Downregulation of the CB₁ Cannabinoid Receptor and Related Molecular Elements of the Endocannabinoid System in Epileptic Human Hippocampus

Gene expression level of cannabinoid receptor-interacting protein CRIP1a, but not CRIP1b, is reduced in the sclerotic epileptic hippocampus. A , CRIP1a mRNA level is significantly decreased in the sclerotic epileptic hippocampus (n = 6) compared with control values (n = 7). In contrast, the decrease in the nonsclerotic hippocampus (n = 7) did not reach significance. The direction and magnitude of expression level changes in the sclerotic hippocampus were identical in parallel experiments using two distinct housekeeping genes, β-actin and GAPDH. B , In contrast, CRIP1b mRNA levels did not differ significantly between nonsclerotic and sclerotic epileptic hippocampi compared with control values. Data are presented as mean expression ratio ± SEM. *p < 0.05.

Gene expression level of DGL-α, the biosynthetic enzyme of 2-AG, is diminished in the sclerotic epileptic hippocampus. A , DGL-α mRNA level is decreased to one-half of its control level in the sclerotic epileptic hippocampus. In contrast, significant difference in mRNA level was not observed between the nonsclerotic hippocampal samples and control subjects. Importantly, the direction and magnitude of expression level changes were identical in parallel experiments using two distinct housekeeping genes, β-actin and GAPDH. B , The related isoenzyme DGL-β is unaffected in the epileptic hippocampus. Real-time PCR measurement did not reveal significant changes in mRNA level either in the nonsclerotic or in the sclerotic epileptic hippocampus as compared with control values. C , MGL, the enzyme responsible for elimination of 2-AG, showed a slight but insignificant decrease in mRNA level in the sclerotic hippocampus [normalized to β-actin (p = 0.171) or to GAPDH (p = 0.079)]. In the nonsclerotic hippocampal samples, there was no indication of any subtle changes in gene expression level. Data are presented as mean expression ratio ± SEM. *p < 0.05.

Metabolic enzymes of anandamide are not downregulated in the epileptic hippocampus. A , Real-time PCR measurement did not reveal alterations in the gene expression level of NAPE-PLD, a key synthetic enzyme of anandamide. The normalized expression level was similar in all three experimental groups and in the parallel experiments, which used β-actin or GAPDH as reference genes. B , FAAH, the degrading enzyme of anandamide, did not show significant expression changes in the epileptic hippocampal samples. Data are presented as mean expression ratio ± SEM.

Immunostaining for CB₁ cannabinoid receptor is reduced in the hippocampus of epileptic patients, particularly in the inner molecular layer of the dentate gyrus (DG). A , Light micrograph illustrating profound CB₁ immunoreactivity throughout the human hippocampal formation of control subjects. By using a highly sensitive guinea pig antibody for CB₁, the immunostaining highlights the different layers and subfields of the hippocampus according to the spatial arrangements of excitatory pathways. B , C , Although the general pattern of CB₁ immunostaining is similar in the nonsclerotic and sclerotic epileptic hippocampi, the density of CB₁ immunoreactivity is reduced in several layers. D–F , The most striking differences between the control and the epileptic hippocampi are visible in the dentate gyrus. The very dense neuropil-like labeling in the inner third of stratum moleculare (str. mol.) is evident in the control sample ( D ), but it is less so in the nonsclerotic epileptic sample ( E ), and it disappears almost completely in the sclerotic epileptic samples ( F ). In contrast, the stratum granulosum (str. gr.) and the hilus remained similar in all three experimental groups. Scattered cell bodies of GABAergic interneurons were also stained for CB₁ (labeled by arrows), but conversely, there was no striking difference either in their distribution pattern or in their number between the control and epileptic human samples. Scale bars: A–C , 500 μm; D–F (in F ), 100 μm.

