Ronald Kim

Summary: Rodent drug self-administration leads to compromised ability of astrocytes to maintain glutamate homeostasis within the brain’s reward circuitry, as well as reductions in surface area, volume, and synaptic colocalization of astrocyte membranes. However, the mechanisms driving astrocyte responses to cocaine are unknown. Here, we report that long-access cocaine self-administration followed by prolonged home cage abstinence results in decreased branching complexity of nucleus accumbens astrocytes, characterized by the loss of peripheral processes. Using a combination of confocal fluorescence microcopy and immuno-gold electron microscopy, we show that alterations in astrocyte structural features are driven by microglia phagocytosis, as labeled astrocyte membranes are found within microglia phagolysosomes. Inhibition of complement C3-mediated phagocytosis using the neutrophil inhibitory peptide (NIF) rescued astrocyte structure and decreased cocaine seeking behavior following cocaine self-administration and abstinence. Collectively, these results provide evidence for microglia pruning of accumbens astrocytes across cocaine abstinence which mediates cocaine craving.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SI_p) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically-encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can “learn” the association between a naïve tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24h later (recall). In the NBM/SI_p cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SI_p cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SI_a cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

The ventral pallidum (VP) mediates motivated behaviors largely via the action of VP GABA and glutamatergic neurons. In addition to these neuronal subtypes, there is a population of cholinergic projection neurons in the VP, whose functional significance remains unclear. To understand the functional role of VP cholinergic neurons, we first examined behavioral responses to an appetitive (APP) odor that elicited approach, and an aversive (AV) odor that led to avoidance. To examine how VP cholinergic neurons were engaged in APP vs. AV responses, we used an immediate early gene marker and in-vivo fiber photometry, examining the activation profile of VP cholinergic neurons in response to each odor. Exposure to each odor led to an increase in the number of cFos counts and increased calcium signaling of VP cholinergic neurons. Activity and cre-dependent viral vectors were designed to label engaged VP cholinergic neurons in two distinct contexts: (1) exposure to the APP odor, (2) followed by subsequent exposure to the AV odor, and vice versa. These studies revealed two distinct, non-overlapping subpopulations of VP cholinergic neurons: one activated in response to the APP odor, and a second distinct population activated in response to the AV odor. These two subpopulations of VP cholinergic neurons are spatially intermingled within the VP, but show differences in electrophysiological properties, neuronal morphology, and projections to the basolateral amygdala. Although VP cholinergic neurons are engaged in behavioral responses to each odor, VP cholinergic signaling is only required for approach behavior. Indeed, inhibition of VP cholinergic neurons not only blocks approach to the APP odor, but reverses the behavior, leading to active avoidance. Our results highlight the functional heterogeneity of cholinergic projection neurons within the VP. These two subpopulations of VP cholinergic neurons differentially encode valence of olfactory stimuli and play unique roles in approach and avoidance behaviors.

Acetylcholine plays an essential role in fundamental aspects of cognition. Studies that have mapped the activity and functional connectivity of cholinergic neurons have shown that the axons of basal forebrain cholinergic neurons innervate the pallium with far more topographical and functional organization than was historically appreciated. Together with the results of studies using new probes that allow release of acetylcholine to be detected with high spatial and temporal resolution, these findings have implicated cholinergic networks in ‘binding’ diverse behaviours that contribute to cognition. Here, we review recent findings on the developmental origins, connectivity and function of cholinergic neurons, and explore the participation of cholinergic signalling in the encoding of cognition-related behaviours.

Accumulating evidence indicates significant consequences for astrocytes associated with drug abuse. For example, reductions in structural features and synaptic colocalization of male rat nucleus accumbens (NAc) astrocytes are observed following short-access (ShA; 2 h/d) self-administration and extinction from cocaine, methamphetamine, and heroin. However, it is unknown whether these observations extend to other rodent models of drug abuse, how enduring these effects may be, and whether similar effects are observed in female rats. Here, we assess the effects of long-access (LgA; 6 h/d) cocaine self-administration and abstinence on NAc astrocytes separately in male and female rats, employing a commonly used behavioral approach to investigate the incubation of cocaine craving. NAc astrocytes from male rats exhibit extensive (∼40%) reductions in surface area, volume, and postsynaptic colocalization 45 d but not 24 h after the last self-administration session. In contrast, no effect of self-administration and abstinence was observed in astrocytes from female rats. Moreover, no effect of LgA self-administration and abstinence was observed on NAc GLT-1 expression in female rats, an effect that has been well described in males. These results indicate striking and sexually dimorphic effects of abstinence subsequent to LgA self-administration on astrocytes. Taken together, these results indicate a pivotal role of prolonged abstinence in the effects of cocaine self-administration on NAc astrocytes, and extend a growing body of evidence regarding sex differences in the cellular consequences of drug self-administration in the brain.