Neuronal circuitries of the subiculum in epileptogenesis

Epilepsy presently affects about 2.6 million patients in Europe and represents a tremendous burden for the affected individuals and for the community. A high proportion of patients do not sufficiently respond to the treatment with the currently available antiepileptic drugs. Thus, there is a high need for better understanding of the pathomechanisms involved in development of epilepsy and identification of sites effective for future drug treatment. There is converging evidence that the subiculum, the major output station of the hippocampal formation, may be causatively involved in the generation of epileptic seizures. Recently we observed a marked loss of parvalbumin-containing GABAergic interneurons in the subiculum of epileptic rats. The amount of cell losses correlated with the number of subsequent spontaneous seizures and thus could be causatively related to epileptogenesis. Since these losses occurred also both in patients with and in patients without Ammon`s horn sclerosis, they seemed to be related to seizure activity and not to the general hippocampal damage caused by seizures. Another striking observation from our previous animal experiments was a marked loss of calretinin- containing glutamatergic projections from thalamic nucleus reuniens to the subiculum. Also degeneration of thalamic-subicular projections correlated with seizure progression in the animal model indicating a similarly important role in seizure generation. To further substantiate these findings and to prove a causative role for epileptogenesis, we now propose experiments in which we will functionally inactivate parvalbumin neurons in the subiculum and calretinin neurons of the nucleus reuniens thalami projecting to the subiculum. We will inject a viral vector leading to local overexpression of tetanus toxin (AAV-FLEX-TeLC) into the subiculum of transgenic mice expressing Cre- recombinase on the parvalbumin- or calretinin-promoter, respectively. Tetanus toxin expression will be induced by Cre present only in parvalbumin or calretinin neurons and will inhibit neurotransmitter release from these neurons in the subiculum and nucleus reuniens, respectively. The effect of these treatments will be investigated by in vitro electrophysiology in slices from the subiculum and by long-time EEG recordings in the freely moving mice. In reverse we will express the artificial mutated muscarinic receptor hM3Dq in the same transgenic mice by injection of a respective flexed viral vector (AAV-hM3Dq-mCherry). Injection of the mice with an otherwise inactive artificial receptor ligand will then specifically activate parvalbumin and calretinin neurons through activation of the artificial receptors expressed only in these neurons. We propose that this treatment increases feed- forward inhibition in the subiculum and results in an anticonvulsive action on pilocarpine-induced seizures and epileptogenesis. Using in situ hybridization and immunohistochemistry, we will also characterize histopathological correlates, notably calretinin nerve terminals in the subiculum of TLE patients with and without Ammon`s horn sclerosis.

Signal transduction in the hippocampus is based on excitatory neuronal pathways (trisynaptic circuitry of the hippocampus). Excitatory transmission is under tight control of inhibitory interneurons. More than 20 subtypes of such interneurons have been characterized by their anatomy, neurochemical marker and functional properties. Among these, 1. parvalbumin expressing basket cells exerting potent feed-forward inhibition upon pyramidal cell somata and 2. somatostatin-containing interneurons projecting from the stratum oriens to pyramidal cell dendrites in the Stratum lacunosum moleculare (O-LM cells) and exerting potent feed- back inhibition are functionally especially prominent. Malfunctioning of either of these neurons has been proposed to cause epilepsy. In our experiments we functionally silenced both subtypes of interneurons and investigated the role on development of epileptic seizures. We used transgenic mice expressing the enzyme cre-recombinase either on the parvalbumin or somatostatin promoter. We locally injected a viral vector into the subiculum of the mice expressing tetanus toxin light chain in a cre-recombinase dependent manner. This treatment resulted in a selective functional silencing (inhibition of GABA release) of these neurons without damaging them. We characterized the selectivity of tetanus toxin expression in the respective neurons and their functional impairment of GABA release by immunohistochemical and electrophysiological experiments. Both, silencing of parvalbumin- containing and of somatostatin-containing GABA neurons resulted in epileptic discharges and spontaneous motor seizures in the mice and were monitored for up to three months by continuous telemetric EEG and video monitoring. The seizure threshold was already reduced before the first motor seizure (after about one week) was observed. Spontaneous seizures developed only after permanent silencing of parvalbumin or somatostatin interneurons but not after their selective transient inhibition. Furthermore, the frequency of spontaneous seizures declined after 4 to 6 weeks. At the same time neuropeptide Y expression increased and that of the cannabinoid receptor CB1 declined indicating possible development of endogenous protective mechanisms.