Neuronal PAS domain 1 identifies a major subpopulation of wakefulness-promoting GABAergic neurons in the basal forebrain.

Here, we describe a group of basal forebrain (BF) neurons expressing neuronal Per-Arnt-Sim (PAS) domain 1 (Npas1), a developmental transcription factor linked to neuropsychiatric disorders. Immunohistochemical staining in Npas1-cre-2A-TdTomato mice revealed BF Npas1+ neurons are distinct from well-studied parvalbumin or cholinergic neurons. Npas1 staining in GAD67-GFP knock-in mice confirmed that the vast majority of Npas1+ neurons are GABAergic, with minimal colocalization with glutamatergic neurons in vGlut1-cre-tdTomato or vGlut2-cre-tdTomato mice. The density of Npas1+ neurons was high, five to six times that of neighboring cholinergic, parvalbumin, or glutamatergic neurons. Anterograde tracing identified prominent projections of BF Npas1+ neurons to brain regions involved in sleep-wake control, motivated behaviors, and olfaction such as the lateral hypothalamus, lateral habenula, nucleus accumbens shell, ventral tegmental area, and olfactory bulb. Chemogenetic activation of BF Npas1+ neurons in the light period increased the amount of wakefulness and the latency to sleep for 2 to 3 h, due to an increase in long wake bouts and short NREM sleep bouts. NREM slow-wave and sigma power, as well as sleep spindle density, amplitude, and duration, were reduced, reminiscent of findings in several neuropsychiatric disorders. Together with previous findings implicating BF Npas1+ neurons in stress responsiveness, the anatomical projections of BF Npas1+ neurons and the effect of activating them suggest a possible role for BF Npas1+ neurons in motivationally driven wakefulness and stress-induced insomnia. Identification of this major subpopulation of BF GABAergic neurons will facilitate studies of their role in sleep disorders, dementia, and other neuropsychiatric conditions involving BF.

Ventral Pallidum and Amygdala Cooperate to Restrain Reward Approach under Threat.

Foraging decisions involve assessing potential risks and prioritizing food sources, which can be challenging when confronted with changing and conflicting circumstances. A crucial aspect of this decision-making process is the ability to actively overcome defensive reactions to threats and focus on achieving specific goals. The ventral pallidum (VP) and basolateral amygdala (BLA) are two brain regions that play key roles in regulating behavior motivated by either rewards or threats. However, it is unclear whether these regions are necessary in decision-making processes involving competing motivational drives during conflict. Our aim was to investigate the requirements of the VP and BLA for foraging choices in conflicts involving overcoming defensive responses. Here, we used a novel foraging task and pharmacological techniques to inactivate either the VP or BLA or to disconnect these brain regions before conducting a conflict test in male rats. Our findings showed that BLA is necessary for making risky choices during conflicts, whereas VP is necessary for invigorating the drive to obtain food, regardless of the presence of conflict. Importantly, our research revealed that the connection between VP and BLA is critical in controlling risky food-seeking choices during conflict situations. This study provides a new perspective on the collaborative function of VP and BLA in driving behavior, aimed at achieving goals in the face of dangers.

Multimodal Interrogation of Ventral Pallidum Projections Reveals Projection-Specific Signatures and Effects on Cocaine Reward.

The ventral pallidum (VP) is a central hub in the reward circuitry with diverse projections that have different behavioral roles attributed mostly to the connectivity with the downstream target. However, different VP projections may represent, as in the striatum, separate neuronal populations that differ in more than just connectivity. In this study, we performed in mice of both sexes a multimodal dissection of four major projections of the VP-to the lateral hypothalamus (VP→LH), ventral tegmental area (VP→VTA), lateral habenula (VP→LHb), and mediodorsal thalamus (VP→MDT)-with physiological, anatomical, genetic, and behavioral tools. We also tested for physiological differences between VP neurons receiving input from nucleus accumbens medium spiny neurons (MSNs) that express either the D1 (D1-MSNs) or the D2 (D2-MSNs) dopamine receptor. We show that each VP projection (1) when inhibited during a cocaine conditioned place preference (CPP) test affects performance differently, (2) receives a different pattern of inputs using rabies retrograde labeling, (3) shows differentially expressed genes using RNA sequencing, and (4) has projection-specific characteristics in excitability and synaptic input characteristics using whole-cell patch clamp. VP→LH and VP→VTA projections have different effects on CPP and show low overlap in circuit tracing experiments, as VP→VTA neurons receive more striatal input, while VP→LH neurons receive more olfactory input. Additionally, VP→VTA neurons are less excitable, while VP→LH neurons are more excitable than the average VP neuron, a difference driven mainly by D2-MSN-responding neurons. Thus, VP→VTA and VP→LH neurons may represent largely distinct populations of VP neurons.

