Basolateral Amygdala Projection Research Articles

Reward contingency gates selective cholinergic suppression of amygdala neurons.

Basal forebrain cholinergic neurons modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, and memory. It is unknown, however, whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of secondary roles in the processing of external stimuli. Using fluorescent imaging, we found that cholinergic neurons are active during behavioral responding for a reward - even prior to reward delivery and in the absence of discrete stimuli. Photostimulation of basal forebrain cholinergic neurons, or their terminals in the basolateral amygdala (BLA), selectively promoted conditioned responding (licking), but not unconditioned behavior nor innate motor outputs. In vivo electrophysiological recordings during cholinergic photostimulation revealed reward-contingency-dependent suppression of BLA neural activity, but not prefrontal cortex. Finally, ex vivo experiments demonstrated that photostimulation of cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling, while also facilitating firing in BLA GABAergic interneurons. Taken together, we show that the neural and behavioral effects of basal forebrain cholinergic activation are modulated by reward contingency in a target-specific manner.

Reward contingency gates selective cholinergic suppression of amygdala neurons

Basal forebrain cholinergic neurons modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, and memory. It is unknown, however, whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of secondary roles in the processing of external stimuli. Using fluorescent imaging, we found that cholinergic neurons are active during behavioral responding for a reward – even prior to reward delivery and in the absence of discrete stimuli. Photostimulation of basal forebrain cholinergic neurons, or their terminals in the basolateral amygdala (BLA), selectively promoted conditioned responding (licking), but not unconditioned behavior nor innate motor outputs. In vivo electrophysiological recordings during cholinergic photostimulation revealed reward-contingency-dependent suppression of BLA neural activity, but not prefrontal cortex. Finally, ex vivo experiments demonstrated that photostimulation of cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling, while also facilitating firing in BLA GABAergic interneurons. Taken together, we show that the neural and behavioral effects of basal forebrain cholinergic activation are modulated by reward contingency in a target-specific manner.

Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens.

In substance use disorders, drug use as unconditioned stimulus (US) reinforces drug taking. Meanwhile, drug-associated cues (conditioned stimulus [CS]) also gain incentive salience to promote drug seeking. The basolateral amygdala (BLA) is implicated in both US- and CS-mediated responses. Here, we show that two genetically distinct BLA neuronal types, expressing Rspo2 versus Ppp1r1b, respectively, project to the nucleus accumbens (NAc) and form monosynaptic connections with both dopamine D1 and D2 receptor-expressing neurons. While intra-NAc stimulation of Rspo2 or Ppp1r1b presynaptic terminals establishes intracranial self-stimulation (ICSS), only Ppp1r1b-stimulated mice exhibit cue-induced ICSS seeking. Furthermore, increasing versus decreasing the Ppp1r1b-to-NAc, but not Rspo2-to-NAc, subprojection increases versus decreases cue-induced cocaine seeking after cocaine withdrawal. Thus, while both BLA-to-NAc subprojections contribute to US-mediated responses, the Ppp1r1b subprojection selectively encodes CS-mediated reward and drug reinforcement. Such differential circuit representations may provide insights into precise understanding and manipulation of drug- versus cue-induced drug seeking and relapse.

Anterior and Posterior Basolateral Amygdala Projections of Cell Type–Specific D1-Expressing Neurons From the Medial Prefrontal Cortex Differentially Control Alcohol-Seeking Behavior

