µ-Opioid Receptors Expressed by Intrinsically Photosensitive Retinal Ganglion Cells Contribute to Morphine-Induced Behavioral Sensitization.

Opioid drugs are the most effective tools for treating moderate to severe pain. Despite their analgesic efficacy, long-term opioid use can lead to drug tolerance, addiction, and sleep/wake disturbances. While the link between opioids and sleep/wake problems is well-documented, the mechanism underlying opioid-related sleep/wake problems remains largely unresolved. Importantly, intrinsically photosensitive retinal ganglion cells (ipRGCs), the cells that transmit environmental light/dark information to the brain's sleep/circadian centers to regulate sleep/wake behavior, express µ-opioid receptors (MORs). In this study, we explored the potential contribution of ipRGCs to opioid-related sleep/circadian disruptions. Using implanted telemetry transmitters, we measured changes in horizontal locomotor activity and body temperature in mice over the course of a chronic morphine paradigm. Mice lacking MORs expressed by ipRGCs (McKO) exhibited reduced morphine-induced behavioral activation/sensitization compared with control littermates with normal patterns of MOR expression. Contrastingly, mice lacking MORs globally (MKO) did not acquire morphine-induced locomotor activation/sensitization. Control mice also showed morphine-induced hypothermia in both the light and dark phases, while McKO littermates only exhibited morphine-induced hypothermia in the dark. Interestingly, only control animals appeared to acquire tolerance to morphine's hypothermic effect. Morphine, however, did not acutely decrease the body temperature of MKO mice. These findings support the idea that MORs expressed by ipRGCs could contribute to opioid-related sleep/wake problems and thermoregulatory changes.

Individual arcuate nucleus proopiomelanocortin neurons project to select target sites.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its efferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their efferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct efferent projections may serve as a way for the POMC population to organize its many functions.

Disruption of GABA or glutamate release from POMC neurons in the adult mouse does not affect metabolic end points.

Proopiomelanocortin (POMC) neurons contribute to the regulation of many physiological processes; the majority of which have been attributed to the release of peptides produced from the POMC prohormone such as α-MSH, which plays key roles in food intake and metabolism. However, it is now clear that POMC neurons also release amino acid transmitters that likely contribute to the overall function of POMC cells. Recent work indicates that constitutive deletion of these transmitters can affect metabolic phenotypes, but also that the expression of GABAergic or glutamatergic markers changes throughout development. The goal of the present study was to determine whether the release of glutamate or GABA from POMC neurons in the adult mouse contributes notably to energy balance regulation. Disturbed release of glutamate or GABA specifically from POMC neurons in adult mice was achieved using a tamoxifen-inducible Cre construct (Pomc-CreERT2) expressed in mice also carrying floxed versions of Slc17a6 (vGlut2) or Gad1 and Gad2, encoding the vesicular glutamate transporter type 2 and GAD67 and GAD65 proteins, respectively. All mice in the experiments received tamoxifen injections, but control mice lacked the tamoxifen-inducible Cre sequence. Body weight was unchanged in Gad1- and Gad2- or vGlut2-deleted female and male mice. Additionally, no significant differences in glucose tolerance or refeeding after an overnight fast were observed. These data collectively suggest that the release of GABA or glutamate from POMC neurons in adult mice does not significantly contribute to the metabolic parameters tested here. In light of prior work, the data also suggest that amino acid transmitter release from POMC cells may contribute to separate functions in the adult versus the developing mouse.

Caloric restriction selectively reduces the GABAergic phenotype of mouse hypothalamic proopiomelanocortin neurons.