Facilitation of Behavioral and Cortical Emergence from Isoflurane Anesthesia by GABAergic Neurons in Basal Forebrain.

General anesthesia shares many similarities with natural sleep in behavior and electroencephalogram (EEG) patterns. The latest evidence suggests that general anesthesia and sleep-wake behavior may share overlapping neural substrates. The GABAergic neurons in the basal forebrain (BF) have recently been demonstrated to play a key role in controlling wakefulness. It was hypothesized that BF GABAergic neurons may participate in the regulation of general anesthesia. Here, using in vivo fiber photometry, we found that the activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia, having obviously decreased during the induction of anesthesia and being gradually restored during the emergence from anesthesia, in Vgat-Cre mice of both sexes. Activation of BF GABAergic neurons with chemogenetic and optogenetic approaches decreased sensitivity to isoflurane, delayed induction, and accelerated emergence from isoflurane anesthesia. Optogenetic activation of BF GABAergic neurons decreased EEG δ power and the burst suppression ratio (BSR) during 0.8% and 1.4% isoflurane anesthesia, respectively. Similar to the effects of activating BF GABAergic cell bodies, photostimulation of BF GABAergic terminals in the thalamic reticular nucleus (TRN) also strongly promoted cortical activation and behavioral emergence from isoflurane anesthesia. Collectively, these results showed that the GABAergic BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthesia via the GABAergic BF-TRN pathway. Our findings may provide a new target for attenuating the depth of anesthesia and accelerating emergence from general anesthesia.SIGNIFICANCE STATEMENT The basal forebrain (BF) is a key brain region controlling sleep-wake behavior. Activation of GABAergic neurons in the BF potently promotes behavioral arousal and cortical activity. Recently, many sleep-wake-related brain structures have been reported to participate in the regulation of general anesthesia. However, it is still unclear what role BF GABAergic neurons play in general anesthesia. In this study, we aim to reveal the role of BF GABAergic neurons in behavioral and cortical emergence from isoflurane anesthesia and elucidate the underlying neural pathways. Understanding the specific role of BF GABAergic neurons in isoflurane anesthesia would improve our understanding of the mechanisms of general anesthesia and may provide a new strategy for accelerating emergence from general anesthesia.

Activation of Basal Forebrain Astrocytes Induces Wakefulness without Compensatory Changes in Sleep Drive.

Mammalian sleep is regulated by a homeostatic process that increases sleep drive and intensity as a function of prior wake time. Sleep homeostasis has traditionally been thought to be a product of neurons, but recent findings demonstrate that this process is also modulated by glial astrocytes. The precise role of astrocytes in the accumulation and discharge of sleep drive is unknown. We investigated this question by selectively activating basal forebrain (BF) astrocytes using designer receptors exclusively activated by designer drugs (DREADDs) in male and female mice. DREADD activation of the Gq-protein-coupled pathway in BF astrocytes produced long and continuous periods of wakefulness that paradoxically did not cause the expected homeostatic response to sleep loss (e.g., increases in sleep time or intensity). Further investigations showed that this was not because of indirect effects of the ligand that activated DREADDs. These findings suggest that the need for sleep is not only driven by wakefulness per se, but also by specific neuronal-glial circuits that are differentially activated in wakefulness.SIGNIFICANCE STATEMENT Sleep drive is controlled by a homeostatic process that increases sleep duration and intensity based on prior time spent awake. Non-neuronal brain cells (e.g., glial astrocytes) influence this homeostatic process, but their precise role is unclear. We used a genetic technique to activate astrocytes in the basal forebrain (BF) of mice, a brain region important for sleep and wake expression and sleep homeostasis. Astroglial activation induced prolonged wakefulness without the expected homeostatic increase in sleep drive (i.e., sleep duration and intensity). These findings indicate that our need to sleep is also driven by non-neuronal cells, and not only by time spent awake.

