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.

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse.

Multiple inhibitory G-protein-coupled receptors resist acute desensitization in the presynaptic but not postsynaptic compartments of neurons.

Acute desensitization is a common property of G(i/o)-coupled receptors. Recent data, however, suggest that, unlike µ-opioid receptors (MORs) located somatodendritically in neurons or expressed in heterologous systems, MORs in the presynaptic compartment of neurons are resistant to acute desensitization. It is not yet clear whether this differential desensitization is a shared property of many G(i/o)-coupled receptors nor whether receptors located presynaptically and postsynaptically in a single cell type display differential desensitization. Here, whole-cell recordings were made from proopiomelanocortin (POMC) neurons in mouse brain slices. Agonists for µ-opioid, nociceptin, and GABA(B) receptors induced postsynaptic currents that desensitized within minutes, whereas inhibition of presynaptic transmitter release mediated by these receptors was maintained throughout agonist exposure. Expression of channelrhodopsin2 in POMC neurons allowed for light-evoked transmitter release from POMC neuron terminals, which was detected by recording postsynaptic currents in downstream neurons. Light-evoked currents were inhibited throughout the application of all agonists tested. Thus, the same receptors that desensitize when expressed in the postsynaptic compartment of POMC neurons resist desensitization when located in the presynaptic compartment. Pharmacologic knockdown of MORs revealed that depletion of receptor reserve does not account for presynaptic resistance to desensitization. In ∼25% of recordings with GABA(B) agonist application, presynaptic GABA(B) receptors desensitized, suggesting that resistance to desensitization is not due to an intrinsic property of the terminals themselves. Together, the results indicate that a variety of presynaptic receptors can continue to function after their postsynaptic counterparts desensitize and suggest that a compartment-specific modification may confer resistance to desensitization.

Regulation of GABA and glutamate release from proopiomelanocortin neuron terminals in intact hypothalamic networks.

Hypothalamic proopiomelanocortin (POMC) neurons and their peptide products mediate important aspects of energy balance, analgesia, and reward. In addition to peptide products, there is evidence that POMC neurons can also express the amino acid transmitters GABA and glutamate, suggesting these neurons may acutely inhibit or activate downstream neurons. However, the release of amino acid transmitters from POMC neurons has not been thoroughly investigated in an intact system. In the present study, the light-activated cation channel channelrhodopsin-2 (ChR2) was used to selectively evoke transmitter release from POMC neurons. Whole-cell electrophysiologic recordings were made in brain slices taken from POMC-Cre transgenic mice that had been injected with a viral vector containing a floxed ChR2 sequence. Brief pulses of blue light depolarized POMC-ChR2 neurons and induced the release of GABA and glutamate onto unidentified neurons within the arcuate nucleus, as well as onto other POMC neurons. To determine whether the release of GABA and glutamate from POMC terminals can be readily modulated, opioid and GABA(B) receptor agonists were applied. Agonists for µ- and κ-, but not δ-opioid receptors inhibited transmitter release from POMC neurons, as did the GABA(B) receptor agonist baclofen. This regulation indicates that opioids and GABA released from POMC neurons may act at presynaptic receptors on POMC terminals in an autoregulatory manner to limit continued transmission. The results show that, in addition to the relatively slow and long-lasting actions of peptides, POMC neurons can rapidly affect the activity of downstream neurons via GABA and glutamate release.