Abstracts

RATIONALE: Dentate granule cells gate the propagation of paroxysmal activity through hippocampal circuits. However, the gating function of the dentate gyrus can collapse during status epilepticus and in temporal lobe epilepsy. This is in part due to altered properties of [gamma]-aminobutyric acid type A (GABA[sub]A[/sub]) receptors, which depends on their subunit composition. More than 10 GABA[sub]A[/sub] receptor subunits are expressed in dentate granule cells, but their subcellular distribution has not been described.
METHODS: The subcellular distribution of GABA[sub]A[/sub] receptor subunits ([alpha]1, [alpha]2, [alpha]4, [beta]1, [beta]2/3, [gamma]2, [delta]) in adult rat dentate granule cells was investigated by immunohistochemistry for subunit protein and for the GABAergic presynaptic marker glutamic acid decarboxylase(GAD) or postsynaptic marker gephyrin. Confocal laser scanning microscopy was used to study the sections.
RESULTS: The [alpha]1, [alpha]2, [beta]1, [beta]2/3 and [gamma]2 immunoreactivity (IR) was in form of clusters that outlined the granule cells. Clusters of [alpha]2 IR were more frequently present on the hilar pole of granule cells. Analysis of 385 [alpha]1 subunit clusters revealed that they ranged in size from 0.05[mu]m² to 0.3[mu]m², with a skewed distribution toward larger size clusters. More than half the clusters were small ([lt] 0.1 [mu]m²). The size distribution of [gamma]2 subunit clusters was similar. Double labeling experiments revealed that the large clusters frequently colocalized with synaptic markers while the small clusters rarely did so. Only 6.7% of 0.05 [mu]m² [alpha]1 subunit IR clusters colocalized with GAD IR while 75 % of 0.16 [mu]m² size clusters, and 100% of 0.22 [mu]m² clusters colocalized with GAD. The rate of colocalization with synaptic markers correlated well with the cluster size (spearman correlation r = 0.92, p [lt]0.0001). The results were similar when [alpha]1 cluster colocalization with gephyrin was studied or that of [gamma]2 subunit with GAD or gephyrin. Some of the clusters for [gamma]2 and [alpha]1 IR colocalized with NMDAR1 clusters. The [alpha]4 and [delta] subunit IR was diffusely distributed throughout the granule cell body and did not colocalize with synaptic markers.
CONCLUSIONS: The GABA[sub]A[/sub] receptor subunits are distributed in synaptic and extrasynaptic compartments of dentate granule cells. Large clusters of [alpha]1, [alpha]2, [beta]1, [beta]2/3, [gamma]2 subunits are frequently present at synapses. Small clusters may be extrasynaptic, or present at excitatory synapses or belong to the trafficking pool. The [alpha]4 and [delta] subunits are diffusely distributed in extrasynaptic compartment.
[Supported by: NINDS grants NS 02081 and NS 40337 and the Epilepsy Foundation through the generous support of the American Epilepsy Society and the Milken Family Foundation.]