Cognitive Aging and the Primate Basal Forebrain Revisited: Disproportionate GABAergic Vulnerability Revealed.

Basal forebrain (BF) projections to the hippocampus and cortex are anatomically positioned to influence a broad range of cognitive capacities that are known to decline in normal aging, including executive function and memory. Although a long history of research on neurocognitive aging has focused on the role of the cholinergic basal forebrain system, intermingled GABAergic cells are numerically as prominent and well positioned to regulate the activity of their cortical projection targets, including the hippocampus and prefrontal cortex. The effects of aging on noncholinergic BF neurons in primates, however, are largely unknown. In this study, we conducted quantitative morphometric analyses in brains from young adult (6 females, 2 males) and aged (11 females, 5 males) rhesus monkeys (Macaca mulatta) that displayed significant impairment on standard tests that require the prefrontal cortex and hippocampus. Cholinergic (ChAT+) and GABAergic (GAD67+) neurons were quantified through the full rostrocaudal extent of the BF. Total BF immunopositive neuron number (ChAT+ plus GAD67+) was significantly lower in aged monkeys compared with young, largely because of fewer GAD67+ cells. Additionally, GAD67+ neuron volume was greater selectively in aged monkeys without cognitive impairment compared with young monkeys. These findings indicate that the GABAergic component of the primate BF is disproportionally vulnerable to aging, implying a loss of inhibitory drive to cortical circuitry. Moreover, adaptive reorganization of the GABAergic circuitry may contribute to successful neurocognitive outcomes.SIGNIFICANCE STATEMENT A long history of research has confirmed the role of the basal forebrain in cognitive aging. The majority of that work has focused on BF cholinergic neurons that innervate the cortical mantle. Codistributed BF GABAergic populations are also well positioned to influence cognitive function, yet little is known about this prominent neuronal population in the aged brain. In this unprecedented quantitative comparison of both cholinergic and GABAergic BF neurons in young and aged rhesus macaques, we found that neuron number is significantly reduced in the aged BF compared with young, and that this reduction is disproportionately because of a loss of GABAergic neurons. Together, our findings encourage a new perspective on the functional organization of the primate BF in neurocognitive aging.

Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons.

Altered activity of the ventral pallidum (VP) underlies disrupted motivation in stress and drug exposure. The VP is a very heterogeneous structure composed of many neuron types with distinct physiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for motivational behavior. While Npas1+ neurons have been characterized in the globus pallidus external, there is limited information on these neurons in the VP. To address this limitation, we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-sequencing on ribosome-associated mRNA from VP Npas1+ neurons to determine their molecular identity. Finally, we used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We used a similar approach in females using the chronic witness defeat stress (CWDS). We identified VP Npas1+ projections to the nucleus accumbens, ventral tegmental area, medial and lateral habenula, lateral hypothalamus, thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct translatome representing distinct biological processes. Chemogenetic activation of hM3D(Gq) receptors in VP Npas1+ neurons increased susceptibility to a subthreshold SDS and anxiety-like behavior in the elevated plus maze and open field while the activation of hM4D(Gi) receptors in VP Npas1+ neurons enhanced resilience to chronic SDS and CWDS. Thus, the activity of VP Npas1+ neurons modulates susceptibility to social stressors and anxiety-like behavior. Our studies provide new information on VP Npas1+ neuron circuitry, molecular identity, and their role in stress response.SIGNIFICANCE STATEMENT The ventral pallidum (VP) is a structure connected to both reward-related and aversive brain centers. It is a key brain area that signals the hedonic value of natural rewards. Disruption in the VP underlies altered motivation in stress and substance use disorder. However, VP is a very heterogeneous area with multiple neuron subtypes. This study characterized the projection pattern and molecular signatures of VP Neuronal PAS 1-positive (Npas1+) neurons. We further used tools to alter receptor signaling in VP Npas1+ neurons in stress to demonstrate a role for these neurons in stress behavioral outcomes. Our studies have implications for understanding brain cell type identities and their role in brain disorders, such as depression, a serious disorder that is precipitated by stressful events.

