Studies of GABA(B) receptors labelled with [(3)H]-CGP62349 in hippocampus resected from patients with temporal lobe epilepsy

Abstract

1 The aim of this study was to investigate the binding of a novel GABA(B) receptor radioligand, [(3)H]-CGP62349, to human post-mortem control and epileptic hippocampal sections using quantitative receptor autoradiography. Utilizing human control hippocampal sections it was shown that [(3)H]-CGP62349 bound with high affinity (K(D) 0.5 nM) to this tissue. 2 Hippocampal slices from surgical specimens obtained from patients with hippocampal sclerosis (HS) and temporal lobe epilepsy (TLE) were compared with neurologically normal post-mortem control subjects for neuropathology and GABA(B) receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, but in the subiculum no significant difference was detected. 3 The localization of GABA(B) receptors with the antagonist [(3)H]-CGP62349 in human control hippocampal sections supported and extended earlier studies using the agonist ligand [(3)H]-GABA. 4 The kinetics of binding to the GABA(B) receptor in human hippocampus using this novel compound was comparable to previous data obtained in rat hippocampal membranes. 5 GABA(B) receptor density (B(max)) was significantly reduced in CA3, hilus, and dentate gyrus (DG); the affinity was increased exclusively in DG. The trend is identical in all the hippocampal subregions with the agonist and the antagonist, although significant differences with the antagonist where recorded in CA3 and hilus, whereas with the agonist a significant reduction was reported in all of the hippocampal subfields. 6 GABA(B) receptor expression per remaining neuron appeared significantly increased in CA3 and hilus. These results suggest altered GABA(B) receptor function may occur in human TLE, possibly as a result of synaptic reorganization, and may contribute to epileptogenesis.

Figure 1

Mean neuronal density in post-mortem control hippocampal samples (n=5) and resected epileptic hippocampal tissues (n=6–11). Three-dimensional cell counts were performed on formaline fixed tissue sections (20 μm thickness). Data are expressed as mean±s.e.mean. Statistical analysis used unpaired Student's t-test (two-tailed), where ^*P<0.05, ^**P<0.01, ^***P<0.001. SUB, subiculum.

Figure 2

Concentrations of [³H]-CGP62349 were in the range 0.125 to 8 nM. Non-specific binding is indicated by closed squares, and total binding by closed triangles. The difference between total and non-specific gives specific binding denoted by dots. Non-specific binding was determined in the presence of 10 μM CGP54626A. Data shown are from a single tissue sample.

Figure 3

Autoradiographic images illustrating film optical density corresponding to total binding site distribution following autoradiography [³H]-CGP62349 (0.75 nM concentration) in sections of hippocampus from a control post-mortem brain numbered in Table 2 case no 5 (A) and an epileptic patient affected by HS (B). An apparent loss of GABA_B receptor can be seen mainly in CA4, DG. The control and TLE hippocampi were processed at the same time in the same conditions. In TLE CA1, CA2 and CA3 were missed because of the surgical procedure. Scale bars represent 4 mm.

Figure 4

Mean receptor affinity (K_D) in post mortem control hippocampal tissue (n=5–8) and resected epileptic hippocampal sample (n=8–13). Data are represented as mean±s.e.mean. Statistical analysis used unpaired Student's t-test (two-tailed), where ^**P<0.01. SUB, subiculum.

Figure 5

Mean receptor population (B_max) in post mortem control hippocampal tissue (n=5–8) and resected epileptic hippocampal sample (n=8–13). Data are represented as mean±s.e.mean. Statistical analysis used unpaired Student's t-test (two-tailed), where ^*P<0.05, ^**P<0.01, ^***P<0.001. SUB, subiculum.

Figure 6

Histogram representing ratio of B_max to neuronal density in tissue sections from post-mortem control and patients with HS. Histogram bars represent the mean ratio from hippocampal subregion and error bars represent s.e.mean. Groups were compared using an unpaired Student's t-test (two-tailed) where ^*P<0.05, ^***P<0.001. SUB, subiculum.