BackgroundAlcohol use disorder is characterized by compulsive alcohol-seeking behavior, which is associated with dysregulation of afferent projections from the medial prefrontal cortex to the basolateral amygdala (BLA). However, the contribution of the cell type–specific mechanism in this neuronal circuit to alcohol-seeking behavior remains unclear. MethodsMice were trained with 2-bottle choice and operant alcohol self-administration procedures. Anterograde and retrograde viral methods traced the connection between dopamine type 1 receptor (D1R) neurons and BLA neurons. Electrophysiology and in vivo optogenetic techniques were used to test the function of neural circuits in alcohol-seeking behavior. ResultsChronic alcohol consumption preferentially changed the activity of posterior BLA (pBLA) neurons but not anterior BLA (aBLA) neurons and overexcited D1R neurons in the medial prefrontal cortex. Interestingly, we found that 2 populations of D1R neurons, anterior and posterior (pD1R) neurons, separately targeted the aBLA and pBLA, respectively, and only a few D1R neurons innervated both aBLA and pBLA neurons. Furthermore, pD1R neurons exhibited more excitability than anterior D1R neurons in alcohol-drinking mice. Moreover, we observed enhanced glutamatergic transmission and an increased NMDA/AMPA receptor ratio in the medial prefrontal cortex inputs from pD1R neurons to the pBLA. Optogenetic long-term depression induction of the pD1R-pBLA circuit reduced alcohol-seeking behavior, while optogenetic long-term depression or long-term potentiation induction of the anterior D1R–aBLA circuit produced no change in alcohol intake. ConclusionsThe pD1R-pBLA circuit mediates chronic alcohol consumption, which may suggest a cell type–specific neuronal mechanism underlying reward-seeking behavior in alcohol use disorder.

Synergistic photoactivation of VTA-catecholaminergic and BLA-glutamatergic projections induces long-term potentiation in the insular cortex

The presentation of novel stimuli induces a reliable dopamine release in the insular cortex (IC) from the ventral tegmental area (VTA). The novel stimuli could be associated with motivational and emotional signals induced by cortical glutamate release from the basolateral amygdala (BLA). Dopamine and glutamate are essential for acquiring and maintaining behavioral tasks, including visual and taste recognition memories. In this study, we hypothesize that the simultaneous activation of dopaminergic and glutamatergic projections to the neocortex can underlie synaptic plasticity. High-frequency stimulation of the BLA-IC circuit has demonstrated a reliable long-term potentiation (LTP), a widely acknowledged synaptic plasticity that underlies memory consolidation. Therefore, the concurrent optogenetic stimulation of the insula’s glutamatergic and dopaminergic terminal fibers would induce reliable LTP. Our results confirmed that combined photostimulation of the VTA and BLA projections to the IC induces a slow-onset LTP. We also found that optogenetically-induced LTP in the IC relies on both glutamatergic NMDA receptors and dopaminergic D1/D5 receptors, suggesting that the combined effects of these neurotransmitters can trigger synaptic plasticity in the neocortex. Overall, our findings provide compelling evidence supporting the essential role of both dopaminergic and glutamatergic projections in modulating synaptic plasticity within the IC. Furthermore, our results suggest that the synergistic actions of these projections have a pivotal influence on the formation of motivational memories.

Harmine exerts anxiolytic effects by regulating neuroinflammation and neuronal plasticity in the basolateral amygdala.

Increasing evidence indicates that an altered immune system is closely linked to the pathophysiology of anxiety disorders, and inhibition of neuroinflammation may represent an effective therapeutic strategy to treat anxiety disorders. Harmine, a beta-carboline alkaloid in various medicinal plants, has been widely reported to display anti-inflammatory and potentially anxiolytic effects. However, the exact underlying mechanisms are not fully understood. Our recent study has demonstrated that dysregulation of neuroplasticity in the basolateral amygdala (BLA) contributes to the pathological processes of inflammation-related anxiety. In this study, using a mouse model of anxiety challenged with Escherichia coli lipopolysaccharide (LPS), we found that harmine alleviated LPS-induced anxiety-like behaviors in mice. Mechanistically, harmine significantly prevented LPS-induced neuroinflammation by suppressing the expression of pro-inflammatory cytokines including IL-1β and TNF-α. Meanwhile, ex vivo whole-cell slice electrophysiology combined with optogenetics showed that LPS-induced increase of medial prefrontal cortex (mPFC)-driven excitatory but not inhibitory synaptic transmission onto BLA projection neurons, thereby alleviating LPS-induced shift of excitatory/inhibitory balance towards excitation. In addition, harmine attenuated the increased intrinsic neuronal excitability of BLA PNs by reducing the medium after-hyperpolarization. In conclusion, our findings provide new evidence that harmine may exert its anxiolytic effect by downregulating LPS-induced neuroinflammation and restoring the changes in neuronal plasticity in BLA PNs.

Anxiety and depression: A top-down, bottom-up model of circuit function.

A functional interplay of bottom-up and top-down processing allows an individual to appropriately respond to the dynamic environment around them. These processing modalities can be represented as attractor states using a dynamical systems model of the brain. The transition probability to move from one attractor state to another is dependent on the stability, depth, neuromodulatory tone, and tonic changes in plasticity. However, how does the relationship between these states change in disease states, such as anxiety or depression? We describe bottom-up and top-down processing from Marr's computational-algorithmic-implementation perspective to understand depressive and anxious disease states. We illustrate examples of bottom-up processing as basolateral amygdala signaling and projections and top-down processing as medial prefrontal cortex internal signaling and projections. Understanding these internal processing dynamics can help us better model the multifaceted elements of anxiety and depression.

Medial prefrontal cortex input to basolateral amygdala controls acute stress-induced short-term anxiety-like behavior in mice.

Anxiety is a normal and transitory emotional state that allows the organisms to cope well with the real or perceived threats, while excessive or prolonged anxiety is a key characteristic of anxiety disorders. We have recently revealed that prolonged anxiety induced by chronic stress is associated with the circuit-varying dysfunction of basolateral amygdala projection neurons (BLA PNs). However, it is not yet known whether similar mechanisms also emerge for acute stress-induced, short-lasting increase of anxiety. Here, using a mouse model of acute restraint stress (ARS), we found that ARS mice showed increased anxiety-like behavior at 2 h but not 24 h after stress, and this effect was accompanied by a transient increase of the activity of BLA PNs. Specifically, ex vivo patch-clamp recordings revealed that the increased BLA neuronal activity did not differ among the distinct BLA neuronal populations, regardless of their projection targets being the dorsomedial prefrontal cortex (dmPFC) or elsewhere. We further demonstrated that such effects were mainly mediated by the enhanced presynaptic glutamate release in dmPFC-to-BLA synapses but not lateral amygdala-to-BLA ones. Furthermore, while optogenetically weakening the presynaptic glutamate release in dmPFC-to-BLA synapses ameliorated ARS-induced anxiety-like behavior, strengthening the release increased in unstressed mice. Together, these findings suggest that acute stress causes short-lasting increase in anxiety-like behavior by facilitating synaptic transmission from the prefrontal cortex to the amygdala in a circuit-independent fashion.

Intrinsic Excitability in Layer IV-VI Anterior Insula to Basolateral Amygdala Projection Neurons Correlates with the Confidence of Taste Valence Encoding.

Avoiding potentially harmful, and consuming safe food is crucial for the survival of living organisms. However, the perceived valence of sensory information can change following conflicting experiences. Pleasurability and aversiveness are two crucial parameters defining the perceived valence of a taste and can be impacted by novelty. Importantly, the ability of a given taste to serve as the conditioned stimulus (CS) in conditioned taste aversion (CTA) is dependent on its valence. Activity in anterior insula (aIC) Layer IV-VI pyramidal neurons projecting to the basolateral amygdala (BLA) is correlated with and necessary for CTA learning and retrieval, as well as the expression of neophobia toward novel tastants, but not learning taste familiarity. Yet, the cellular mechanisms underlying the updating of taste valence representation in this specific pathway are poorly understood. Here, using retrograde viral tracing and whole-cell patch-clamp electrophysiology in trained mice, we demonstrate that the intrinsic properties of deep-lying Layer IV-VI, but not superficial Layer I-III aIC-BLA neurons, are differentially modulated by both novelty and valence, reflecting the subjective predictability of taste valence arising from prior experience. These correlative changes in the profile of intrinsic properties of LIV-VI aIC-BLA neurons were detectable following both simple taste experiences, as well as following memory retrieval, extinction learning, and reinstatement.

Glucocorticoid receptor, a potential mediator of differential regulation on amygdala neurons by chronic stress

The amygdala, a key target for chronic stress, forms the hub of the brain’s emotional circuits with the medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC). The structural and functional remodeling of the amygdala, especially the basolateral amygdala (BLA), caused by chronic stress is closely related to attendant changes in mood disorders such as anxiety disorders. Our previous study found that chronic stress differentially regulated BLA projection neurons (PNs) in different circuits, as evidenced by the fact that spine density and glutamatergic signaling only in BLA PNs targeting vHPC (BLA→vHPC PNs) were remodeled by chronic restraint stress (CRS), whereas BLA PNs targeting mPFC (BLA→mPFC PNs) and nucleus accumbens (BLA→NAc PNs) were not markedly altered. However, the underlying mechanisms are unclear. These BLA PNs integrated in different neural circuits are in an almost-the-same microenvironment, but triggered different reactions to stress, suggesting that they may have different signal receiving or transmitting mechanisms. Considering the critical role of glucocorticoids in the modulation of brain structure and function by stress, we hypothesize that glucocorticoid receptor (GR) may be a potential mediator of the differential regulation of BLA neurons by chronic stress. In this study, we sought to clarify whether the expression levels of GR on BLA PNs in different circuits and their response patterns to CRS are different. And the results showed that CRS significantly enhanced the GR expression of BLA→vHPC PNs, but had no obvious effect on the GR expression of BLA→mPFC PNs and BLA→NAc PNs. Moreover, the increase of corticosterone concentration in mice caused by prolonged stress only selectively caused the elevated response of GR in BLA→vHPC PNs. Taken together, our findings hint that the GR may be a potential and essential mediator of chronic stress-caused differential regulation on BLA PN.

Amygdala-cortical collaboration in reward learning and decision making.

Adaptive reward-related decision making requires accurate prospective consideration of the specific outcome of each option and its current desirability. These mental simulations are informed by stored memories of the associative relationships that exist within an environment. In this review, I discuss recent investigations of the function of circuitry between the basolateral amygdala (BLA) and lateral (lOFC) and medial (mOFC) orbitofrontal cortex in the learning and use of associative reward memories. I draw conclusions from data collected using sophisticated behavioral approaches to diagnose the content of appetitive memory in combination with modern circuit dissection tools. I propose that, via their direct bidirectional connections, the BLA and OFC collaborate to help us encode detailed, outcome-specific, state-dependent reward memories and to use those memories to enable the predictions and inferences that support adaptive decision making. Whereas lOFC→BLA projections mediate the encoding of outcome-specific reward memories, mOFC→BLA projections regulate the ability to use these memories to inform reward pursuit decisions. BLA projections to lOFC and mOFC both contribute to using reward memories to guide decision making. The BLA→lOFC pathway mediates the ability to represent the identity of a specific predicted reward and the BLA→mOFC pathway facilitates understanding of the value of predicted events. Thus, I outline a neuronal circuit architecture for reward learning and decision making and provide new testable hypotheses as well as implications for both adaptive and maladaptive decision making.

Examining ventral subiculum and basolateral amygdala projections to the nucleus accumbens shell: Differential expression of VGLuT1, VGLuT2 and VGaT in the rat

Projections to the striatum are well-identified. For example, in the ventral striatum, two major inputs to the medial nucleus accumbens shell include the ventral subiculum and basolateral amygdala. However, the chemical phenotype(s) of these projection neurons remain unclear. In this study, we examined amygdalostriatal and corticostriatal connectivity in rats using injections of the retrograde tracer cholera toxin b into the nucleus accumbens shell. To determine the neurotransmitter identity of projection neurons, we combined retrograde tracing with RNAscope in-situ hybridization, using mRNA probes against vesicular transporters associated with glutamatergic (VGluT1 – Slc17a7, VGluT2 – Slc17a6) or GABAergic (VGaT – Slc32a1) neurotransmission. Confocal imaging was used to examine vesicular transporter mRNA expression in the ventral subiculum and basolateral amygdala inputs to the nucleus accumbens shell. Both projections contained mostly VGluT1-expressing neurons. Interestingly, almost a quarter of ventral subiculum to nucleus accumbens shell projections co-expressed VGluT1 and VGluT2 compared to a relatively small number (∼3%) that were co-expressed in basolateral amygdala to nucleus accumbens shell afferents. However, almost a quarter of basolateral amygdala to nucleus accumbens shell projections were VGaT-positive. These findings highlight the diverse proportions of glutamatergic and GABAergic afferents in two major projections to the nucleus accumbens shell and raise important questions for functional studies.

Metabotropic Glutamate Receptor 5 in Amygdala Target Neurons Regulates Susceptibility to Chronic Social Stress

BackgroundMetabotropic glutamate receptor 5 (mGluR5) has been implicated in stress-related psychiatric disorders, particularly major depressive disorder. Although growing evidence supports the proresilient role of mGluR5 in corticolimbic circuitry in the depressive-like behaviors following chronic stress exposure, the underlying neural mechanisms, including circuits and molecules, remain unknown. MethodsWe measured the c-Fos expression and probability of neurotransmitter release in and from basolateral amygdala (BLA) neurons projecting to the medial prefrontal cortex (mPFC) and to the ventral hippocampus (vHPC) after chronic social defeat stress. The role of BLA projections in depressive-like behaviors was assessed using optogenetic manipulations, and the underlying molecular mechanisms of mGluR5 and downstream signaling were investigated by Western blotting, viral-mediated gene transfer, and pharmacological manipulations. ResultsChronic social defeat stress disrupted neural activity and glutamatergic transmission in both BLA projections. Optogenetic activation of BLA projections reversed the detrimental effects of chronic social defeat stress on depressive-like behaviors and mGluR5 expression in the mPFC and vHPC. Conversely, inhibition of BLA projections of mice undergoing subthreshold social defeat stress induced a susceptible phenotype and mGluR5 reduction. These two BLA circuits appeared to act in an independent way. We demonstrate that mGluR5 overexpression in the mPFC or vHPC was proresilient while the mGluR5 knockdown was prosusceptible and that the proresilient effects of mGluR5 are mediated through distinctive downstream signaling pathways in the mPFC and vHPC. ConclusionsThese findings identify mGluR5 in the mPFC and vHPC that receive BLA inputs as a critical mediator of stress resilience, highlighting circuit-specific signaling for depressive-like behaviors.

Early life stress induces anxiety-like behavior during adulthood through dysregulation of neuronal plasticity in the basolateral amygdala

AimsEarly life stress (ELS) increases the risk of psychiatric diseases such as anxiety disorders and depression in later life. Hyperactivation of the basolateral amygdala (BLA) neurons plays a pivotal role in the pathogenesis of stress-related diseases. However, the functional roles of BLA neurons in ELS-induced anxiety disorders are not completely understood. Main methodsMice were subjected to maternal separation (MS) during postnatal days 3 to 21 to mimic ELS. Anxiety-like behavior was tested by open field test (OFT), elevated plus maze (EPM), and novelty suppressed feeding (NSF). Then, c-fos expression, a proxy for neuronal activity, was evaluated by immunofluorescence. Finally, synaptic transmission and intrinsic excitability were measured by whole-cell patch-clamp recordings. Key findingsMS significantly increased anxiety-like behavior in adulthood, as indicated by less time spent in the center area of the OFT, less time spent in and fewer entries to the open arms of the EPM, and increased latency to feed in NSF. Mechanistically, MS increased the expression of c-fos in BLA. MS enhanced the excitatory, but not inhibitory, synaptic transmission onto BLA projection neurons (PNs), which was caused by enhanced presynaptic glutamate release. Moreover, MS also markedly increased the intrinsic neuronal excitability of BLA PNs, probably due to the reduced medium afterhyperpolarization (mAHP) in BLA PNs. SignificanceOur results suggest that the changes of neuronal activity and synaptic transmission in the BLA PNs may play a crucial role in ELS-induced anxiety-like behavior, and these findings provide new insights into the pathological mechanisms of stress-related anxiety disorders.

The Medial Orbitofrontal Cortex–Basolateral Amygdala Circuit Regulates the Influence of Reward Cues on Adaptive Behavior and Choice

Adaptive reward-related decision making requires accurate prospective consideration of the specific outcome of each option and its current desirability. Often this information must be inferred based on the presence of predictive environmental events. The basolateral amygdala (BLA) and medial orbitofrontal cortex (mOFC) are two key nodes in the circuitry supporting such outcome expectations, but very little is known about the function of direct connections between these regions. Here, in male rats, we first anatomically confirmed the existence of bidirectional, direct projections between the mOFC and BLA and found that BLA projections to mOFC are largely distinct from those to lateral OFC (lOFC). Next, using pathway-specific chemogenetic inhibition and the outcome-selective Pavlovian-to-instrumental transfer and devaluation tests, we interrogated the function of the bidirectional mOFC-BLA connections in reward-directed behavior. We found evidence that the mOFC→BLA pathway mediates the use of environmental cues to understand which specific reward is predicted, information needed to infer which action to choose, and how desirable that reward is to ensure adaptive responses to the cue. By contrast, the BLA→mOFC pathway is not needed to use the identity of an expected reward to guide choice but does mediate adaptive responses to cues based on the current desirability of the reward they predict. These functions differ from those we previously identified for the lOFC-BLA circuit. Collectively, the data reveal the mOFC-BLA circuit as critical for the cue-dependent reward outcome expectations that influence adaptive behavior and decision making.SIGNIFICANCE STATEMENT To make good decisions we evaluate how advantageous a particular course of action would be. This requires understanding what rewarding outcomes can be expected and how desirable they currently are. Such prospective considerations are critical for adaptive decision making but disrupted in many psychiatric diseases. Here, we reveal that direct connections between the medial orbitofrontal cortex and basolateral amygdala mediate these functions. These findings are especially important in light of evidence of dysfunction in this circuit in substance use disorder and mental illnesses marked by poor decision making.

A bidirectional corticoamygdala circuit for the encoding and retrieval of detailed reward memories.

Adaptive reward-related decision making often requires accurate and detailed representation of potential available rewards. Environmental reward-predictive stimuli can facilitate these representations, allowing one to infer which specific rewards might be available and choose accordingly. This process relies on encoded relationships between the cues and the sensory-specific details of the rewards they predict. Here, we interrogated the function of the basolateral amygdala (BLA) and its interaction with the lateral orbitofrontal cortex (lOFC) in the ability to learn such stimulus-outcome associations and use these memories to guide decision making. Using optical recording and inhibition approaches, Pavlovian cue-reward conditioning, and the outcome-selective Pavlovian-to-instrumental transfer (PIT) test in male rats, we found that the BLA is robustly activated at the time of stimulus-outcome learning and that this activity is necessary for sensory-specific stimulus-outcome memories to be encoded, so they can subsequently influence reward choices. Direct input from the lOFC was found to support the BLA in this function. Based on prior work, activity in BLA projections back to the lOFC was known to support the use of stimulus-outcome memories to influence decision making. By multiplexing optogenetic and chemogenetic inhibition we performed a serial circuit disconnection and found that the lOFC→BLA and BLA→lOFC pathways form a functional circuit regulating the encoding (lOFC→BLA) and subsequent use (BLA→lOFC) of the stimulus-dependent, sensory-specific reward memories that are critical for adaptive, appetitive decision making.

Calcium-permeable AMPA receptor activity and GluA1 trafficking in the basolateral amygdala regulate operant alcohol self-administration.

Addiction is viewed as maladaptive glutamate-mediated neuroplasticity that is regulated, in part, by calcium-permeable AMPA receptor (CP-AMPAR) activity. However, the contribution of CP-AMPARs to alcohol-seeking behavior remains to be elucidated. We evaluated CP-AMPAR activity in the basolateral amygdala (BLA) as a potential target of alcohol that also regulates alcohol self-administration in C57BL/6J mice. Operant self-administration of sweetened alcohol increased spontaneous EPSC frequency in BLA neurons that project to the nucleus accumbens as compared with behavior-matched sucrose controls indicating an alcohol-specific upregulation of synaptic activity. Bath application of the CP-AMPAR antagonist NASPM decreased evoked EPSC amplitude only in alcohol self-administering mice indicating alcohol-induced synaptic insertion of CP-AMPARs in BLA projection neurons. Moreover, NASPM infusion in the BLA dose-dependently decreased the rate of operant alcohol self-administration providing direct evidence for CP-AMPAR regulation of alcohol reinforcement. As most CP-AMPARs are GluA1-containing, we asked if alcohol alters the activation state of GluA1-containing AMPARs. Immunocytochemistry results showed elevated GluA1-S831 phosphorylation in the BLA of alcohol as compared with sucrose mice. To investigate mechanistic regulation of alcohol self-administration by GluA1-containing AMPARs, we evaluated the necessity of GluA1 trafficking using a TET-ON AAV encoding a dominant-negative GluA1 c-terminus (GluA1ct) that blocks activity-dependent synaptic delivery of native GluA1-containing AMPARs. GluA1ct expression in the BLA reduced alcohol self-administration with no effect on sucrose controls. These results show that CP-AMPAR activity and GluA1 trafficking in the BLA mechanistically regulate the reinforcing effects of sweetened alcohol. Pharmacotherapeutic targeting these mechanisms of maladaptive neuroplasticity may aid medical management of alcohol use disorder.

Sex differences in the Role of Basolateral Amygdala Projections to the Bed Nucleus of the Stria Terminalis in Anticipatory Anxiety

Sustained anxiety in the absence of a threat can lead to pathological manifestations like post traumatic stress disorder (PTSD) and anxiety disorders. Recent data suggest that the bed nucleus of the stria terminalis (BST) is implicated in the shift from a phasic to sustained anxiety state, but the specific mechanisms by which this occurs remains unknown. We developed a model for sustained fear conditioning to a long duration cue (8 min) paired with intermittent foot shocks in order to better understand the neurocircuitry of anticipatory anxiety. Sex differences in the prevalence of anxiety disorders exist and a secondary goal of this work was to examine if sex differences exist in anticipatory anxiety during prolonged cues and the circuitry that contributes to this behavior. One of the major inputs to the BST activated during sustained fear learning is the basolateral amygdala (BLA), which also differs in activity between sexes. To determine a role for BLA projecting BST neurons (BLA-BST) in this sustained anxiety paradigm, we examined DREADD-induced suppression of the BLA-BST pathway during prolonged cued fear conditioning in both male and female rats. In males, inhibition of BLA-BST during extinction hastened the extinction of freezing compared to male controls, but had no effect on freezing behavior in females. Inhibition of BLA-BST did not alter exploration of the central area in the open field task, a test of general anxiety-like behavior, in either sex. Since the BLA-BST pathway also mediates social anxiety-like behavior in humans and animals, we assessed potential sex differences in social interaction (SI) following suppression of this pathway. Inhibition of the BLA-BST pathway increased SI time and reduced the latency to interact in male, but not female rats. Together, these results suggest that the BLA-BST projection does not modulate generalized anxiety-like behavior, but is specific to anticipatory anxiety induced by prolonged cued fear or an unpredictable social environment and differs between sexes. To examine potential sex differences in BLA-BST neurons, we implemented in vivo and in vitro electrophysiological approaches to determine the activity of these neurons. While spontaneous general BLA neuronal activity was augmented in females compared to males, sex differences were noted in the activity of BLA-BST projections. In males, the BLA-BST projection neurons were more active and excitable which coincided with a smaller after hyperpolarization (AHP) amplitude compared to other non-BST-projecting BLA neurons whereas in females, BLA-BST neurons were less excitable and had larger AHP amplitudes compared to non-BST-projecting BLA neurons. These physiological data support our behavioral experiment findings in which increased activity of BLA-BST neurons mediates anticipatory anxiety-like behavior in males, but not females. These novel findings suggest BLA-BST neurons contribute to anticipatory anxiety in male rats, but not females. Moreover, these sex differences in neurocircuitry may explain the sex disparity in anxiety disorders.

