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

Abstract

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.

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

“…Thus, the expression of Gad1 can be a reliable indicator of terminal GABA release, as was recently shown to be the case in AgRP neurons (Dicken et al . ). In AgRP neurons, fasting increased the expression of Gad1 and enhanced the readily‐releasable pool of GABA from AgRP neurons, whereas the inhibition of GAD activity decreased GABA release.…”

“…One study did examine GABA release from AgRP neurons in response to caloric restriction and found that Gad1 mRNA and GABA release was increased under this condition, which correlated well with increased GABA release from these cells (Dicken et al . ). This is consistent with the expected general increase in AgRP neuron activity when the animal is in a state of hunger (Hahn et al .…”

“…The finding that Gad1 is especially sensitive to changes in physiological state was also found for hypothalamic AgRP neurons where Gad1 mRNA expression was increased in response to fasting, without altering the percentage of AgRP neurons expressing Gad2 (Dicken et al . ).…”

“…; Dicken et al . ), which is primarily regulated by the activity of the GABA synthetic enzyme glutamate decarboxylase (67 kDa isoform) (GAD67; encoded by Gad1 mRNA; Lau & Murthy, ). Although both GAD67 and glutamate decarboxylase (65 kDa isoform) (GAD65; encoded by Gad2 mRNA) can produce GABA from the decarboxylation of glutamate, GAD67 is responsible for over 90% of brain GABA levels and is indispensable for most synaptic GABA transmission (Asada et al .…”

In Utero One‐Carbon Metabolism Interplay and Metabolic Syndrome in Cardiovascular Disease Risk Reduction

“…Liver [74] Sprague-Dawley rats [11,14] is in turn involved in appetite regulation. [88,89] Also, offspring of rat dams fed methyl (folate and vitamin B 12 )-deficient diets presented with NAFLD following postnatal consumption of HFD. [90] Gene transcription analysis revealed a significant increase in the expression of nuclear factor B (NF B) and TGF-1.…”