SUMMARY thesis Else A.Tolner

Anatomical and functional reorganization in the parahippocampal region during temporal lobe epilepsy

Research in epilepsy in the last years led to the recognition that both the hippocampal formation and the parahippocampal regions play an important role in the process of epileptogenesis in the mesial temporal lobe (MTLE). Selective cell loss and axonal reorganization is clearly present in the hippocampus proper and both processes have been linked to hyperexcitability underlying the development of chronic epilepsy. In the entorhinal cortex (EC) a clear pathology is observed in MTLE patients, specifically in layer III of the medial EC (MEA-III). Several descriptive studies concerning the EC in epilepsy have been complemented by functional studies both in patients and animal models, suggesting that the EC is actively involved in seizure generation and propagation throughout the temporal lobe.

In this thesis, anatomical and physiological alterations were examined in the hippocampal and parahippocampal region of rats after induction of a status epilepticus (SE). One of the main goals (see 1.6) was to examine whether functional reorganization of connections occurs in the MEA of chronic epileptic rats. In the dentate gyrus (DG) a well-known form of reorganization, the process of mossy fibre sprouting (MFS), is observed both in MTLE patients and in animal models. The first series of experiments ( chapters 2 and 3 ) were conducted to examine changes in expression of axonal growth-related molecules in relation to the process of mossy fibre sprouting (MFS). First, in chapter 2 the mRNA expression profile of the growth-repelling molecule Sema3A and the axonal growth-associated protein GAP-43 were examined after electrically induced SE in rats (self-sustaining limbic SE, SSLSE model). Induction of SE caused a rapid (within 24 hours) but temporary (recovery after 1 week) down-regulation of Sema3A mRNA expression in layer II of the EC, which was strongest in MEA. Together with down-regulation of Sema3A, GAP-43 mRNA was up-regulated in dentate granule cells. These changes occurred before MFS was detectable. When induction of SE was incomplete, down-regulation of Sema3A mRNA did not occur and no significant MFS was observed, although up-regulation of GAP-43 mRNA in granule cells was still displayed. These findings indicate that Sema3A mRNA down-regulation in MEA is correlated in time with the occurrence of MFS. It appears that loss of Sema3A protein in the dentate molecular layers facilitates sprouting of the mossy fibres into this area. GAP-43 mRNA up-regulation in granule cells, however, is not directly linked to MFS. This conclusion is in contrast with previous reports in the literature that suggest GAP-43 expression in the DG to be a good marker for MFS. Therefore in chapter 3 a more detailed examination was performed of GAP-43 mRNA and protein expression in the DG in relation with the occurrence of MFS in chronic epileptic rats from both the SSLSE and kainate (KA) models. No link was found between GAP-43 protein expression and MFS. The findings suggested that in control rats the expression of GAP-43 in the DG inner molecular layer (IML) is the result of presence of GAP-43 in axons from hilar neurons and not of the presence of GAP-43 in mossy fibres. With loss of hilar neurons after epilepsy, GAP-43 expression is lost in the IML. In the parahippocampal region no significant changes were observed in GAP-43 expression after SE, except for mRNA changes linked to neuronal loss in MEA-III. The presence of GAP-43 protein in a degenerated MEA-III suggests preservation of connections in this area after epilepsy. Nevertheless it does not provide evidence defining GAP-43 as a marker for axonal reorganization, and thus the presence of GAP-43 does not give a clue as to whether synaptic reorganization might have occurred.

In chapters 4, 5, and 6 different aspects of the parahippocampal circuitry were studied in chronic epileptic rats (KA model) employing both electrophysiological ( in vivo field recordings and in vitrofield and intracellular recordings) and anatomical techniques (anatomical tracings and immunocytochemistry). The central question in these studies was: ‘How does the SE-induced neuronal loss in MEA-III affect the anatomical and functional connectivity with and within the MEA?' In Chapter 4, the output from the subiculum to MEA was studied in vivo in anesthetized chronic KA rats. In chronic epileptic rats displaying extensive neuronal loss in MEA-III, subiculum stimulation evoked oscillations in the b / g -frequency range that were confined to superficial layers of MEA. Further, increased activation was observed at the level of MEA-II apical dendrites in the form of an increased current sink that was associated with antidromic activation of MEA-II. This increase was not observed in controls or in rats with minor MEA-III loss that did not display evoked oscillations in superficial layers. The findings suggest that in epileptic rats alterations in inhibition and/or connectivity occur in superficial MEA layers, resulting in hyperexcitability of the superficial MEA. Since a direct input to MEA-III arises from the presubiculum (prS), we hypothesized ( chapter 5 ) that the effect of MEA-III loss after epilepsy might result in a reorganization of prS-MEA connectivity. Surprisingly, anatomical tracings revealed that in chronic epileptic rats, presubicular fibres still target MEA-III specifically, in spite of extensive MEA-III neurodegeneration. In vivo recordings were performed in MEA of anesthetized chronic KA rats to examine the functional characteristics of the prS-MEA connection. Complementary recordings were performed in the hippocampus. Similar as for subiculum stimulation (chapter 4), prS stimulation evoked b / g -oscillations that were confined to superficial layers of the MEA. These oscillations were not observed in deep layers of the MEA or in the hippocampus and were only observed in rats in which MEA-III was degenerated. In some rats prS stimulation evoked deep layer oscillations that appeared unrelated to the events in superficial layers. Deep layer oscillations were of lower frequency ( q -range) than the superficial oscillations and were in line with similar oscillations observed in the subiculum and DG. The observation of distinct oscillations in superficial and deep MEA (and hippocampus) indicates that specific alterations occur in superficial versus deep layers of the MEA after chronic epilepsy. In chapter 6 in vitrorecordings were performed in hippocampal-entorhinal slices from control and chronic epileptic rats to examine which cellular and/or network alterations underlie the pathophysiological alterations in superficial MEA after chronic epilepsy. NeuN and GAD67 immunocytochemistry and GAD-65/67 in situ hybridization revealed that in spite of significant neuronal loss in MEA-III after epilepsy, there was preservation of GAD-positive interneurons. A slight decrease, however, of GAD-positive neurons was observed in deep MEA. E xtracellular responses to prS, parasubiculum (paraS) and deep MEA stimulation revealed high frequency (100-300 Hz) field potential transients in superficial layers that were not seen in controls. Application of the GABA-A receptor antagonist bicuculline had only minor effects on these evoked field potentials in chronic KA rats, in contrast to strong evoked effects in control slices. Intracellular recordings in MEA-II and MEA-III revealed prolonged excitatory post-synaptic potentials (EPSPs) in the KA tissue superimposed on IPSPs. These findings support the view (chapter 4 and 5) that synaptic connectivity with and within superficial layers of MEA is altered in chronic epileptic rats and that inhibition seems to be impaired. In addition, the results from chapter 6 suggest that functional disinhibition may occur in superficial MEA after chronic epilepsy, leading to increased hyperexcitability.