Elevated branched-chain amino acids (BCAAs) are associated with obesity and insulin resistance. How long-term dietary BCAAs impact late-life health and lifespan is unknown. Here, we show that when dietary BCAAs are varied against a fixed, isocaloric macronutrient background, long-term exposure to high BCAA diets leads to hyperphagia, obesity and reduced lifespan. These effects are not due to elevated BCAA per se or hepatic mammalian target of rapamycin activation, but instead are due to a shift in the relative quantity of dietary BCAAs and other amino acids, notably tryptophan and threonine. Increasing the ratio of BCAAs to these amino acids results in hyperphagia and is associated with central serotonin depletion. Preventing hyperphagia by calorie restriction or pair-feeding averts the health costs of a high-BCAA diet. Our data highlight a role for amino acid quality in energy balance and show that health costs of chronic high BCAA intakes need not be due to intrinsic toxicity but instead are a consequence of hyperphagia driven by amino acid imbalance.

Fear and emotional learning are modulated by endogenous opioids but the cellular basis for this is unknown. The intercalated cells (ITCs) gate amygdala output and thus regulate the fear response. Here we find endogenous opioids are released by synaptic stimulation to act via two distinct mechanisms within the main ITC cluster. Endogenously released opioids inhibit glutamate release through the δ-opioid receptor (DOR), an effect potentiated by a DOR-positive allosteric modulator. Postsynaptically, the opioids activate a potassium conductance through the μ-opioid receptor (MOR), suggesting for the first time that endogenously released opioids directly regulate neuronal excitability. Ultrastructural localization of endogenous ligands support these functional findings. This study demonstrates a new role for endogenously released opioids as neuromodulators engaged by synaptic activity to regulate moment-to-moment neuronal communication and excitability. These distinct actions through MOR and DOR may underlie the opposing effect of these receptor systems on anxiety and fear.

Endogenous opioid peptides in the amygdala regulate many of our behaviors and emotional responses. In particular, the endogenous opioid enkephalin plays a significant role in regulating amygdala activity, but its action is strongly limited by peptidases, which degrade enkephalin into inactive fragments. Inhibiting peptidases may be an attractive method to enhance endogenous opioid signaling; however, we do not know which specific peptidase(s) to target. Using inhibition of glutamate release onto the intercalated cells of the amygdala as an assay for enkephalin activity, we applied specific peptidase inhibitors to determine which peptidase(s) regulate enkephalin signaling in this region. Thiorphan (10 mM), captopril (1 mM), or bestatin (10 mM) were used to inhibit the activity of neprilysin, angiotensin-converting enzyme, or aminopeptidase N, respectively. In rat brain slices containing the intercalated cells, we found that inhibition of glutamate release by a submaximal concentration of enkephalin was doubled by application of all three peptidase inhibitors combined. Then, we tested inhibitors individually and found that inhibition of neprilysin alone could enhance enkephalin responses to the same extent as inhibitors of all three peptidases combined. This indicates neprilysin is the predominant peptidase responsible for degrading enkephalins in the intercalated cells of the amygdala. This differs from the striatum, locus coeruleus, and spinal cord, where multiple peptidases metabolize enkephalin. These data highlight the importance of knowing which specific peptidase(s) control opioid actions in the relevant neural circuit and how they change in disease states to allow rational choices of drugs targeting the specific peptidase of interest. SIGNIFICANCE STATEMENT Endogenous opioids modulate many of our emotional and behavioral responses. In the amygdala, they modulate our pain, fear, and addictive behaviors. Their actions are terminated when they are catabolized into inactive fragments by at least three different peptidases. In this study, we found that neprilysin selectively controls endogenous opioid concentrations at synapses in the intercalated cells of the amygdala. This peptidase may be a target for regulation of endogenous opioid modulation of amygdala-mediated emotional and behavioral responses.

Neural circuits in the amygdala are important for associating the positive experience of drug taking with the coincident environmental cues. During abstinence, cue re-exposure activates the amygdala, increases dopamine release in the amygdala and stimulates relapse to drug use in an opioid dependent manner. Neural circuits in the amygdala and the learning that underlies these behaviours are inhibited by GABAergic synaptic inhibition. A specialised subtype of GABAergic neurons in the amygdala are the clusters of intercalated cells. We focussed on the main-island of intercalated cells because these neurons, located ventromedial to the basolateral amygdala, express very high levels of dopamine D1-receptor and μ-opioid receptor, release enkephalin and are densely innervated by the ventral tegmental area. However, where these neurons project to was not fully described and their regulation by opioids and dopamine was incomplete. To address this issue we electrically stimulated in the main-island of the intercalated cells in rat brain slices and made patch-clamp recordings of GABAergic synaptics from amygdala neurons. We found that main-island neurons had a strong GABAergic inhibitory output to pyramidal neurons of the basolateral nucleus and the medial central nucleus, the major output zones of the amygdala. Opioids inhibited both these synaptic outputs of the intercalated neurons and thus would disinhibit these target zones. Additionally, dopamine acting at D1-receptors inhibited main-island neuron synapses onto other main-island neurons. This data indicates that the inhibitory projections from the main-island neurons could influence multiple aspects of addiction and emotional processing in an opioid and dopamine dependent manner.

While the physical signs of opioid withdrawal are most readily observable, withdrawal insidiously drives relapse, and contributes to compulsive drug use, by disrupting brain emotional learning circuits. How these circuits become disrupted during withdrawal is poorly understood. Because amygdala neurons mediate relapse, and are highly opioid-sensitive, we hypothesised that opioid withdrawal would induce adaptations in these neurons, opening a window of disrupted emotional learning circuit function. Under normal physiological conditions, synaptic transmission between the basolateral amygdala (BLA) and the neighbouring main island (Im) of GABAergic intercalated cells (ITCs) is strongly inhibited by endogenous opioids. Using patch-clamp electrophysiology in brain slices prepared from male rats, we reveal that opioid withdrawal abruptly reduces the ability of these peptides to inhibit neurotransmission – a direct consequence of a protein kinase A-driven increase in the synaptic activity of peptidases. Reduced peptide control of neurotransmission in the amygdala shifts the excitatory/inhibitory balance of inputs onto accumbens-projecting amygdala cells involved in relapse. These findings provide novel insights into how peptidases control synaptic activity within the amygdala and presents restoration of endogenous peptide activity during withdrawal as a viable option to mitigate withdrawal-induced disruptions in emotional learning circuits and rescue the relapse behaviours exhibited during opioid withdrawal and beyond into abstinence.SIGNIFICANCE STATEMENT:We find that opioid withdrawal dials down inhibitory neuropeptide activity in the amygdala. This disrupts both GABAergic and glutamatergic transmission through amygdala circuits, including reward-related outputs to the nucleus accumbens. This likely disrupts peptide-dependent emotional learning processes in the amygdala during withdrawal and may direct behaviour towards compulsive drug use.