Glucose metabolism links astroglial mitochondria to cannabinoid effects.

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1-5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5-7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB1) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

GAP43 Located on Corticostriatal Terminals Restrains Novelty-Induced Hyperactivity in Mice.

Growth-associated protein of 43 kDa (GAP43) is a key cytoskeleton-associated component of the presynaptic terminal that facilitates neuroplasticity. Downregulation of GAP43 expression has been associated to various psychiatric conditions in humans and evokes hippocampus-dependent memory impairments in mice. Despite the extensive studies conducted on hippocampal GAP43 in past decades, however, very little is known about its roles in modulating the excitatory versus inhibitory balance in other brain regions. We recently generated conditional knock-out mice in which the Gap43 gene was selectively inactivated in either telencephalic glutamatergic neurons (Gap43fl/fl ;Nex1Cre mice, hereafter Glu-GAP43-/- mice) or forebrain GABAergic neurons (Gap43fl/fl ;Dlx5/6Cre mice, hereafter GABA-GAP43-/- mice). Here, we show that Glu-GAP43-/- but not GABA-GAP43-/- mice of either sex show a striking hyperactive phenotype when exposed to a novel environment. This behavioral alteration of Glu-GAP43-/- mice was linked to a selective activation of dorsal-striatum neurons, as well as to an enhanced corticostriatal glutamatergic transmission and an abrogation of corticostriatal endocannabinoid-mediated long-term depression. In line with these observations, GAP43 was abundantly expressed in corticostriatal glutamatergic terminals of wild-type mice. The novelty-induced hyperactive phenotype of Glu-GAP43-/- mice was abrogated by chemogenetically inhibiting corticostriatal afferences with a Gi-coupled "designer receptor exclusively activated by designer drugs" (DREADDs), thus further supporting that novelty-induced activity is controlled by GAP43 at corticostriatal excitatory projections. Taken together, these findings show an unprecedented regulatory role of GAP43 in the corticostriatal circuitry and provide a new mouse model with a delimited neuronal-circuit alteration for studying novelty-induced hyperactivity, a phenotypic shortfall that occurs in diverse psychiatric diseases.

Differential expression of the neuronal CB1 cannabinoid receptor in the hippocampus of male Ts65Dn Down syndrome mouse model.

Down syndrome (DS) or Trisomy 21 is the most common genetic cause of mental retardation with severe learning and memory deficits. DS is due to the complete or partial triplication of human chromosome 21 (HSA21) triggering gene overexpression and protein synthesis alterations responsible for a plethora of mental and physical phenotypes. Among the diverse brain target systems that affect hippocampal-dependent learning and memory deficit impairments in DS, the upregulation of the endocannabinoid system (ECS), and notably the overexpression of the cannabinoid type-1 receptor (CB1), seems to play a major role. Combining various protein and gene expression targeted approaches using western blot, qRT-PCR and FISH techniques, we investigated the expression pattern of ECS components in the hippocampus (HPC) of male Ts65Dn mice. Among all the molecules that constitute the ECS, we found that the expression of the CB1 is altered in the HPC of Ts65Dn mice. CB1 distribution is differentially segregated between the dorsal and ventral part of the HPC and within the different cell populations that compose the HPC. CB1 expression is upregulated in GABAergic neurons of Ts65Dn mice whereas it is downregulated in glutamatergic neurons. These results highlight a complex regulation of the CB1 encoding gene (Cnr1) in Ts65Dn mice that could open new therapeutic solutions for this syndrome.

A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

A restricted population of CB1 cannabinoid receptors with neuroprotective activity.

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.

Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB1 receptor blockade.

Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of ß-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral ß-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors.

Cannabinoids regulate an insula circuit controlling water intake.

The insular cortex, or insula, is a large brain region involved in the detection of thirst and the regulation of water intake. However, our understanding of the topographical, circuit, and molecular mechanisms for controlling water intake within the insula remains parcellated. We found that type-1 cannabinoid (CB1) receptors in the insular cortex cells participate in the regulation of water intake and deconstructed the circuit mechanisms of this control. Topographically, we revealed that the activity of excitatory neurons in both the anterior insula (aIC) and posterior insula (pIC) increases in response to water intake, yet only the specific removal of CB1 receptors in the pIC decreases water intake. Interestingly, we found that CB1 receptors are highly expressed in insula projections to the basolateral amygdala (BLA), while undetectable in the neighboring central part of the amygdala. Thus, we recorded the neurons of the aIC or pIC targeting the BLA (aIC-BLA and pIC-BLA) and found that they decreased their activity upon water drinking. Additionally, chemogenetic activation of pIC-BLA projection neurons decreased water intake. Finally, we uncovered CB1-dependent short-term synaptic plasticity (depolarization-induced suppression of excitation [DSE]) selectively in pIC-BLA, compared with aIC-BLA synapses. Altogether, our results support a model where CB1 receptor signaling promotes water intake by inhibiting the pIC-BLA pathway, thereby contributing to the fine top-down control of thirst responses.

