Abstracts

Rationale: Dravet syndrome (DS) is a childhood-onset epilepsy leading to severe and permanent cognitive impairment. In most patients, anti-epileptic treatments fail to ameliorate cognitive deficits, indicating that mechanisms other than seizures may be involved. As many as 85% of cases of DS are linked with loss-of-function mutations in the SCN1A gene, coding for the type I voltage-gated sodium channel (Nav1.1). However, the impact of Nav1.1 loss of function on neural networks involved in cognition has not been studied. Here, we develop an RNAi approach to investigate the direct effects of Nav1.1 down-regulation on specific neural networks in vivo. Methods: We first screened siRNA constructs in vitro for efficient suppression of SCN1A expression by real-time PCR and western blot. We then examined the effects of siRNA injections in vivo by targeting the basal forebrain, a region known to be critical for learning and memory in rodents. Short-term effects on hippocampal depth EEG and spatial memory were tested in a reaction-to-novelty task. Because siRNA induces only a transient knockdown, a second approach was then developed to achieve a stable suppression of SCN1A expression. Using the same nucleotide sequence targeted to SCN1A, we constructed a lentiviral vector expressing short hairpin RNA (shRNA), and we subsequently evaluated its longer-term effects on SCN1A expression. Results: Transfection of siRNA complexes into rat neuroblastoma cells transiently suppressed SCN1A expression by 71% compared to a negative control siRNA construct (F(2,5)=28.24, p<.01, n=3 per group) with an equivalent reduction (73%) at the protein level after 48 hours (t(4)=6.15, p<.01, n=3 per group). When evaluated in vivo, knockdown of Nav1.1 in the basal forebrain region induced a spatial memory deficit. Notably, spatial memory performance was significantly related to the frequency of hippocampal theta oscillations in controls (R2=0.76, p<.05, n=6). However, this relationship was abolished after Nav1.1 knockdown (R2=0.005, p>.05, n=6). Lentiviral expression of shRNA similarly suppressed SCN1A expression in rat neuroblastoma cells. Despite a relatively low infection efficiency in this cell line (less than 50% of cells revealed GFP+ fluorescence), SCN1A expression was reduced by 51% (t(10)=3.25, p<.01, n=6 per group). In contrast to the transient effects of siRNA, this effect was stable and was observed after two weeks from the time of infection. Conclusions: We describe a novel approach to investigate the effects of Nav1.1 loss of function that has precise temporal and spatial control in vivo. Secondly, we show that Nav1.1 down-regulation in the basal forebrain region causes a dysregulation of oscillations that is related to spatial cognition. New strategies employing lentiviral expression of shRNA to stably suppress Nav1.1 expression will allow further elucidation of the neural mechanisms responsible for the dysfunction of such networks. Importantly, our results to this point strongly suggest that the loss of function of Nav1.1 in Dravet syndrome may directly impact cognition through mechanisms other than seizures.