Abstract:
One major hallmark of alcohol use disorder (AUD) is the persistence of drinking despite negative consequences. Among indicators of AUD vulnerability, binge drinking has been identified as one of the strongest risk factors. Although the lifetime prevalence of both binge drinking and AUD has historically been higher in men than women, this gap has dramatically narrowed in the last decade. Additionally, sex differences in AUD and binge drinking have been found in clinical and preclinical studies. At the neurobiological level, the insular cortex plays an important role in AUD, with the anterior (aIC) and posterior (pIC) divisions supporting different functions. However, the contributions of the aIC and pIC sections in sexual dimorphism of alcohol binge drinking and the persistence of alcohol drinking despite aversion remain to be uncovered. Using the drinking in the dark model in mice, we validated that female mice have a higher binge ethanol intake compared to males. To evaluate persistent ethanol consumption despite aversion, we supplemented ethanol with the bitter compound quinine, and found a higher persistent drinking in females compared to males. Using fiber photometry recordings, we revealed that aIC activity was increased during binge and persistent ethanol consumption independently of sex, whereas pIC glutamatergic neuron activity was higher during persistent ethanol drinking, specifically in female mice. Using chemogenetics, we revealed that inhibition of aIC glutamatergic neurons reduced intake of bitter solutions independently of the solvent (ethanol or water) in both sexes. In addition, inhibition of pIC glutamatergic neurons exclusively reduced persistent ethanol drinking in females, while decreasing quinine consumption only in males. These findings suggest a sex-dependent function of the pIC in the persistence of ethanol consumption, providing a starting point in understanding sex-specific functions of the insular cortex in the neurobiology of AUD.

Abstract:
The insular cortex (or insula), and particularly its anterior region, plays a crucial role in the control of emotional valence and anxiety (Etkin & Wager, 2007; Méndez-Ruette et al., 2019; Nicolas et al., 2023). While dopamine neurotransmission is known to modulate anxiety levels in humans (Hjorth et al., 2021) and animal models (de la Mora et al., 2010; Bananej et al., 2012; Zarrindast & Khakpai, 2015; DeGroot et al., 2020; Godino et al., 2023), its regulatory effects on the anterior insula remained unexplored. Here, using a multifaceted approach, we uncovered how dopamine shapes anterior insula function in anxiety and valence processing. First, we revealed a high density of neurons expressing type-1 dopamine receptors (D1) in the insula, particularly important in the anterior insula, and seven times greater than the density of neurons expressing type-2 dopamine receptors (D2). Few neurons co-expressed Drd1 and Drd2 mRNAs in the anterior and posterior insula, and the density of Drd1+ neurons in the anterior insula was twice higher among inhibitory neurons than excitatory neurons. Second, we found that pharmacological activation of D1 in the anterior insula is anxiogenic, suggesting a direct link between insular dopamine signaling and anxiety-related behaviors. Using fiber-photometry recordings, we identified that the amplitude of dopamine release onto D1+ neurons in the anterior insula while mice were in anxiogenic spaces or receiving mild foot shocks was both positively correlated with mice level of trait anxiety. Population dynamics and deep-learning analyses of anterior insula single-unit recordings uncovered distinct coding patterns of anxiety-provoking and safe environments, as well as tastants of positive and negative valence. Remarkably, systemic D1 activation, which heightens anxiety- related behaviors, dampens this coding dichotomy by increasing coding variability for protected spaces while increasing the coding reliability for anxiogenic spaces. Interestingly, the coding reliability of anxiogenic areas was positively correlated with mice level of trait anxiety, and we observed a trend towards a positive correlation between the coding reliability of a negative tastants, and mice level of anxiety. Altogether, our findings provide a new model of neural population coding of anxiety and emotional valence and unravel D1-dependent coding mechanisms in the mouse anterior insula.

Abstract:
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 (CB(1)) 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 CB(1) receptors in the pIC decreases water intake. Interestingly, we found that CB(1) 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 CB(1)-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 CB(1) receptor signaling promotes water intake by inhibiting the pIC-BLA pathway, thereby contributing to the fine top-down control of thirst responses.

Abstract:
Responses of the insular cortex (IC) and amygdala to stimuli of positive and negative valence are altered in patients with anxiety disorders. However, neural coding of both anxiety and valence by IC neurons remains unknown. Using fiber photometry recordings in mice, we uncover a selective increase of activity in IC projection neurons of the anterior (aIC), but not posterior (pIC) section, when animals are exploring anxiogenic spaces, and this activity is proportional to the level of anxiety of mice. Neurons in aIC also respond to stimuli of positive and negative valence, and the strength of response to strong negative stimuli is proportional to mice levels of anxiety. Using ex vivo electrophysiology, we characterized the IC connection to the basolateral amygdala (BLA), and employed projection-specific optogenetics to reveal anxiogenic properties of aIC-BLA neurons. Finally, we identified that aIC-BLA neurons are activated in anxiogenic spaces, as well as in response to aversive stimuli, and that both activities are positively correlated. Altogether, we identified a common neurobiological substrate linking negative valence with anxiety-related information and behaviors, which provides a starting point to understand how alterations of these neural populations contribute to psychiatric disorders.

Abstract: