Amylin Modulates a Ventral Tegmental Area-to-Medial Prefrontal Cortex Circuit to Suppress Food Intake and Impulsive Food-Directed Behavior.

BACKGROUND: A better understanding of the neural mechanisms regulating impaired satiety to palatable foods is essential to treat hyperphagia linked with obesity. The satiation hormone amylin signals centrally at multiple nuclei including the ventral tegmental area (VTA). VTA-to-medial prefrontal cortex (mPFC) projections encode food reward information to influence behaviors including impulsivity. We hypothesized that modulation of VTA-to-mPFC neurons underlies amylin-mediated decreases in palatable food-motivated behaviors. METHODS: We used a variety of pharmacological, behavioral, genetic, and viral approaches (n = 4-16/experiment) to investigate the anatomical and functional circuitry of amylin-controlled VTA-to-mPFC signaling in rats. RESULTS: To first establish that VTA amylin receptor (calcitonin receptor) activation can modulate mPFC activity, we showed that intra-VTA amylin decreased food-evoked mPFC cFos. VTA amylin delivery also attenuated food-directed impulsive behavior, implicating VTA amylin signaling as a regulator of mPFC functions. Palatable food activates VTA dopamine and mPFC neurons. Accordingly, dopamine receptor agonism in the mPFC blocked the hypophagic effect of intra-VTA amylin, and VTA amylin injection reduced food-evoked phasic dopamine levels in the mPFC, supporting the idea that VTA calcitonin receptor activation decreases dopamine release in the mPFC. Surprisingly, calcitonin receptor expression was not found on VTA-to-mPFC projecting neurons but was instead found on GABAergic (gamma-aminobutyric acidergic) interneurons in the VTA that provide monosynaptic inputs to this pathway. Blocking intra-VTA GABA signaling, through GABA receptor antagonists and DREADD (designer receptor exclusively activated by designer drugs)-mediated GABAergic neuronal silencing, attenuated intra-VTA amylin-induced hypophagia. CONCLUSIONS: These results indicate that VTA amylin signaling stimulates GABA-mediated inhibition of dopaminergic projections to the mPFC to mitigate impulsive consumption of palatable foods.

Liposaccharide-induced sustained mild inflammation fragments social behavior and alters basolateral amygdala activity.

RATIONALE: Conditions with sustained low-grade inflammation have high comorbidity with depression and anxiety and are associated with social withdrawal. The basolateral amygdala (BLA) is critical for affective and social behaviors and is sensitive to inflammatory challenges. Large systemic doses of lipopolysaccharide (LPS) initiate peripheral inflammation, increase BLA neuronal activity, and disrupt social and affective measures in rodents. However, LPS doses commonly used in behavioral studies are high enough to evoke sickness syndrome, which can confound interpretation of amygdala-associated behaviors. OBJECTIVES AND METHODS: The objectives of this study were to find a LPS dose that triggers mild peripheral inflammation but not observable sickness syndrome in adult male rats, to test the effects of sustained mild inflammation on BLA and social behaviors. To accomplish this, we administered single doses of LPS (0-100 µg/kg, intraperitoneally) and measured open field behavior, or repeated LPS (5 µg/kg, 3 consecutive days), and measured BLA neuronal firing, social interaction, and elevated plus maze behavior. RESULTS: Repeated low-dose LPS decreased BLA neuron firing rate but increased the total number of active BLA neurons. Repeated low-dose LPS also caused early disengagement during social bouts and less anogenital investigation and an overall pattern of heightened social caution associated with reduced gain of social familiarity over the course of a social session. CONCLUSIONS: These results provide evidence for parallel shifts in social interaction and amygdala activity caused by prolonged mild inflammation. This effect of inflammation may contribute to social symptoms associated with comorbid depression and chronic inflammatory conditions.

The medial orbitofrontal cortex governs reward-related circuits in an age-dependent manner.

Nucleus accumbens (NAc) neurons integrate excitatory inputs from cortical and limbic structures, contributing to critical cognitive functions, including decision-making. As these afferents mature from adolescence through adulthood, incoming signals to the NAc may summate differently between age groups. Decision-making evaluates both reward and risk before action selection, suggesting an interplay between reward- and risk-related circuits. Medial orbitofrontal cortex (MO)-NAc circuits permit risk assessment behaviors and likely underlie risk information incorporation. As adolescents make reward-centric choices regardless of risk, we hypothesized the impact of MO activity alters reward-related NAc circuits in an age-dependent manner. To test this hypothesis, we used single-unit electrophysiology to measure MO train stimulation's effect on reward-related pathways, specifically the basolateral amygdala (BLA)-NAc circuit, in adult and adolescent rats. MO train stimulation altered the strength but not the timing of BLA-NAc interactions in a frequency-dependent manner. In adults, MO train stimulation produced a frequency-dependent, bidirectional effect on BLA-evoked NAc AP probability. Contrastingly, MO train stimulation uniformly attenuated BLA-NAc interactions in adolescents. While the mature MO can govern reward-related circuits in an activity-dependent manner, perhaps to adapt to positive or negative decision-making outcomes, the adolescent MO may be less able to bidirectionally impact reward-related pathways resulting in biased decision-making.

Medial orbitofrontal cortex and nucleus accumbens mediation in risk assessment behaviors in adolescents and adults.

Risk assessment behaviors are necessary for gathering risk information and guiding decision-making. Risky decision-making heightens during adolescence, possibly as a result of low risk awareness and an increase in sensitivity to reward-associated cues and experiences. Higher adolescent engagement in high-risk behaviors may be, in part, due to developing circuits that contribute to risk assessment behaviors. Nucleus accumbens (NAc) activity is linked to risky decision-making and receives inputs carrying sensory and emotional information. Namely, the medial orbitofrontal cortex (MO) contributes to behavior guided by reward probability and sends direct projections to the NAc (MO→NAc), which may permit risk assessment in a mature circuit. Here, we evaluated risk assessment behaviors in adult and adolescent rats during elevated plus maze (EPM) exploration, including stretch and attend postures, head dips, and rears. We found that adolescents exhibited fewer EPM risk assessment behaviors than adults. We also quantified MO→NAc projections using a fluorescent anterograde tracer, Fluoro-Ruby, in both age groups. Labeled MO→NAc pathways exhibited greater total fluorescence in adults than in adolescents, indicating MO→NAc fibers increase over development. Using a disconnection approach to measure the contribution of the MO-NAc pathway in adults, we found that ipsilateral inactivation of the MO-NAc did not alter risk assessment behavior; however, MO-NAc disconnection reduced the number of stretch-and-attend postures. Together, this work suggests that the development of MO-NAc pathways can contribute to age-dependent differences in risk assessment.

Shifts in Medial Orbitofrontal Cortex Activity from Adolescence to Adulthood.

Adolescents are characterized by a propensity for risky and impulsive behaviors, likely due to immature frontostriatal circuits. The medial orbitofrontal cortex (MO) is linked to risk and reward prediction during decision-making. Identifying age-dependent differences in MO activity and its inputs to downstream regions can elucidate the neural substrates that permit the transition from high-risk adolescent behaviors to increased risk assessment in adulthood. Action selection biased by information gathered by the MO is likely carried out by efferents into the nucleus accumbens (NAc), which guides reward-directed behaviors. Despite the large age dependency of risk-based decision-making, there is nothing known about adolescent MO activity. Here, we recorded action potentials of MO neurons from anesthetized adult and adolescent rats in vivo. On average, adolescent MO neurons fire faster and within narrower ranges than adults, and adolescents have more active MO neurons than adults. Using antidromic stimulation of axon terminals to identify MO neurons that project to NAc (MO→NAc), we found that adolescent MO→NAc neurons have a narrower range of firing frequencies than non-NAc-projecting MO neurons and adult MO→NAc neurons. These age-dependent differences in MO and MO→NAc populations may result from the fine-tuning of circuits between adolescence and adulthood that promote specific age-dependent behaviors.

Developmental Shifts in Amygdala Activity during a High Social Drive State.

Amygdala abnormalities characterize several psychiatric disorders with prominent social deficits and often emerge during adolescence. The basolateral amygdala (BLA) bidirectionally modulates social behavior and has increased sensitivity during adolescence. We tested how an environmentally-driven social state is regulated by the BLA in adults and adolescent male rats. We found that a high social drive state caused by brief social isolation increases age-specific social behaviors and increased BLA neuronal activity. Chemogenetic inactivation of BLA decreased the effect of high social drive on social engagement. High social drive preferentially enhanced BLA activity during social engagement; however, the effect of social opportunity on BLA activity was greater during adolescence. While this identifies a substrate underlying age differences in social drive, we then determined that high social drive increased BLA NMDA GluN2B expression and sensitivity to antagonism increased with age. Further, the effect of a high social drive state on BLA activity during social engagement was diminished by GluN2B blockade in an age-dependent manner. These results demonstrate the necessity of the BLA for environmentally driven social behavior, its sensitivity to social opportunity, and uncover a maturing role for BLA and its GluN2B receptors in social engagement.SIGNIFICANCE STATEMENT Social engagement during adolescence is a key component of healthy development. Social drive provides the impetus for social engagement and abnormalities underlie social symptoms of depression and anxiety. While adolescence is characterized by transitions in social drive and social environment sensitivity, little is known about the neural basis for these changes. We found that amygdala activity is uniquely sensitive to social environment during adolescence compared with adulthood, and is required for expression of heightened social drive. In addition, the neural substrates shift toward NMDA dependence in adulthood. These results are the first to demonstrate a unique neural signature of higher social drive and begin to uncover the underlying factors that heighten social engagement during adolescence.

Fear Learning Enhances Prefrontal Cortical Suppression of Auditory Thalamic Inputs to the Amygdala in Adults, but Not Adolescents.

Adolescence is characterized by increased susceptibility to the development of fear- and anxiety-related disorders. Adolescents also show elevated fear responding and aversive learning that is resistant to behavioral interventions, which may be related to alterations in the circuitry supporting fear learning. These features are linked to ongoing adolescent development of medial prefrontal cortical (PFC) inputs to the basolateral amygdala (BLA) that regulate neural activity and contribute to the refinement of fear responses. Here, we tested the hypothesis that the extent of PFC inhibition of the BLA following fear learning is greater in adults than in adolescents, using anesthetized in vivo recordings to measure local field potentials (LFPs) evoked by stimulation of PFC or auditory thalamic (MgN) inputs to BLA. We found that BLA LFPs evoked by stimulation of MgN inputs were enhanced in adults following fear conditioning. Fear conditioning also led to reduced summation of BLA LFPs evoked in response to PFC train stimulation, and increased the capacity of PFC inhibition of MgN inputs in adults. These data suggest that fear conditioning recruits additional inhibitory capacity by PFC inputs to BLA in adults, but that this capacity is weaker in adolescents. These results provide insight into how the development of PFC inputs may relate to age differences in memory retention and persistence following aversive learning.

Repeated stress induces a pro-inflammatory state, increases amygdala neuronal and microglial activation, and causes anxiety in adult male rats.

A link exists between immune function and psychiatric conditions, particularly depressive and anxiety disorders. Psychological stress is a powerful trigger for these disorders and stress influences immune state. However, the nature of peripheral immune changes after stress conflicts across studies, perhaps due to the focus on few measures of pro-inflammatory or anti-inflammatory processes. The basolateral amygdala (BLA) is critical for emotion, and plays an important role in the effects of stress on anxiety. As such, it may be a primary central nervous system (CNS) mediator for the effects of peripheral immune changes on anxiety after stress. Therefore, this study aimed to delineate the influence of stress on peripheral pro-inflammatory and anti-inflammatory aspects, BLA immune activation, and its impact on BLA neuronal activity. To produce a more encompassing view of peripheral immune changes, this study used a less restrictive approach to categorize and group peripheral immune changes. We found that repeated social defeat stress in adult male Sprague-Dawley rats increased the frequencies of mature T-cells positive for intracellular type 2-like cytokine and serum pro-inflammatory cytokines. Principal component analysis and hierarchical clustering was used to guide grouping of T-cells and cytokines, producing unique profiles. Stress shifted the balance towards a specific set that included mostly type 2-like T-cells and pro-inflammatory cytokines. Within the CNS component, repeated stress caused an increase of activated microglia in the BLA, increased anxiety-like behaviors across several assays, and increased BLA neuronal firing in vivo that was prevented by blockade of microglia activation. Because repeated stress can trigger anxiety states by actions in the BLA, and altered immune function can trigger anxiety, these results suggest that repeated stress may trigger anxiety-like behaviors by inducing a pro-inflammatory state in the periphery and the BLA. These results begin to uncover how stress may recruit the immune system to alter the function of brain regions critical to emotion.