Probes for INS

Cue-reward associations form distinct memories that can drive appetitive behaviors and are involved in craving for both drugs and natural rewards. Distinct sets of neurons, so called neuronal ensembles, in the infralimbic area (IL) of the medial prefrontal cortex play a key role in alcohol seeking. Whether this ensemble is specific for alcohol or controls reward seeking in general remains unclear. Here, we compared IL ensembles formed upon recall of drug (alcohol) or natural reward (saccharin) memories in male Wistar rats. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal, we found that cue-induced seeking of either alcohol or saccharin activated ensembles of similar size and organization, whereby these ensembles consist of largely overlapping neuronal populations. Thus, the IL seems to act as a general integration hub for reward seeking behavior, but also contains subsets of neurons that encode for the different rewards.

SIGNIFICANCE STATEMENT

Cue-reward associations form distinct memories that can act as drivers of appetitive behaviors and are involved in craving for natural rewards as well as for drugs. Distinct sets of neurons, so called neuronal ensembles, in the infralimbic area of the medial prefrontal cortex play a key role in cue-triggered reward seeking. However, it is unclear whether these ensembles act as broadly tuned controllers of approach behavior or represent the learned associations between specific cues and rewards. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal we find largely overlapping neuronal populations. Repeated activation by two distinct events could reflect the linking of the two memory traces within the same neuron.

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via . GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In the present study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse cross-breeding strategy revealed that fewer than 1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not utilize GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Negative affect is a core symptom domain associated with an array of neurological and psychiatric disorders and is only partially targeted by current therapies, highlighting the need for better, more targeted treatment options. This study focuses on negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by forced abstinence (CDFA), prolonged alcohol abstinence increased c-fos expression and spontaneous glutamatergic neurotransmission in the dorsal bed nucleus of the stria terminalis (dBNST), a region heavily implicated in negative affect in both humans and rodents. Further, pharmacologically enhancing eCBs with JZL184 prevents abstinence-induced increases in dBNST neuronal activity, underscoring the therapeutic potential of drugs targeting the brain's eCB system. Next, we used a channelrhodopsin-assisted mapping strategy to identify excitatory inputs to the dBNST that could contribute to CDFA-induced negative affect. We identified the insular cortex (insula), a region involved in regulating interoception, as a dense, functional, endocannabinoid-sensitive input to the dBNST. Using a chemogenetic strategy to locally mimic eCB signaling, we demonstrate that the insula strongly influences CDFA behavioral and BNST neuronal activity. Lastly, we used viral anterograde transsynaptic expression in combination with a Gq-DREADD to selectively recruit dBNST neurons receiving insula projections. Chemogenetic recruitment of these neurons mimicked behavioral and c-fos responses observed in CDFA. Collectively, this study supports a role for the insula-BNST neural circuit in negative affective disturbances and highlights the therapeutic potential of the endocannabinoid system for treating negative affective disorders.

Stress contributes to numerous psychiatric disorders. CRF signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and β-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α2A-AR is a Gi-linked GPCR, we assessed the impact of activating the Gi-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, utilizing Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the co-expression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.SIGNIFICANCE STATEMENTStress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well-tolerated α2A-AR agonist guanfacine reduces activity of these cells in vivo, and reduces excitatory PBN inputs onto these cells ex vivo Additionally, we uncover a novel sex-dependent co-expression of Prkcd with Crh in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between NE and CRF, and point to an action by which guanfacine may reduce negative affective responses.

Objective

Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism.

Methods

Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1r^loxP/loxP mice and control littermates.

Results

Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture.

Conclusion

These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.