection of AngII than do vehicle-treated controls given the same test injection. The present studies were designed to test three potential explanations for the reduced dipsogenic potency of AngII after repeated administration. To this end, we tested for motor impairment caused by repeated injections of AngII, for a possible role of visceral distress or illness, and for differences in the pressor response to the final test injection of AngII. We found that repeated injections of AngII neither affected drinking stimulated by carbachol nor did they produce a conditioned flavor avoidance. Furthermore, we found no evidence that differences in the pressor response to the final test injection of AngII accounted for the difference in intake. In light of these findings, we are able to reject these three explanations for the observed behavioral desensitization, and, we suggest instead that the mechanism for this phenomenon may be at the level of the receptor.

A single central injection of angiotensin II (AngII) potently increases water intake; however, a growing body of research suggests that repeated, acute intracerebroventricular injections of AngII cause a reduction in the dipsogenic response to subsequent AngII. This AngII-induced behavioral desensitization is specific to the effects of angiotensin and mediated by the angiotensin type-1 (AT1) receptor. The neuroanatomical substrate for this phenomenon, however, remains unknown. The anteroventral third ventricle region (AV3V) is an important site for the behavioral and physiological actions of AngII. Therefore, we hypothesized that this region also mediates the effects of repeated central AngII administration. In support of this hypothesis, we found that repeated injections of AngII into the AV3V reduced water intake stimulated by a test injection of AngII given into this region. Moreover, repeated AngII injections in the AV3V reduced water intake after AngII was injected into the lateral ventricle. These studies also demonstrate that activation of the AT1 receptor within the AV3V is required for AngII-induced behavioral desensitization because direct injection of the AT1 receptor antagonist, losartan, into the AV3V blocked the desensitizing effect of repeated AngII injections into the lateral ventricle. These findings provide additional support for a role of the AV3V in the dipsogenic actions of AngII, and suggest that this region is critical for the desensitization that occurs after acute repeated central injections of AngII.

Angiotensin II (AngII) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to AngII in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioral correlate of this desensitization has been shown in rats after repeated central injections of AngII. Specifically, rats given repeated injections of AngII drink less water than controls after a subsequent test injection of AngII. Under the same conditions, however, repeated injections of AngII have no effect on AngII-induced saline intake. Given earlier studies indicating that separate intracellular signaling pathways mediate AngII-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in AngII-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an AngII test injection in rats given prior treatment with repeated injections of vehicle, AngII, or Sar1,Ile4,Ile8-AngII (SII), an AngII analog that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated AngII injections on water intake. Furthermore, AngII-induced water intake was reduced similarly by repeated injections of AngII or SII. The results suggest that G protein-independent signaling is sufficient to produce behavioral desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

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