Ventral Pallidal GABAergic Neuron Calcium Activity Encodes Cue-Driven Reward Seeking and Persists in the Absence of Reward Delivery.

Reward-seeking behavior is often initiated by environmental cues that signal reward availability. This is a necessary behavioral response; however, cue reactivity and reward-seeking behavior can become maladaptive. To better understand how cue-elicited reward seeking becomes maladaptive, it is important to understand the neural circuits involved in assigning appetitive value to rewarding cues and actions. Ventral pallidum (VP) neurons are known to contribute to cue-elicited reward-seeking behavior and have heterogeneous responses in a discriminative stimulus (DS) task. The VP neuronal subtypes and output pathways that encode distinct aspects of the DS task remain unknown. Here, we used an intersectional viral approach with fiber photometry to record bulk calcium activity in VP GABAergic (VP GABA) neurons in male and female rats as they learned and performed the DS task. We found that VP GABA neurons are excited by reward-predictive cues but not neutral cues and that this response develops over time. We also found that this cue-evoked response predicts reward-seeking behavior and that inhibiting this VP GABA activity during cue presentation decreases reward-seeking behavior. Additionally, we found increased VP GABA calcium activity at the time of expected reward delivery, which occurred even on trials when reward was omitted. Together, these findings suggest that VP GABA neurons encode reward expectation, and calcium activity in these neurons encodes the vigor of cue-elicited reward seeking.SIGNIFICANCE STATEMENT VP circuitry is a major driver of cue-evoked behaviors. Previous work has found that VP neurons have heterogenous responses and contributions to reward-seeking behavior. This functional heterogeneity is because of differences of neurochemical subtypes and projections of VP neurons. Understanding the heterogenous responses among and within VP neuronal cell types is a necessary step in further understanding how cue-evoked behavior becomes maladaptive. Our work explores the canonical GABAergic VP neuron and how the calcium activity of these cells encodes components of cue-evoked reward seeking, including the vigor and persistence of reward seeking.

Sex differences in the structure and function of the vasopressin system in the ventral pallidum are associated with the sex-specific regulation of social play behavior in juvenile rats.

Vasopressin (AVP) regulates various social behaviors, often in sex-specific ways, including social play behavior, a rewarding behavior displayed primarily by juveniles. Here, we examined whether and how AVP acting in the brain's reward system regulates social play behavior in juvenile rats. Specifically, we focused on AVP signaling in the ventral pallidum (VP), a brain region that is a part of the reward system. First, we examined the organization of the VP-AVP system in juvenile rats and found sex differences, with higher density of both AVP-immunoreactive fibers and AVP V1a receptor (V1aR) binding in males compared to females while females show a greater number of V1aR-expressing cells compared to males. We further found that, in both sexes, V1aR-expressing cells co-express a GABA marker to a much greater extent (approx. 10 times) than a marker for glutamate. Next, we examined the functional involvement of V1aR-expressing VP cells in social play behavior. We found that exposure to social play enhanced the proportion of activated V1aR-expressing VP cells in males only. Finally, we showed that infusion of a specific V1aR antagonist into the VP increased social play behaviors in juvenile male rats while decreasing these behaviors in juvenile female rats. Overall, these findings reveal structural and functional sex differences in the AVP-V1aR system in the VP that are associated with the sex-specific regulation of social play behavior.

Ventral pallidum regulates the default mode network, controlling transitions between internally and externally guided behavior.

Daily life requires transitions between performance of well-practiced, automatized behaviors reliant upon internalized representations and behaviors requiring external focus. Such transitions involve differential activation of the default mode network (DMN), a group of brain areas associated with inward focus. We asked how optogenetic modulation of the ventral pallidum (VP), a subcortical DMN node, impacts task switching between internally to externally guided lever-pressing behavior in the rat. Excitation of the VP dramatically compromised acquisition of an auditory discrimination task, trapping animals in a DMN state of automatized internally focused behavior and impairing their ability to direct attention to external sensory stimuli. VP inhibition, on the other hand, facilitated task acquisition, expediting escape from the DMN brain state, thereby allowing rats to incorporate the contingency changes associated with the auditory stimuli. We suggest that VP, instant by instant, regulates the DMN and plays a deterministic role in transitions between internally and externally guided behaviors.

