Acute and chronic changes in glycogen phosphorylase in hippocampus and entorhinal cortex after status epilepticus in the adult male rat

Abstract

Glial cells provide energy substrates to neurons, in part from glycogen metabolism, which is influenced by glycogen phosphorylase (GP). To gain insight into the potential subfield and laminar-specific expression of GP, histochemistry can be used to evaluate active GP (GPa) or totalGP (GPa + GPb). Using this approach, we tested the hypothesis that changes in GP would occur under pathological conditions that are associated with increased energy demand, i.e. severe seizures (status epilepticus or 'status'). We also hypothesized that GP histochemistry would provide insight into changes in the days and weeks after status, particularly in the hippocampus and entorhinal cortex, where there are robust changes in structure and function. One hour after the onset of pilocarpine-induced status, GPa staining was reduced in most regions of the hippocampus and entorhinal cortex relative to saline-injected controls. One week after status, there was increased GPa and totalGP, especially in the inner molecular layer, where synaptic reorganization of granule cell mossy fibre axons occurs (mossy fibre sprouting). In addition, patches of dense GP reactivity were evident in many areas. One month after status, levels of GPa and totalGP remained elevated in some areas, suggesting an ongoing role of GP or other aspects of glycogen metabolism, possibly due to the evolution of intermittent, recurrent seizures at approximately 3-4 weeks after status. Taken together, the results suggest that GP is dynamically regulated during and after status in the adult rat, and may have an important role in the pilocarpine model of epilepsy.

Fig. 1

Glycogen phosphorylase a (GPa) and total glycogen phosphorylase (totalGP) histochemistry in the rat hippocampus after status. Representative examples of GPa (left column, A, C, E and G) and totalGP (right column, B, D, F and H) in saline-treated control rats (A and B), compared to pilocarpine-treated rats that were killed one hour after the onset of status (C and D), one week after status (E and F), or one month after status (G and H). Asterisks in E and F indicate regions of tissue damage. Tissue damage was due to seizure-induced neuronal loss, and in addition, tissue sections were processed without fixation. Therefore, some sections have cracks (e.g. Fig. 3E and F). Fixation was avoided because it is incompatible with GP histochemistry. DG, dentate gyrus. Calibration bar, 500 μm.

Fig. 2

GPa and totalGP in the entorhinal cortex after status. Representative examples of GPa (left column, A, C, E and G) and totalGP (right column, B, D, F and H) in saline-treated control rats (A and B), compared to pilocarpine-treated rats that were killed one hour after the onset of status (C and D), one week after status (E and F), or one month after status (G and H). I, II, III-LD and V–VI refer to entorhinal cortical layers. LD, lamina dissecans; PreS, presubiculum; PaS, parasubiculum. Scale bar, 500 μm.

Fig. 3

Changes in GPa in the inner molecular layer of the dentate gyrus after status. GPa histochemistry (A, C and E) and NeuN/GFAP immunohistochemistry on adjacent fresh tissue sections (B, D and F) in the dentate gyrus in a control rat (A and B) compared to pilocarpine-treated rats that had status one week earlier (C and D) or one month (E and F) earlier. NeuN (blue) and GFAP (brown) illustrate colocalized GPa and GFAP in the inner molecular layer (arrows), just above the granule cell layer. IML, inner molecular layer; GCL, granule cell layer. Scale bar, 100 μm.

Fig. 4

Changes in GPa in the dentate gyrus and entorhinal cortex after status. GPa histochemistry is compared for a control rat (A), a pilocarpine-treated rat that was examined one week after status (B) and a pilocarpine-treated rat that was evaluated one month after status (C). Intense GPa reactivity is marked by the arrows. IML, inner molecular layer; GCL, granule cell layer. Note that at 1 month, the hilus appears to have expanded, an observation that has been made previously (McCloskey et al., 2006). (D) Intense GPa reactive structures, similar to the dentate gyrus hilus, were present in the entorhinal cortex (EC) one week after status (arrows). II, III, V refer to entorhinal cortical layers. Scale bar, 100 μm (A–C); 200 μm.

Fig. 5

GP-dense structures in hippocampal cell layers after status. GPa in CA1 (left column, A–C) and CA3 (right column, D–F) of the hippocampus in saline-injected controls (A and D) and pilocarpine-injected animals one week (B and E) or one month (C and F) after status. Arrows indicate GPa-intense structures found throughout the layers of CA1 (B) and CA3 (E) one week after status and one month after status (C and F). Scale bars, 200 μm (A–C) and 100 μm (D–F).