Density of glutamatergic axon terminals bearing presynaptic CB₁ cannabinoid receptors is decreased in the epileptic human hippocampus. A–C , The electron micrograph demonstrates a robust accumulation of strong CB₁ immunoreactivity within axon terminals in the inner third of the stratum moleculare of control subjects. These CB₁-positive boutons (b) form the classic asymmetric synapses (arrowheads) with an extensive postsynaptic density on dendritic spine heads. In control samples, nearly all axon terminals with asymmetric synapses are positive for CB₁, whereas in the nonsclerotic ( B ) and sclerotic ( C ) samples, the number of CB₁-positive asymmetric synapses drops noticeably. Note that a lack of staining does not necessarily mean the complete absence of CB₁ receptors, but it means that the antigen level fails to reach detection threshold in these CB₁-negative boutons (depicted by asterisks). Scale bar, 0.5 μm.

Quantitative analysis of the ratio and density of CB₁-positive excitatory axon terminals in the inner molecular layer of the human dentate gyrus. A , The number of excitatory axon terminals either positive or negative for CB₁ cannabinoid receptor was established using an unbiased stereological estimation method (Geinisman et al., 1996). Altogether, 1092 disector pairs were analyzed, which resulted in 327 terminals in control, 224 terminals in nonsclerotic epileptic, and 197 terminals in sclerotic epileptic patients. The number of CB₁-positive terminals decreased from 235 terminals in control subjects to 106 or 40 terminals in the nonsclerotic or sclerotic epileptic samples, respectively. In contrast, the number of CB₁-negative terminals increased from 92 terminals in control subjects to 118 or 157 terminals in nonsclerotic or sclerotic epileptic samples, respectively. In the quantitative analysis, tissue samples from three individuals from each experimental group were used. B , The ratio of CB₁-positive excitatory axon terminals versus all excitatory axon terminals was 72.8 ± 2.1% in control, 50 ± 2.8% in nonsclerotic epileptic, and 21 ± 3.8% in sclerotic epileptic samples (mean ± SEM). The difference between control and epileptic samples was highly significant (χ² test, ***p < 0.001 both for nonsclerotic and sclerotic epileptic samples). C , The estimated numerical density of CB₁-positive axon terminals in the inner molecular layer of the dentate gyrus of control subjects (0.648 ± 0.075/μm³) was strongly decreased in nonsclerotic epileptic patients (0.3 ± 0.051/μm³) and in sclerotic epileptic patients (0.112 ± 0.021/μm³) as well (values are mean ± SEM). This sharp decline in the density of CB₁-positive axon terminals was statistically significant (ANOVA, p < 0.001). Significance exists between control values and both nonsclerotic and sclerotic epileptic patients (Dunnett's post hoc test, ***p < 0.001). D , The estimated numerical density of CB₁-negative axon terminals was elevated in epileptic samples [density in control samples, 0.248 ± 0.038/μm³; in nonsclerotic samples, 0.326 ± 0.062/μm³; and in sclerotic samples, 0.454 ± 0.07/μm³ (mean ± SEM)]. Increase was significant between analyzed groups (ANOVA, p = 0.04); density of CB₁-negative axon terminals in sclerotic epileptic patients was significantly increased compared with control values (Dunnett's post hoc test, *p = 0.037), but not in nonsclerotic patients (Dunnett's post hoc test, p = 0.546).

CB₁-positive GABAergic axon terminals are intact in the epileptic human hippocampus. A–C , The electron micrographs show striking CB₁ immunoreactivity within GABAergic axon terminals (b_GABA) forming symmetric synapses (open arrowheads) in the inner third of stratum moleculare of the dentate gyrus. Dense accumulation of the end product of immunoperoxidase reaction (DAB) indicates that these GABAergic axon terminals are fully equipped with CB₁ receptors in both the control and the epileptic hippocampi. Glutamatergic boutons terminate on dendritic spine heads with typical asymmetric synapses (closed arrowheads), which is characterized by broad postsynaptic density. In the control sample, the glutamatergic axon terminal (b_GLU) is positive for CB₁ ( A ), whereas CB₁-negative boutons (asterisks) forming asymmetric synapses are shown in electron micrographs taken from the nonsclerotic ( B ) and sclerotic ( C ) samples. Note that CB₁-positive axon terminals forming inhibitory synapses are larger than those giving excitatory synapses. Scale bars, 0.5 μm.