Late-Onset, Short-Term Intermittent Fasting Reverses Age-Related Changes in Calcium Buffering and Inhibitory Synaptic Transmission in Mouse Basal Forebrain Neurons.

Aging is often associated with cognitive decline and recurrent cellular and molecular impairments. While life-long caloric restriction (CR) may delay age-related cognitive deterioration as well as the onset of neurologic disease, recent studies suggest that late-onset, short-term intermittent fasting (IF), may show comparable beneficial effects as those of life-long CR to improve brain health. We used a new optogenetic aging model to study the effects of late-onset (>18 months), short-term (four to six weeks) IF on age-related changes in GABAergic synaptic transmission, intracellular calcium (Ca2+) buffering, and cognitive status. We used male mice from a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R [VGAT-ChR2(H134R)-EYFP] in a reduced synaptic preparation that allows for specific optogenetic light stimulation on GABAergic synaptic terminals across aging. We performed quantal analysis using the method of failures in this model and show that short-term IF reverses the age-related decrease in quantal content of GABAergic synapses. Likewise, short-term IF also reversed age-related changes in Ca2+ buffering and spontaneous GABAergic synaptic transmission in basal forebrain (BF) neurons of aged mice. Our findings suggest that late-onset short-term IF can reverse age-related physiological impairments in mouse BF neurons but that four weeks IF is not sufficient to reverse age-related cognitive decline.SIGNIFICANCE STATEMENT Here, we demonstrate plasticity of the aging brain and reversal of well-defined hallmarks of brain aging using short-term intermittent fasting (IF) initiated later in life. Few therapeutics are currently available to treat age-related neurologic dysfunction although synaptic dysfunction occurs during aging and neurologic disease is a topic of intense research. Using a new reduced synaptic preparation and optogenetic stimulation we are able to study age-related synaptic mechanisms in greater detail. Several neurophysiological parameters including quantal content were altered during aging and were reversed with short-term IF. These methods can be used to identify potential therapies to reverse physiological dysfunction during aging.

Optogenetic stimulation of basal forebrain cholinergic neurons prevents neuroinflammation and neuropsychiatric manifestations in pristane induced lupus mice.

BACKGROUND: Neuroinflammation has been identified as one of the primary pathogenic factors of neuropsychiatric systemic lupus erythematosus (NPSLE). However, there are no dedicated treatments available in clinics to alleviate neuroinflammation in NPSLE. It has been proposed that stimulating basal forebrain (BF) cholinergic neurons may provide potent anti-inflammatory effects in several inflammatory diseases, but its potential role in NPSLE remains unexplored. This study aims to investigate whether and how stimulating BF cholinergic neurons has a protective effect on NPSLE. RESULTS: Optogenetic stimulation of BF cholinergic neurons significantly ameliorated olfactory dysfunction and anxiety- and depression-like phenotype in pristane induced lupus (PIL) mice. The increased expression of adhesion molecules (P-selectin and vascular cell adhesion molecule-1 (VCAM-1)), leukocyte recruitment, blood-brain barrier (BBB) leakage were significantly decreased. Notably, the brain histopathological changes, including the elevated levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), IgG deposition in the choroid plexus and lateral ventricle wall and lipofuscin accumulation in the cortical and hippocampal neurons, were also significantly attenuated. Furthermore, we confirmed the colocalization between the BF cholinergic projections and the cerebral vessels, and the expression of α7-nicotinic acetylcholine receptor (α7nAChR) on the cerebral vessels. CONCLUSION: Our data indicate that stimulation of BF cholinergic neurons could play a neuroprotective role in the brain through its cholinergic anti-inflammatory effects on cerebral vessels. Therefore, this may be a promising preventive target for NPSLE.