Olfactory bulb astrocytes link social transmission of stress to cognitive adaptation in male mice.

Emotions and behavior can be affected by social chemosignals from conspecifics. For instance, olfactory signals from stressed individuals induce stress-like physiological and synaptic changes in naïve partners. Direct stress also alters cognition, but the impact of socially transmitted stress on memory processes is currently unknown. Here we show that exposure to chemosignals produced by stressed individuals is sufficient to impair memory retrieval in unstressed male mice. This requires astrocyte control of information in the olfactory bulb mediated by mitochondria-associated CB1 receptors (mtCB1). Targeted genetic manipulations, in vivo Ca2+ imaging and behavioral analyses reveal that mtCB1-dependent control of mitochondrial Ca2+ dynamics is necessary to process olfactory information from stressed partners and to define their cognitive consequences. Thus, olfactory bulb astrocytes provide a link between social odors and their behavioral meaning.

Single cell tracing of Pomc neurons reveals recruitment of 'Ghost' subtypes with atypical identity in a mouse model of obesity.

The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are 'invisible' to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice.

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of D-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter D-serine, by its precursor L-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards L-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration.

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited ß-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

Striatopallidal cannabinoid type-1 receptors mediate amphetamine-induced sensitization.

Repeated exposure to psychostimulants, such as amphetamine, causes a long-lasting enhancement in the behavioral responses to the drug, called behavioral sensitization.1 This phenomenon involves several neuronal systems and brain areas, among which the dorsal striatum plays a key role.2 The endocannabinoid system (ECS) has been proposed to participate in this effect, but the neuronal basis of this interaction has not been investigated.3 In the CNS, the ECS exerts its functions mainly acting through the cannabinoid type-1 (CB1) receptor, which is highly expressed at terminals of striatal medium spiny neurons (MSNs) belonging to both the direct and indirect pathways.4 In this study, we show that, although striatal CB1 receptors are not involved in the acute response to amphetamine, the behavioral sensitization and related synaptic changes require the activation of CB1 receptors specifically located at striatopallidal MSNs (indirect pathway). These results highlight a new mechanism of psychostimulant sensitization, a phenomenon that plays a key role in the health-threatening effects of these drugs.

Mitochondrial cannabinoid receptors gate corticosterone impact on novel object recognition.

Corticosteroid-mediated stress responses require the activation of complex brain circuits involving mitochondrial activity, but the underlying cellular and molecular mechanisms are scantly known. The endocannabinoid system is implicated in stress coping, and it can directly regulate brain mitochondrial functions via type 1 cannabinoid (CB1) receptors associated with mitochondrial membranes (mtCB1). In this study, we show that the impairing effect of corticosterone in the novel object recognition (NOR) task in mice requires mtCB1 receptors and the regulation of mitochondrial calcium levels in neurons. Different brain circuits are modulated by this mechanism to mediate the impact of corticosterone during specific phases of the task. Thus, whereas corticosterone recruits mtCB1 receptors in noradrenergic neurons to impair NOR consolidation, mtCB1 receptors in local hippocampal GABAergic interneurons are required to inhibit NOR retrieval. These data reveal unforeseen mechanisms mediating the effects of corticosteroids during different phases of NOR, involving mitochondrial calcium alterations in different brain circuits.

Expression of Functional Cannabinoid Type-1 (CB1) Receptor in Mitochondria of White Adipocytes.

Via activation of the cannabinoid type-1 (CB1) receptor, endogenous and exogenous cannabinoids modulate important biochemical and cellular processes in adipocytes. Several pieces of evidence suggest that alterations of mitochondrial physiology might be a possible mechanism underlying cannabinoids' effects on adipocyte biology. Many reports suggest the presence of CB1 receptor mRNA in both white and brown adipose tissue, but the detailed subcellular localization of CB1 protein in adipose cells has so far been scarcely addressed. In this study, we show the presence of the functional CB1 receptor at different subcellular locations of adipocytes from epididymal white adipose tissue (eWAT) depots. We observed that CB1 is located at different subcellular levels, including the plasma membrane and in close association with mitochondria (mtCB1). Functional analysis in tissue homogenates and isolated mitochondria allowed us to reveal that cannabinoids negatively regulate complex-I-dependent oxygen consumption in eWAT. This effect requires mtCB1 activation and consequent regulation of the intramitochondrial cAMP-PKA pathway. Thus, CB1 receptors are functionally present at the mitochondrial level in eWAT adipocytes, adding another possible mechanism for peripheral regulation of energy metabolism.

Astroglial ER-mitochondria calcium transfer mediates endocannabinoid-dependent synaptic integration.

