Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice

Abstract

The basal ganglia, a brain structure critical for sensorimotor and motivational aspects of behavior, contain very high levels of CB1 cannabinoid receptors. These receptors are activated by endogenous lipophilic ligands, and they are thought to mediate behavioral effects of cannabinoid drugs. To evaluate the role of the endogenous cannabinoid system in the regulation of basal ganglia pathways, we have investigated the effects of targeted deletion of CB1 receptors on gene expression of various neuropeptides and transmitter-related enzymes in basal ganglia neurons. Mice without CB1 receptors are extremely hypoactive in a test for exploratory behavior (open-field test), showing markedly reduced locomotion and rearing. These CB1 mutants display significantly increased levels of substance P, dynorphin, enkephalin, and GAD 67 mRNAs in neurons of the two output pathways of the striatum that project to the substantia nigra and the globus pallidus. Our findings demonstrate that elimination of CB1 receptors results in behavioral abnormalities and functional reorganization of the basal ganglia.

Figure 1

Behavioral effects of CB1 receptor mutation. (A) Open-field behavior in CB1^−/− mice. Animals (CB1^+/+, n = 11; CB1^−/−, n = 8) were tested for 40 min in an automated open field. Ambulation counts (mean ± SEM), ambulation time, vertical counts (rearing), and local counts (e.g., grooming, shifting) are presented (Upper). Illustrations of the path of ambulation during such a test are shown for a CB1^+/+ (Lower Left) and a CB1^−/− (Lower Right) animal. (B) Rotarod performance in CB1^−/− mice. The average latency to fall (mean ± SEM) for three 5-min trials (Left) and the fall latencies for each of these three trials (Right) are shown for CB1^+/+ (n = 9) and CB1^−/− mice (n = 11). An accelerating-beam procedure (4–40 rpm over 5 min) was used. ∗∗, P < 0.01; ∗, P < 0.05.

Figure 2

Gene expression in the striatum in CB1^−/− mutants. Film autoradiograms depict CB1, dynorphin, substance P, enkephalin, and GAD 67 mRNA expression in coronal sections from the midstriatal level in CB1^+/+ (Upper) and CB1^−/− mice (Lower). Maximal hybridization signal is black. In wild-type (+/+) animals, CB1 receptor expression is maximal in the lateral striatum (arrows) and minimal in the medial striatum. In CB1^−/− mice, increases in dynorphin and substance P mRNA expression (both in striatonigral neurons) are also maximal in the lateral striatum (arrows) and minimal in the medial striatum (for quantitative results, see Table 1). In contrast, increases in enkephalin mRNA expression (in striatopallidal neurons) are similar or even greater in some medial than lateral regions in CB1^−/− mutants. GAD 67 mRNA expression (in both types of projection neurons) subsumes the changes seen for the neuropeptides, with an increase in most striatal regions that is somewhat stronger laterally. (Lower Left) The absence of CB1 mRNA in CB1^−/− mutants and the sample areas in the middle striatum in which gene expression was measured are shown.

Figure 3

Relationship between increases in neuropeptide mRNA expression in the striatum of CB1^−/− mutants and CB1 receptor expression in wild-type mice (CB1^+/+). Dynorphin (A), substance P (B), enkephalin (C), and GAD 67 mRNA levels (D) were measured in 11 striatal regions of CB1^−/− mice (n = 7) and expressed as percentage of levels in CB1^+/+ mice (n = 7). These data were correlated with CB1 mRNA levels in CB1^+/+ animals. Significant positive correlations were found for increases in dynorphin and substance P expression (both in striatonigral neurons), but not for enkephalin (in striatopallidal neurons) or GAD 67 expression (in both neuronal types). ∗∗, P < 0.01; ∗, P < 0.05.

Figure 4

Expression of tyrosine hydroxylase mRNA in the ventral midbrain of CB1^+/+ and CB1^−/− animals. (A) Film autoradiograms depict CB1 (Left) and tyrosine hydroxylase expression (Right) in coronal brain sections containing the substantia nigra and ventral tegmental area for the two genotypes. Note the absence of CB1 mRNA expression in the area of tyrosine hydroxylase labeling (dopamine neurons) in wild-type (+/+) animals. The substantia nigra pars reticulata (SNr) was outlined by using Nissl-stained and GAD 67-labeled adjacent sections. HC, hippocampus; IP, interpeduncular nucleus; SNc, substantia nigra pars compacta; VTA, ventral tegmental area. (B) Mean density (mean ± SEM) (Left) and area (number of pixels with density values above background) (Right) for tyrosine hydroxylase expression in the substantia nigra/ventral tegmental area are given for CB1^+/+ (n = 7) and CB1^−/− mice (n = 7). Gene expression was measured in sections from seven rostrocaudal levels, and average values are presented.