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

Eur J Neurosci. 2015 Nov;42(9):2644-53. doi: 10.1111/ejn.13076. Epub 2015 Oct 19.

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.

Keywords: AgRP; in situ hybridization; mouse; proopiomelanocortin; readily releasable pool.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agouti-Related Protein / metabolism
  • Animals
  • Fasting / metabolism
  • Female
  • Glutamate Decarboxylase / metabolism*
  • Hypothalamus / enzymology*
  • Hypothalamus / metabolism
  • Hypothalamus / physiology
  • Inhibitory Postsynaptic Potentials*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Neurons / enzymology*
  • Neurons / metabolism
  • Neurons / physiology
  • Neuropeptide Y / metabolism
  • Pro-Opiomelanocortin / metabolism
  • RNA, Messenger / metabolism
  • gamma-Aminobutyric Acid / metabolism*

Substances

  • Agouti-Related Protein
  • Neuropeptide Y
  • RNA, Messenger
  • gamma-Aminobutyric Acid
  • Pro-Opiomelanocortin
  • Glutamate Decarboxylase
  • glutamate decarboxylase 1