Cortical glutamic acid decarboxylase 67 deficiency results in lower cannabinoid 1 receptor messenger RNA expression: implications for schizophrenia

Abstract

Background: Levels of cannabinoid 1 receptor (CB1R) messenger RNA (mRNA) and protein, which are expressed most heavily in the cholecystokinin class of γ-aminobutyric acid (GABA) neurons, are lower in the dorsolateral prefrontal cortex in schizophrenia, and the magnitude of these differences is strongly correlated with that for glutamic acid decarboxylase 67 (GAD(67)) mRNA, a synthesizing enzyme for GABA. However, whether this correlation reflects a cause-effect relationship is unknown.

Methods: Using quantitative in situ hybridization, we measured CB1R, GAD(67), and diacylglycerol lipase alpha (the synthesizing enzyme for the endocannabinoid 2-arachidonoylglycerol) mRNA levels in the medial prefrontal cortex of genetically engineered GAD(67) heterozygous (GAD(67)(+/-)), CB1R heterozygous (CB1R(+/-)), CB1R knockout (CB1R(-/-)), and matched wild-type mice.

Results: In GAD(67)(+/-) mice, GAD(67) and CB1R mRNA levels were significantly reduced by 37% and 16%, respectively, relative to wild-type mice and were significantly correlated across animals (r = .61; p = .01). In contrast, GAD(67) mRNA levels were unaltered in CB1R(+/-) andCB1R(-/-) mice. Expression of diacylglycerol lipase alpha mRNA, which is not altered in schizophrenia, was also not altered in any of the genetically engineered mice.

Conclusions: The findings that reduced GAD(67) mRNA expression can induce lower CB1R mRNA expression support the hypothesis that lower cortical levels of CB1Rs in schizophrenia may partially compensate for deficient GAD(67)-mediated GABA synthesis by reducing endogenous cannabinoid suppression of GABA release.

Figure 1

Transcript levels in the mPFC of adult wild-type and GAD₆₇^+/− mice. Representative film autoradiograms illustrating the expression of GAD₆₇ (A, B), CB1R (D, E), and DAGLα (G, H) mRNAs. The density of hybridization signal for each transcript is presented in pseudocolor according to the calibration bars below B, E, and H. Expression of GAD₆₇ and CB1R mRNA in GAD₆₇^+/− mice (B, E) appears lower than in wild-type mice (A, D), whereas DAGLα (G, H) does not appear to differ across the two conditions. Note that CB1R mRNA signal is most pronounced in the superficial cortical layers, consistent with the laminar distribution of CCK-containing GABA neurons that heavily express CB1R mRNA and that are the principal CB1R mRNA expressing neuron type in the cortex(4). White contours denote the quantified region of the mPFC. Comparison of cortical GAD₆₇ (C), CB1R (F), and DAGLα (I) mRNA levels by film optical density (OD) in wild-type (diamonds) and GAD₆₇^+/− (triangles) mice. Mean values for each genetic condition are indicated by hash marks. Scale bar (1mm) in H applies to all panels.

Figure 2

Positive correlation between levels of CB1R and GAD₆₇ mRNAs in adult wild-type and GAD₆₇^+/− mice. These findings suggest that changes in CB1R mRNA expression parallel changes in GAD₆₇ mRNA expression.

Figure 3

Cellular expression of CB1R mRNA. Representative photomicrograph of Nissl-counterstained, emulsion-exposed tissue section showing silver grains representing CB1R mRNA clustered over a subset of neuronal cell bodies (A). Scale bar = 30 μm. Expression of CB1R mRNA is lower in GAD₆₇^+/− mice relative to wild-type mice (B).

Figure 4

Transcript levels in the mPFC of adult wild-type, CB1R^+/−, and CB1R^−/− mice. Representative film autoradiograms illustrating the expression of CB1R (A, B, C), GAD₆₇ (E, F, G), and DAGLα (I, J, K) mRNA. The density of hybridization signal for each transcript is presented in pseudocolor according to the calibration bars below C, G, and K. Expression of CB1R mRNA is markedly reduced in CB1R^+/− mice (B) and nearly undetectable in CB1R^−/− mice (C) compared to wild-type mice (A). Expression of GAD₆₇ (E, F, G) and DAGLα (I, J, K) mRNA appear unaltered in either genetic condition compared to wild-type mice. White contours denote the quantified region of the mPFC. Comparison of cortical CB1R (D), GAD₆₇ (H), and DAGLα (L) mRNA levels by film optical density (OD) in wild-type (diamonds), CB1R^+/− (triangles), and CB1R^−/− (circles) mice. Mean values for each condition are indicated by hash marks. Scale bar (1mm) in K applies to all panels.