Selective impairment of circuits between prefrontal cortex glutamatergic neurons and basal forebrain cholinergic neurons in a tauopathy mouse model.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder linked to cognitive decline. To understand how specific neuronal circuits are impaired in AD, we have used optogenetic and electrophysiological approaches to reveal the functional changes between prefrontal cortex (PFC) and basal forebrain (BF), 2 key regions controlling cognitive processes, in a tauopathy mouse model. We found that the glutamatergic synaptic responses in BF cholinergic neurons from P301S Tau mice (6-8 months old) were markedly diminished. The attenuated long-range PFC to BF pathway in the AD model significantly increased the failure rate of action potential firing of BF cholinergic neurons triggered by optogenetic stimulations of glutamatergic terminals from PFC. In contrast, the projection from PFC to other regions, such as amygdala and striatum, was largely unaltered. On the other hand, optogenetic stimulation of cholinergic terminals from BF induced a persistent reduction of the excitability of PFC pyramidal neurons from Tau mice, instead of the transient reduction exhibited in wild-type mice. Taken together, these data have revealed a selective aberration of the pathway between PFC pyramidal neurons and BF cholinergic neurons in a tauopathy mouse model. This circuit deficit may underlie the loss of attention and executive function in AD.

Human decisions about when to act originate within a basal forebrain-nigral circuit.

Decisions about when to act are critical for survival in humans as in animals, but how a desire is translated into the decision that an action is worth taking at any particular point in time is incompletely understood. Here we show that a simple model developed to explain when animals decide it is worth taking an action also explains a significant portion of the variance in timing observed when humans take voluntary actions. The model focuses on the current environment's potential for reward, the timing of the individual's own recent actions, and the outcomes of those actions. We show, by using ultrahigh-field MRI scanning, that in addition to anterior cingulate cortex within medial frontal cortex, a group of subcortical structures including striatum, substantia nigra, basal forebrain (BF), pedunculopontine nucleus (PPN), and habenula (HB) encode trial-by-trial variation in action time. Further analysis of the activity patterns found in each area together with psychophysiological interaction analysis and structural equation modeling suggested a model in which BF integrates contextual information that will influence the decision about when to act and communicates this information, in parallel with PPN and HB influences, to nigrostriatal circuits. It is then in the nigrostriatal circuit that action initiation per se begins.

Ventral Pallidum GABA Neurons Mediate Motivation Underlying Risky Choice.

Pursuing rewards while avoiding danger is an essential function of any nervous system. Here, we examine a new mechanism helping rats negotiate the balance between risk and reward when making high-stakes decisions. Specifically, we focus on GABA neurons within an emerging mesolimbic circuit nexus: the ventral pallidum (VP). These neurons play a distinct role from other VP neurons in simple motivated behaviors in mice, but their role in more complex motivated behaviors is unknown. Here, we interrogate the behavioral functions of VPGABA neurons in male and female transgenic GAD1:Cre rats (and WT littermates), using a reversible chemogenetic inhibition approach. Using a behavioral assay of risky decision-making, and of the food-seeking and shock-avoidance components of this task, we show that engaging inhibitory Gi/o signaling specifically in VPGABA neurons suppresses motivation to pursue highly salient palatable foods, and possibly also motivation to avoid being shocked. In contrast, inhibiting these neurons did not affect seeking of low-value food, free consumption of palatable food, or unconditioned affective responses to shock. Accordingly, when rats considered whether to pursue food despite potential for shock in a risky decision-making task, inhibiting VPGABA neurons caused them to more readily select a small but safe reward over a large but dangerous one, an effect not seen in the absence of shock threat. Together, results indicate that VPGABA neurons are critical for high-stakes adaptive responding that is necessary for survival, but which may also malfunction in psychiatric disorders.SIGNIFICANCE STATEMENT In a dynamic world, it is essential to implement appropriate behaviors under circumstances involving rewards, threats, or both. Here, we demonstrate a crucial role for VPGABA neurons in high-stakes motivated behavior of several types. We show that this VPGABA role in motivation impacts decision-making, as inhibiting these neurons yields a conservative, risk-averse strategy not seen when the task is performed without threat of shock. These new roles for VPGABA neurons in behavior may inform future strategies for treating addiction, and other disorders of maladaptive decision-making.

Basal Forebrain Cholinergic Neurons Selectively Drive Coordinated Motor Learning in Mice.

