Abstract
Impaired Excitability of Somatostatin- and Parvalbumin-Expressing Cortical Interneurons in a Mouse Model of Dravet Syndrome.
Tai C, Abe Y, Westenbroek RE, Scheuer T, Catterall WA. Proc Natl Acad Sci U S A 2014;111:E3139–E3148.
Haploinsufficiency of the voltage-gated sodium channel NaV1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of NaV1.1 in forebrain GAB-Aergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the NaV1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.
Commentary
Dravet syndrome is an infantile-onset epileptic encephalopathy that results in drug-refractory epilepsy, delays of cognitive and motor development, and increased mortality risk (1). In more than 80% of cases, Dravet syndrome is caused by heterozygous mutation of the SCN1A gene that encodes the NaV1.1 voltage-gated sodium channel (2). Approximately half of Dravet syndrome SCN1A mutations result in loss of NaV1.1 protein due to nonsense, splice site, or frameshift mutations, indicating that SCN1A is haploinsufficient. Mice with heterozygous deletion of Scn1a provide a genetic model of Dravet syndrome and recapitulate many of the clinical features observed in patients, including spontaneous seizures, cognitive deficits, and premature lethality (3, 4). While mice with Scn1a+/− global deletion provide the most accurate model for Dravet syndrome, mice with conditional Scn1a deletion in particular neuron types have provided valuable information about which neurons are most profoundly affected by reduced NaV1.1 function. Deletion of Scn1a in only cortical excitatory neurons does not result in an observable phenotype, while deletion in cortical interneurons results in a phenotype that is similar or worse than global deletion in Scn1a+/− mice (5–7). This suggested that NaV1.1 function is particularly important for signaling in inhibitory neurons, lending support to the hypothesis that Dravet syndrome is an “interneuronopathy.” Further dissection of interneuron subtypes by conditional deletion in parvalbumin-expressing interneurons showed that impaired NaV1.1 function in these interneurons contributes to the phenotype of Dravet mice (6, 7). However, the phenotype was slightly milder suggesting that additional interneuron subtypes are likely involved.
Within the cortex, inhibitory GABAergic interneurons are heterogeneous with many different subtypes defined by distinct morphological, physiological, and molecular characteristics (8, 9). Two major interneuron subtypes in the neocortex are fast-spiking interneurons that express parvalbumin and Martinotti cells that express somatostatin. Together, they make up approximately 80% of cortical interneurons. These two subtypes differ in their axonal targets and, therefore, exert different inhibitory functions. Fast-spiking parvalbumin-expressing interneurons target the perisomatic region of pyramidal cells where they exert powerful feed-forward inhibition. Martinotti cells target the apical dendrites of layer V tufted pyramidal neurons and mediate disynaptic inhibition and recurrent feedback inhibition. The dendritic targeting of Martinotti cell inhibition predominantly modulates incoming pyramidal cell inputs, while perisomatic-targeting PV interneurons have a greater influence on pyramidal cell outputs.
In the current study, Tai and colleagues sought to dissect the contribution of these neocortical GABAergic interneuron subtypes to the pathophysiology of Dravet syndrome. To characterize the functional effects of NaV1.1 deletion in parvalbumin-expressing, fast-spiking (PV) interneurons and Martinotti cells, they performed electrophysiological recording in brain slices prepared from somatosensory cortex of Scn1a+/− mice and wild-type Scn1a+/+ controls. The slices were isolated at postnatal days 20–22, during a critical vulnerable period when the Scn1a+/− Dravet mice have numerous spontaneous seizures and are at highest risk for sudden unexpected death.
Recordings of layer V PV interneurons showed a significant reduction of excitability in Scn1a+/− compared to wild-type. In response to depolarizing current injections, Scn1a+/− PV interneurons fired fewer action potentials at lower frequencies than wild-type PV interneurons. Although the general morphology of single action potentials did not differ, the action potential threshold was significantly increased and the spike amplitude was significantly decreased in Scn1a+/− PV interneurons compared to wild-type. In response to trains of current injection, Scn1a+/− PV interneurons showed an increased AP failure rate at higher frequencies relative to wild-type, indicating that NaV1.1 channels are required for reliable spike firing in response to high frequency stimulation. Taken together, these results suggest that Scn1a+/− PV interneurons are less capable of mediating inhibition and would likely fail under conditions of repetitive stimulation.
The authors also looked at how PV interneurons respond to synaptic inputs from neighboring excitatory pyramidal neurons using dual current-clamp recordings. In response to pyramidal neuron stimulation, amplitudes of excitatory post-synaptic potentials (EPSPs) in Scn1a+/− PV interneurons were approximately half those observed in wild-type. Conversely, stimulation of PV interneurons resulted in similar pyramidal neurons responses in Scn1a+/− and wild-type, as long as the PV interneurons were stimulated equivalently. Overall, this indicates that the synaptic communication between PV and pyramidal neurons is not impaired but rather that the Scn1a+/− PV interneurons may be intrinsically less excitable.
Although much attention in Dravet models has focused on PV interneurons, there are many other important interneuron classes that have not previously been examined. To address the potential contribution of other interneuron classes, the authors examined the function of layer V Martinotti cells in neocortical slices from Scn1a+/− Dravet mice. Similar to the PV interneurons, Scn1a+/− Martinotti cells fired fewer action potentials at lower frequencies than wild-type in response to current injections. Single action potential characteristics were similar between Scn1a+/− and wild-type Martinotti cells, except that Scn1a+/− cells had a significantly higher action potential threshold. In response to trains of current injection, Scn1a+/− Martinotti cells had an increased failure rate at higher frequencies compared to wild-type. This indicates that NaV1.1 channels are critical for reliable firing of Martinotti cells, and would likely fail to mediate inhibition under conditions of high frequency stimulation.
Martinotti cells are important mediators of frequency-dependent disynaptic inhibition (FDDI), whereby high frequency firing of a pyramidal cell leads to inhibition of neighboring pyramidal cells via a strongly facilitating synapse. To examine the effect of NaV1.1 loss on FDDI, the authors performed dual current clamp recording in neighboring layer V pyramidal cells. The probability of observing GABAergic-dependent FDDI between neighboring pyramidal neurons was significantly reduced in Scn1a+/− slices compared to wild-type controls at all stimulus frequencies. This would likely result in a significant deficit in cortical circuit inhibition. To examine monosynaptic transmission, the authors performed dual current recordings on pairs of pyramidal cells and Martinotti cells. They observed that in response to pyramidal cell stimulation, EPSP amplitudes were significantly reduced in Scn1a+/− Martinotti cells relative to wild-type. However, there was no difference in pyramidal cell response to Martinotti cell inputs of equivalent strength. Similar to the PV interneurons, the data suggest that the observed impairments are the result of Scn1a+/− Martinotti cells being less excitable.
The identification of functional impairment in a second major class of inhibitory interneurons adds another dimension to the Dravet syndrome neuronal phenotype: These two interneuron subtypes are major mediators of inhibition in the neocortex and impairments in both classes have previously been associated with nervous system disorders, including epilepsy and schizophrenia (8).
Overall, this study is a step forward in our understanding of the neuronal phenotypes in Dravet syndrome. However, we are still in the early days of understanding the pathophysiology. There are many other GABAergic interneuron subtypes to explore, and there may be regional and age-dependent variation in specific dysfunction. Furthermore, while cortical interneurons exert powerful inhibitory control on pyramidal neurons, they also synapse on one another and coordinate inhibition that underlies cortical oscillations. Additional levels of analysis are needed to integrate this information into the cortical network to understand how dysfunctional neurons result in seizures, as well as cognitive and motor deficits.