GAD mRNA in Orbital Prefrontal Cortex and Anterior Cingulate Cortex in Alcoholics Compared with Nonpsychiatric Controls: A Negative Postmortem Study

Abstract

Alcohol increases inhibitory neurotransmission, an effect mediated through GABA receptors. With chronic alcohol exposure, the inhibitory effects diminish. Glutamic acid decarboxylase (GAD) catalyzes glutamate in the synthesis of GABA. We sought to determine the amount of GAD_65/67 mRNA in anterior cingulate cortex (BA24) and orbital prefrontal cortex (BA45) of medication-free alcoholics and nonpsychiatric controls postmortem. Studies were performed in 16 pairs of nonpsychiatric controls and alcoholics, matched for age, sex and PMI. DSM-IV diagnosis of alcohol use disorder (AUD) was made by the SCID I in a psychological autopsy. Frozen blocks of BA24 or BA45 were sectioned (10 µm) for in situ hybridization of ³⁵S-labelled riboprobe for GAD_65/67 mRNA and autoradiograms were analyzed by quantitative densitometry. Three isodensity bands of labeling were evident, with different relative amounts of GAD₆₅ and GAD₆₇ (outer and inner, predominantly GAD₆₅, intermediate predominantly GAD₆₇), and the isodensity bands were analyzed separately. GAD_65/67 mRNA levels were not different between alcoholics and controls in the gray matter of BA24 (p = 0.53) or BA45 (p = 0.84) or in any of the three isodensity bands in which the GAD_65/67 mRNA was distributed. GAD_65/67 mRNA in white matter underlying either region was also not different in alcoholics (p > 0.05). GAD_65/67 mRNA levels did not correlate with age, sex or duration of alcoholism in either BA24 or BA45. Effects on inhibitory neurotransmission in alcoholics do not appear to be associated with change in the levels of GAD₆₅ or GAD₆₇ mRNA.

Keywords: GABA; alcohol; human; in situ hybridization; postmortem.

Figure 1

GAD_65/67 mRNA expression in BA24 (A, B) and BA45/47 (C, D) in a representative control (A, C) and alcoholic (B, D) visualized by in situ hybridization histochemistry. Note the labeled GAD_65/67 mRNA is localized throughout the gray matter and is greater in BA45 than in BA24. Scale bar = 2 mm.

Figure 2

Photomicrographs of tissue section hybridized with [³⁵S]-labeled ribonucleotide probe and developed for silver grains in a representative control (A, B) and alcoholic (C, D). Note the silver grains over cells.

Figure 3

Bar plots of GAD_65/67 density in Brodmann Areas 24 and 45/47. Data are presented as mean ± S.E.M. Note the lack of distinction between controls and AUD cases regardless of region sampled.