KEY POINTS: Hypothalamic proopiomelanocortin (POMC) neurons release peptide products that potently inhibit food intake and reduce body weight. These neurons also release the amino acid transmitter GABA, which can inhibit downstream neurons. Although the release of peptide transmitters from POMC neurons is regulated by energy state, whether similar regulation of GABA release might occur had not been examined. The present results show that the GABAergic phenotype of POMC neurons is decreased selectively by caloric deficit and not altered by high-fat diet or stress. The fact the GABAergic phenotype of POMC neurons is sensitive to energy state suggests a dynamic physiological role for this transmitter and highlights the importance of determining the functional consequence of GABA released from POMC neurons in terms of the regulation of normal energy balance. ABSTRACT: In addition to peptide transmitters, hypothalamic neurons, including proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, also release amino acid transmitters that can alter energy balance regulation. While recent studies show that the GABAergic nature of AgRP neurons is increased by caloric restriction, whether the GABAergic phenotype of POMC neurons is also regulated in an energy-state-dependent manner has not been previously examined. The present studies used fluorescence in situ hybridization to detect Gad1 and Gad2 mRNA in POMC neurons, as these encode the glutamate decarboxylase enzymes GAD67 and GAD65, respectively. The results show that both short-term fasting and chronic caloric restriction significantly reduce the percentage of POMC neurons expressing Gad1 mRNA in both male and female mice, with less of an effect on Gad2 expression. Neither acute nor chronic intermittent restraint stress altered Gad1 expression in POMC neurons. Maintenance on a high-fat diet also did not affect the portion POMC neurons expressing Gad1, suggesting that the GABAergic phenotype of POMC neurons is particularly sensitive to energy deficit. Because changes in Gad1 expression have been previously shown to correlate with altered terminal GABA release, fasting is likely to cause a decrease in GABA release from POMC neurons. Altogether, the present results show that the GABAergic nature of POMC neurons can be dynamically regulated by energy state in a manner opposite to that in AgRP neurons and suggest the importance of considering the functional role of GABA release in addition to the peptide transmitters from POMC neurons.

Age-dependent changes in amino acid phenotype and the role of glutamate release from hypothalamic proopiomelanocortin neurons.

Hypothalamic proopiomelanocortin (POMC) neurons are important regulators of energy balance. Recent studies indicate that in addition to their peptides, POMC neurons can release either the amino acid (AA) transmitter gamma-aminobutyric acid (GABA) or glutamate. A small subset of POMC neurons appears to have a dual AA phenotype based on coexpression of mRNA for the vesicular glutamate transporter (vGlut2) and the GABA synthetic enzyme Gad67. To determine whether the colocalization of GABAergic and glutamatergic markers may be indicative of a switch in AA transmitter phenotype, fluorescent in situ hybridization was used to detect vGlut2 and Gad mRNA in POMC neurons during early postnatal development. The percentage of POMC neurons expressing vGlut2 mRNA in POMC neurons progressively decreased from ∼40% at day 1 to less than 10% by 8 weeks of age, whereas Gad67 was only expressed in ∼10% of POMC neurons at day 1 and increased until ∼45% of POMC neurons coexpressed Gad67 at 8 weeks of age. To determine whether the expression of vGlut2 may play a role in energy balance regulation, genetic deletion of vGlut2 in POMC neurons was accomplished using Cre-lox technology. Male, but not female, mice lacking vGlut2 in POMC neurons were unable to maintain energy balance to the same extent as control mice when fed a high-fat diet. Altogether, the results indicate that POMC neurons are largely glutamatergic early in life and that the release of glutamate from these cells is involved in sex- and diet-specific regulation of energy balance.

Relative number and distribution of murine hypothalamic proopiomelanocortin neurons innervating distinct target sites.

Proopiomelanocortin (POMC) neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences.

Proopiomelanocortin expression in both GABA and glutamate neurons.

Proopiomelanocortin (POMC) neurons have been intensively studied because of their essential role in regulating energy balance and body weight. Many effects of POMC neurons can be attributed to their release of cognate neuropeptides from secretory granules in axon terminals. However, these neurons also synaptically release non-peptide neurotransmitters. The aim of this study was to settle the controversy whether there are separate populations of POMC neurons that release GABA or glutamate. Transgenic mice expressing a red fluorescent protein [Discosoma red (DsRed)] driven by Pomc neuronal regulatory elements (POMC-DsRed) were crossed to mice that expressed green fluorescent protein (gfp) in GABAergic neurons (GAD67-gfp). Approximately 40% of POMC neurons in the arcuate nucleus of the double-transgenic mice expressed the GAD67-gfp transgene. In vitro neurotransmitter release was detected using whole-cell electrophysiologic recordings in cultured GAD67-gfp-positive and GAD67-gfp-negative POMC neurons that had formed recurrent synapses (autapses). Autapses from GAD67-gfp-positive neurons were uniformly GABAergic. In contrast, autapses from the GAD67-gfp-negative POMC neurons exclusively exhibited postsynaptic currents mediated by glutamate. Together, these results indicate that there are two subpopulations of POMC neurons in the arcuate nucleus differentiated by their amino acid neurotransmitter phenotype. Whole-cell voltage-clamp recordings from POMC neurons in live brain slices indicated that GABAergic and glutamatergic POMC neurons are under similar presynaptic and postsynaptic regulation, although the GABAergic POMC neurons are smaller and have higher input resistance. GABAergic and glutamatergic POMC neurons may mediate distinct aspects of POMC neuron function, including the regulation of energy homeostasis.