Replacement of Asymmetric Synaptic Profiles in the Molecular Layer of Dentate Gyrus Following Cycloheximide in the Pilocarpine Model in Rats

Abstract

Mossy fiber sprouting is among the best-studied forms of post-lesional synaptic plasticity and is regarded by many as contributory to seizures in both humans and animal models of epilepsy. It is not known whether mossy fiber sprouting increases the number of synapses in the molecular layer or merely replaces lost contacts. Using the pilocarpine (Pilo) model of status epilepticus to induce mossy fiber sprouting, and cycloheximide (CHX) to block this sprouting, we evaluated at the ultrastructural level the number and type of asymmetric synaptic contacts in the molecular layer of the dentate gyrus. As expected, whereas Pilo-treated rats had dense silver grain deposits in the inner molecular layer (IML) (reflecting mossy fiber sprouting), pilocarpine + cycloheximide (CHX + Pilo)-treated animals did not differ from controls. Both groups of treated rats (Pilo group and CHX + Pilo group) had reduced density of asymmetric synaptic profiles (putative excitatory synaptic contacts), which was greater for CHX-treated animals. For both treated groups, the loss of excitatory synaptic contacts was even greater in the outer molecular layer than in the best-studied IML (in which mossy fiber sprouting occurs). These results indicate that mossy fiber sprouting tends to replace lost synaptic contacts rather than increase the absolute number of contacts. We speculate that the overall result is more consistent with restored rather than with increased excitability.

Keywords: asymmetric synaptic profiles; cycloheximide; epilepsy; mossy fiber sprouting; synaptic plasticity.

Figure 1

Electron micrograph of the inner molecular layer showing asymmetric synaptic profiles and their localization in dendritic spines or shafts (A). (B–D) are higher magnification views of different synaptic contacts: PSD1 [(B) – non-perforated type 1, synaptic profiles with a single-synaptic bouton]; PSD2 [(C) – perforated type 2, synaptic profiles with two postsynaptic densities]; and PSD3 [(D) – perforated type 3, synaptic profiles with more than two postsynaptic densities]. Note the spherical presynaptic vesicles and mitochondrion (*) in the axon terminal (a) contacting a dendritic spine (e) and a dendritic shaft (s) with a mitochondria (^#). Scale bars, 1.25 μm.

Figure 2

Electron micrographs of the dentate gyrus molecular layer in controls (A,B), Pilo-treated (C,D) and CHX + Pilo-treated animals (E,F). (A,C,E) represent the inner molecular layer (IML). (B,D,F) represent the outer molecular layer (OML). Silver grain dots in the IML were only observed in Pilo-treated animals (C). These profiles have not been found in the outer molecular layer of any of the groups (B,D,F). Scale bar, 150 nm.

Figure 3

(A) Density of silver grains staining per 100 μm² within dentate molecular layer of control, pilocarpine (Pilo)- and cycloheximide + pilocarpine (CHX + Pilo)-treated animals. IML, inner molecular layer; OML, outer molecular layer. *P < 0.001; compared to controls; ^#P < 0.001; compared to the Pilo group. (B) Sections at the level of the inner molecular layer staining for mossy fiber sprouting of Pilo-treated animal. Note the greater silver grains staining in the molecular layer more proximal (IMLp) to the granule cell layer (Gr) as compared to the more distal portion of the inner molecular layer (IMLd). In these higher magnification views, histochemically reactive silver grains could easily be localized on the asymmetric synapse contacts. Axon terminal (a) and dendritic shaft (s) with mitochondria (*). IMLp, Inner molecular layer proximal; IMLd, inner molecular layer distal. Scale bars, 1.25 μm.

Figure 4

Means and 95% confidence interval of the total number of synapses obtained using six photomicrographs per layer (IML and OML) in five animals per group. IML, inner molecular layer; OML, outer molecular layer.

Figure 5

Schematic view of the changes in the synaptic profiles of the different experimental groups as compared to controls. Synaptic profiles located on the shaft or spine of dendrites in inner (IML) or outer (OML) molecular layer were identified as PSD1, PSD2, or PSD3. Red denotes type and location of synaptic profile showing a significant increase in density as compared to control animals. Blue represents synaptic types and location that were significantly decreased as compared to control animals. Pilo – animals subjected to pilocarpine-induced status epilepticus. CHX + Pilo – animals subjected to pilocarpine-induced status epilepticus and co-injected with cycloheximide.