Motor control requires precise temporal and spatial encoding across distinct motor centers that is refined through the repetition of learning. The recruitment of motor regions requires modulatory input to shape circuit activity. Here, we identify a role for the baso-cortical cholinergic pathway in the acquisition of a coordinated motor skill in mice. Targeted depletion of basal forebrain cholinergic neurons results in significant impairments in training on the rotarod task of coordinated movement. Cholinergic neuromodulation is required during training sessions as chemogenetic inactivation of cholinergic neurons also impairs task acquisition. Rotarod learning is known to drive refinement of corticostriatal neurons arising in both medial prefrontal cortex (mPFC) and motor cortex, and we have found that cholinergic input to both motor regions is required for task acquisition. Critically, the effects of cholinergic neuromodulation are restricted to the acquisition stage, as depletion of basal forebrain cholinergic neurons after learning does not affect task execution. Our results indicate a critical role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning.SIGNIFICANCE STATEMENT Acetylcholine release from basal forebrain cholinergic neuron terminals rapidly modulates neuronal excitability, circuit dynamics, and cortical coding; all processes required for processing complex sensory information, cognition, and attention. We found that depletion or transient silencing of cholinergic inputs to anatomically isolated motor areas, medial prefrontal cortex (mPFC) and motor cortex, selectively led to significant impairments on coordinated motor learning; disrupting this baso-cortical network after acquisition elicited no effect on task execution. Our results indicate a pivotal role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning. These findings support the concept that cognitive components (such as attention) are indispensable in the adjustment of motor output and training-induced improvements in motor performance.

Basal forebrain projections to the lateral habenula modulate aggression reward.

Maladaptive aggressive behaviour is associated with a number of neuropsychiatric disorders and is thought to result partly from the inappropriate activation of brain reward systems in response to aggressive or violent social stimuli. Nuclei within the ventromedial hypothalamus, extended amygdala and limbic circuits are known to encode initiation of aggression; however, little is known about the neural mechanisms that directly modulate the motivational component of aggressive behaviour. Here we established a mouse model to measure the valence of aggressive inter-male social interaction with a smaller subordinate intruder as reinforcement for the development of conditioned place preference (CPP). Aggressors develop a CPP, whereas non-aggressors develop a conditioned place aversion to the intruder-paired context. Furthermore, we identify a functional GABAergic projection from the basal forebrain (BF) to the lateral habenula (lHb) that bi-directionally controls the valence of aggressive interactions. Circuit-specific silencing of GABAergic BF-lHb terminals of aggressors with halorhodopsin (NpHR3.0) increases lHb neuronal firing and abolishes CPP to the intruder-paired context. Activation of GABAergic BF-lHb terminals of non-aggressors with channelrhodopsin (ChR2) decreases lHb neuronal firing and promotes CPP to the intruder-paired context. Finally, we show that altering inhibitory transmission at BF-lHb terminals does not control the initiation of aggressive behaviour. These results demonstrate that the BF-lHb circuit has a critical role in regulating the valence of inter-male aggressive behaviour and provide novel mechanistic insight into the neural circuits modulating aggression reward processing.

A cholinergic basal forebrain feeding circuit modulates appetite suppression.

Atypical food intake is a primary cause of obesity and other eating and metabolic disorders. Insight into the neural control of feeding has previously focused mainly on signalling mechanisms associated with the hypothalamus, the major centre in the brain that regulates body weight homeostasis. However, roles of non-canonical central nervous system signalling mechanisms in regulating feeding behaviour have been largely uncharacterized. Acetylcholine has long been proposed to influence feeding owing in part to the functional similarity between acetylcholine and nicotine, a known appetite suppressant. Nicotine is an exogenous agonist for acetylcholine receptors, suggesting that endogenous cholinergic signalling may play a part in normal physiological regulation of feeding. However, it remains unclear how cholinergic neurons in the brain regulate food intake. Here we report that cholinergic neurons of the mouse basal forebrain potently influence food intake and body weight. Impairment of cholinergic signalling increases food intake and results in severe obesity, whereas enhanced cholinergic signalling decreases food consumption. We found that cholinergic circuits modulate appetite suppression on downstream targets in the hypothalamus. Together our data reveal the cholinergic basal forebrain as a major modulatory centre underlying feeding behaviour.