Neuroinflammation induces anxiety- and depressive-like behavior by modulating neuronal plasticity in the basolateral amygdala

Increasing evidence indicates that excessive inflammatory responses play a crucial role in the pathophysiology of psychiatric diseases, including depression and anxiety disorders. The dysfunctional neural plasticity in amygdala has long been proposed as the vital cause for the progression of psychiatric disorders. However, the effect of neuroinflammation on the functional changes of the amygdala remains largely unknown. Here, by using a mouse model of inflammation induced by lipopolysaccharide (LPS) injection, we investigated the effect of LPS-induced neuroinflammation on the synaptic and non-synaptic plasticity in basolateral amygdala (BLA) projection neurons (PNs) and their contribution to the LPS-induced anxiety- and depressive-like behavior. The results showed that LPS treatment led to the activation of microglia and production of proinflammatory cytokines in the BLA. Furthermore, LPS treatment increased excitatory but not inhibitory synaptic transmission due to the enhanced presynaptic glutamate release, thus leading to the shift of excitatory/inhibitory balance towards excitatory. In addition, the intrinsic neuronal excitability of BLA PNs was also increased by LPS treatment through the loss of expression and function of small-conductance, calcium-activated potassium channel. Chronic fluoxetine pretreatment significantly prevented these neurophysiological changes induced by LPS, and alleviated anxiety and depressive-like behavior, indicating that LPS-induced neuronal dysregulation of BLA PNs may contribute to the development of psychiatry disorders. Collectively, these findings provide evidence that dysregulation of synaptic and non-synaptic transmission in the BLA PNs may account for neuroinflammation-induced anxiety- and depressive-like behavior.

Chemogenetic Stimulation and Silencing of the Insula, Amygdala, Nucleus Accumbens, and Their Connections Differentially Modulate Alcohol Drinking in Rats.

The anterior insular cortex is hypothesized to represent interoceptive effects of drug reward in the service of goal-directed behavior. The insula is richly connected, but the insula circuitry in addiction remains poorly characterized. We examined the involvement of the anterior insula, amygdala, and nucleus accumbens, as well as the projections of the anterior insula to the central amygdala, basolateral amygdala (BLA), and nucleus accumbens core in voluntary alcohol drinking. We trained alcohol-preferring Alko Alcohol (AA) rats to drink alcohol during intermittent 2-h sessions. We then expressed excitatory or inhibitory designer receptors [designer receptors exclusively activated by designer drugs (DREADDs)] in the anterior insula, nucleus accumbens, or amygdala by means of adenovirus-mediated gene transfer and activated the DREADDs with clozapine-N-oxide (CNO) prior to the drinking sessions. Next, to examine the role of specific insula projections, we expressed FLEX-DREADDs in the efferent insula → nucleus accumbens core, insula → central amygdala, and insula → BLA projections by means of a retrograde AAV-Cre vector injected into the insula projection areas. In the anterior insula and amygdala, excitatory Gq-DREADDs significantly attenuated alcohol consumption. In contrast, in the nucleus accumbens, the Gq-DREADD stimulation increased alcohol drinking, and the inhibitory Gi-DREADDs suppressed it. The Gq-DREADDs expressed in the insula → nucleus accumbens core and insula → central amygdala projections increased alcohol intake, whereas inhibition of these projections had no effect. These data demonstrate that the anterior insula, along with the amygdala and nucleus accumbens, has a key role in controlling alcohol drinking by providing excitatory input to the central amygdala and nucleus accumbens to enhance alcohol reward.