Probes for INS

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive PACAP knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in wild-type mice that do not occur in knock-out mice (aPRGs). Comparing constitutive PACAP knock-out mice to a variety of temporally and regionally specific PACAP knock-outs, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knock-out mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Behaviors associated with distress can affect the anxiety-like states in observers and this social transfer of affect shapes social interactions among stressed individuals. We hypothesized that social reactions to stressed individuals engage the serotonergic dorsal raphe nucleus (DRN) which promotes anxiety-like behavior via postsynaptic action of serotonin at serotonin 2C (5-HT2C) receptors in the forebrain. First, we inhibited the DRN by administering an agonist (8-OH-DPAT, 1 μg in 0.5 μL) for the inhibitory 5-HT1A autoreceptors which silences 5-HT neuronal activity. 8-OH-DPAT prevented the approach and avoidance, respectively, of stressed juvenile (PN30) or stressed adult (PN60) conspecifics in the social affective preference (SAP) test in rats. Similarly, systemic administration of a 5-HT2C receptor antagonist (SB242084, 1 mg/kg, i.p.) prevented approach and avoidance of stressed juvenile or adult conspecifics, respectively. Seeking a locus of 5-HT2C action, we considered the posterior insular cortex which is critical for social affective behaviors and rich with 5-HT2C receptors. SB242084 administered directly into the insular cortex (5 μM in 0.5 μL bilaterally) interfered with the typical approach and avoidance behaviors observed in the SAP test. Finally, using fluorescent in situ hybridization, we found that 5-HT2C receptor mRNA (htr2c) is primarily colocalized with mRNA associated with excitatory glutamatergic neurons (vglut1) in the posterior insula. Importantly, the results of these treatments were the same in male and female rats. These data suggest that interactions with stressed others require the serotonergic DRN and that serotonin modulates social affective decision-making via action at insular 5-HT2C receptors.

Social interaction allows for the transfer of affective states among individuals, and the behaviors and expressions associated with pain and fear can evoke anxiety-like states in observers which shape subsequent social interactions. We hypothesized that social reactions to stressed individuals engage the serotonergic dorsal raphe nucleus (DRN) which promotes anxiety-like behavior via postsynaptic action of serotonin at serotonin 2C (5-HT 2C ) receptors in the forebrain. First, we inhibited the DRN by administering an agonist (8-OH-DPAT, 1µg in 0.5µL) for the inhibitory 5-HT 1A autoreceptors which silences 5-HT neuronal activity via G-protein coupled inward rectifying potassium channels. 8-OH-DPAT prevented the approach and avoidance, respectively, of stressed juvenile (PN30) or stressed adult (PN50) conspecifics in the social affective preference (SAP) test in rats. Similarly, systemic administration of a 5-HT 2C receptor antagonist (SB242084, 1mg/kg, i.p.) prevented approach and avoidance of stressed juvenile or adult conspecifics, respectively. Seeking a locus of 5-HT 2C action, we considered the posterior insular cortex which is critical for social affective behaviors and rich with 5-HT 2C receptors. SB242084 administered directly into the insular cortex (5µM bilaterally in 0.5µL ) interfered with the typical approach and avoidance behaviors observed in the SAP test. Finally, using fluorescent in situ hybridization, we found that 5-HT 2C receptor mRNA ( htr2c) is primarily colocalized with mRNA associated with excitatory glutamatergic neurons ( vglut1 ) in the posterior insula. Importantly, the results of these treatments were the same in male and female rats. These data suggest that interactions with stressed others require the serotonergic DRN and that serotonin modulates social affective decision-making via action at insular 5-HT 2C receptors.

Dopamine acts on neurons in the arcuate nucleus (ARC) of the hypothalamus, which controls homeostatic feeding responses. Here we demonstrate a differential enrichment of dopamine receptor 1 (Drd1) expression in food intake-promoting agouti related peptide (AgRP)/neuropeptide Y (NPY) neurons and a large proportion of Drd2-expressing anorexigenic proopiomelanocortin (POMC) neurons. Owing to the nature of these receptors, this translates into a predominant activation of AgRP/NPY neurons upon dopamine stimulation and a larger proportion of dopamine-inhibited POMC neurons. Employing intersectional targeting of Drd2-expressing POMC neurons, we reveal that dopamine-mediated POMC neuron inhibition is Drd2 dependent and that POMCDrd2+ neurons exhibit differential expression of neuropeptide signaling mediators compared with the global POMC neuron population, which manifests in enhanced somatostatin responsiveness of POMCDrd2+ neurons. Selective chemogenetic activation of POMCDrd2+ neurons uncovered their ability to acutely suppress feeding and to preserve body temperature in fasted mice. Collectively, the present study provides the molecular and functional characterization of POMCDrd2+ neurons and aids our understanding of dopamine-dependent control of homeostatic energy-regulatory neurocircuits.