Metaplasticity in the Ventral Pallidum as a Potential Marker for the Propensity to Gain Weight in Chronic High-Calorie Diet.

A major driver of obesity is the increasing palatability of processed foods. Although reward circuits promote the consumption of palatable food, their involvement in obesity remains unclear. The ventral pallidum (VP) is a key hub in the reward system that encodes the hedonic aspects of palatable food consumption and participates in various proposed feeding circuits. However, there is still no evidence for its involvement in developing diet-induced obesity. Here we examine, using male C57BL6/J mice and patch-clamp electrophysiology, how chronic high-fat high-sugar (HFHS) diet changes the physiology of the VP and whether mice that gain the most weight differ in their VP physiology from others. We found that 10-12 weeks of HFHS diet hyperpolarized and decreased the firing rate of VP neurons without a major change in synaptic inhibitory input. Within the HFHS group, the top 33% weight gainers (WGs) had a more hyperpolarized VP with longer latency to fire action potentials on depolarization compared with bottom 33% of weight gainers (i.e., non-weight gainers). WGs also showed synaptic potentiation of inhibitory inputs both at the millisecond and minute ranges. Moreover, we found that the tendency to potentiate the inhibitory inputs to the VP might exist in overeating mice even before exposure to HFHS, thus making it a potential property of being an overeater. These data point to the VP as a critical player in obesity and suggest that hyperpolarized membrane potential of, and potentiated inhibitory inputs to, VP neurons may play a significant role in promoting the overeating of palatable food.SIGNIFICANCE STATEMENT In modern world, where highly palatable food is readily available, overeating is often driven by motivational, rather than metabolic, needs. It is thus conceivable that reward circuits differ between obese and normal-weight individuals. But is such difference, if it exists, innate or does it develop with overeating? Here we reveal synaptic properties in the ventral pallidum, a central hub of reward circuits, that differ between mice that gain the most and the least weight when given unlimited access to highly palatable food. We show that these synaptic differences also exist without exposure to palatable food, potentially making them innate properties that render some more susceptible than others to overeat. Thus, the propensity to overeat may have a strong innate component embedded in reward circuits.

Complementary Roles for Ventral Pallidum Cell Types and Their Projections in Relapse.

The ventral pallidum (VP) is a key node in the neural circuits controlling relapse to drug seeking. How this role relates to different VP cell types and their projections is poorly understood. Using male rats, we show how different forms of relapse to alcohol-seeking are assembled from VP cell types and their projections to lateral hypothalamus (LH) and ventral tegmental area (VTA). Using RNAScope in situ hybridization to characterize activity of different VP cell types during relapse to alcohol-seeking provoked by renewal (context-induced reinstatement), we found that VP Gad1 and parvalbumin (PV), but not vGlut2, neurons show relapse-associated changes in c-Fos expression. Next, we used retrograde tracing, chemogenetic, and electrophysiological approaches to study the roles of VPGad1 and VPPV neurons in relapse. We show that VPGad1 neurons contribute to contextual control over relapse (renewal), but not to relapse during reacquisition, via projections to LH, where they converge with ventral striatal inputs onto LHGad1 neurons. This convergence of striatopallidal inputs at the level of individual LHGad1 neurons may be critical to balancing propensity for relapse versus abstinence. In contrast, VPPV neurons contribute to relapse during both renewal and reacquisition via projections to VTA. These findings identify a double dissociation in the roles for different VP cell types and their projections in relapse. VPGad1 neurons control relapse during renewal via projections to LH. VPPV neurons control relapse during both renewal and reacquisition via projections to VTA. Targeting these different pathways may provide tailored interventions for different forms of relapse.SIGNIFICANCE STATEMENT Relapse to drug or reward seeking after a period of extinction or abstinence remains a key impediment to successful treatment. The ventral pallidum, located in the ventral basal ganglia, has long been recognized as an obligatory node in a 'final common pathway' for relapse. Yet how this role relates to the considerable VP cellular and circuit heterogeneity is not well understood. We studied the cellular and circuit architecture for VP in relapse control. We show that different forms of relapse have complementary VP cellular and circuit architectures, raising the possibility that targeting these different neural architectures may provide tailored interventions for different forms of relapse.