Intracellular calcium signaling underlies the astroglial control of synaptic transmission and plasticity. Mitochondria-endoplasmic reticulum contacts (MERCs) are key determinants of calcium dynamics, but their functional impact on astroglial regulation of brain information processing is unexplored. We found that the activation of astrocyte mitochondrial-associated type-1 cannabinoid (mtCB1) receptors determines MERC-dependent intracellular calcium signaling and synaptic integration. The stimulation of mtCB1 receptors promotes calcium transfer from the endoplasmic reticulum to mitochondria through a specific molecular cascade, involving the mitochondrial calcium uniporter (MCU). Physiologically, mtCB1-dependent mitochondrial calcium uptake determines the dynamics of cytosolic calcium events in astrocytes upon endocannabinoid mobilization. Accordingly, electrophysiological recordings in hippocampal slices showed that conditional genetic exclusion of mtCB1 receptors or dominant-negative MCU expression in astrocytes blocks lateral synaptic potentiation, through which astrocytes integrate the activity of distant synapses. Altogether, these data reveal an endocannabinoid link between astroglial MERCs and the regulation of brain network functions.

Functional heterogeneity of POMC neurons relies on mTORC1 signaling.

Hypothalamic pro-opiomelanocortin (POMC) neurons are known to trigger satiety. However, these neuronal cells encompass heterogeneous subpopulations that release γ-aminobutyric acid (GABA), glutamate, or both neurotransmitters, whose functions are poorly defined. Using conditional mutagenesis and chemogenetics, we show that blockade of the energy sensor mechanistic target of rapamycin complex 1 (mTORC1) in POMC neurons causes hyperphagia by mimicking a cellular negative energy state. This is associated with decreased POMC-derived anorexigenic α-melanocyte-stimulating hormone and recruitment of POMC/GABAergic neurotransmission, which is restrained by cannabinoid type 1 receptor signaling. Electrophysiology and optogenetic studies further reveal that pharmacological blockade of mTORC1 simultaneously activates POMC/GABAergic neurons and inhibits POMC/glutamatergic ones, implying that the functional specificity of these subpopulations relies on mTORC1 activity. Finally, POMC neurons with different neurotransmitter profiles possess specific molecular signatures and spatial distribution. Altogether, these findings suggest that mTORC1 orchestrates the activity of distinct POMC neurons subpopulations to regulate feeding behavior.

Hypothalamic bile acid-TGR5 signaling protects from obesity.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

Subcellular specificity of cannabinoid effects in striatonigral circuits.

Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.

Synaptic Functions of Type-1 Cannabinoid Receptors in Inhibitory Circuits of the Anterior Piriform Cortex.

In the olfactory system, the endocannabinoid system (ECS) regulates sensory perception and memory. A major structure involved in these processes is the anterior piriform cortex (aPC), but the impact of ECS signaling in aPC circuitry is still scantly characterized. Using ex vivo patch clamp experiments in mice and neuroanatomical approaches, we show that the two major forms of ECS-dependent synaptic plasticity, namely depolarization-dependent suppression of inhibition (DSI) and long-term depression of inhibitory transmission (iLTD) are present in the aPC. Interestingly, iLTD expression depends on layer localization of the inhibitory neurons associated with the expression of the neuropeptide cholecystokinin. Conversely, the decrease of inhibitory transmission induced by exogenous cannabinoid agonists or DSI do not seem to be impacted by these factors. Altogether, these results indicate that CB1 receptors exert an anatomically specific and differential control of inhibitory plasticity in the aPC, likely involved in spatiotemporal regulation of olfactory processes.

A Novel Cortical Mechanism for Top-Down Control of Water Intake.

Water intake is crucial for maintaining body fluid homeostasis and animals' survival [1-4]. In the brain, complex processes trigger thirst and drinking behavior [1-5]. The anterior wall of the third ventricle formed by the subfornical organ (SFO), the median preoptic nucleus, and the organum vasculosum of the lamina terminalis (OVLT) constitute the primary structures sensing thirst signals and modulating water intake [6-10]. These subcortical regions are connected with the neocortex [11]. In particular, insular and anterior cingulate cortices (IC and ACC, respectively) have been shown to receive indirect innervations from the SFO and OVLT in rats [11] and to be involved in the control of water intake [12-15]. Type-1 cannabinoid receptors (CB1) modulate consummatory behaviors, such as feeding [16-26]. However, the role of CB1 receptors in the control of water intake is still a matter of debate [27-31]. Here, we show that endogenous activation of CB1 in cortical glutamatergic neurons of the ACC promotes water intake. Notably, presynaptic CB1 receptors of ACC glutamatergic neurons are abundantly located in the basolateral amygdala (BLA), a key area in the regulation of water intake. The selective expression of CB1 receptors in the ACC-to-BLA-projecting neurons is sufficient to stimulate drinking behavior. Moreover, chemogenetic stimulation of these projecting neurons suppresses drinking behavior, further supporting the role of this neuronal population in the control of water intake. Altogether, these data reveal a novel cortico-amygdalar mechanism involved in the regulation of drinking behavior.