Abstracts

Rationale: Experimental febrile seizures, and kainate- or pilocarpine-induced epilepsy in rats all produce a persistent down-regulation in the expression of the hyperpolarization-activated HCN1 channel, a key determinant of intrinsic neuronal excitability. However, it is unclear whether the decrease in HCN1 expression contributes to increased excitability in limbic circuitry, or provides a homeostatic response to limit excitability by driving neuronal resting potential to more hyperpolarized values and/or reducing rebound firing. To address this question, we sought to determine the seizure susceptibility of genetically modified (knock-out) mice carrying a deletion of the HCN1 gene, using two different experimental paradigms: amygdala kindling and pilocarpine injection. Methods: Mice were obtained by breeding heterozygote HCN1+/- animals (129SvEv x C57BL/6J, F1) to obtain HCN1-/- and HCN1+/+ littermates (F2). Males, 3-4 months, were stereotactically implanted with bipolar electrodes in right amygdala for kindling or injected with 300 mg/kg pilocarpine i.p. (preceded by 5 mg/kg atropine). Seizure class rating was according to Butler 1995 PNAS 92:6852 (for kindling) or Winawer 2006 Mamm Genome 18:23 (for pilocarpine). Results: The HCN1 KO mice show no detectable spontaneous seizure activity. In the amygdala kindling model, the mutant mice showed no significant differences with wild-type littermates in afterdischarge threshold, seizure class per stimulus number, or number of stimuli needed to obtain full kindling (defined as three consecutive class 5 seizures or higher). However, a total of 5/9 animals in the KO group presented with tonic hindlimb extensions culminating in death (class 8) compared to none in the wild-type group (0/12 animals). Thus, lack of HCN1 expression was associated with more severe seizures and increased seizure-related mortality with no change in seizure threshold. Similarly, following pilocarpine exposure the average seizure class reached at 30 min from the time of injection was significantly higher for the KO group (3.2 + 0.18; n=15) compared with the wild-type littermates (2.3 + 0.19; n=14; p<0.01). All HCN1 KO animals used in this study displayed an abnormal baseline neurological phenotype, characterized by altered hindlimb tone, periodic back arching, and slow sideways motions of the head (paroxysmal dystonia). The presence of such abnormalities provides an intriguing link between certain movement disorders and seizure disorders. However, it may affect our ability to accurately assess seizure intensity in HCN1 KO mice based on purely behavioral manifestations. Conclusions: Lack of HCN1 expression is associated with more severe seizure behavior, both in the case of amygdala kindling and pilocarpine injection. While these findings suggest a net increase in seizure susceptibility following global HCN1 down-regulation, further experiments using restricted HCN1 deletions are needed to control for altered motor behaviors in the KO mice and to address the relative contribution of different brain structures and neuronal subtypes (glutamatergic vs GABAergic) to the seizure phenotype.