Abstract
Temperature-Sensitive Cav1.2 Calcium Channels Support Intrinsic Firing of Pyramidal Neurons and Provide a Target for the Treatment of Febrile Seizures
Radzicki D, Yau H-J, Pollema-Mays SL, Mlsna L, Cho K, Koh S, Martina M. J Neurosci 2013;33(24):9920–9931.
Febrile seizures are associated with increased brain temperature and are often resistant to treatments with antiepileptic drugs, such as carbamazepine and phenytoin, which are sodium channel blockers. Although they are clearly correlated with the hyperthermic condition, the precise cellular mechanisms of febrile seizures remain unclear. We performed patch-clamp recordings from pyramidal cells in acute rat brain slices at temperatures up to 40°C and found that, at ≥37°C, L-type calcium channels are active at unexpectedly hyperpolarized potentials and drive intrinsic firing, which is also supported by a temperature-dependent, gadolinium-sensitive sodium conductance. Pharmacological data, RT-PCR, and the current persistence in Cav1.3 knock-out mice suggested a critical contribution of Cav1.2 subunits to the temperature-dependent intrinsic firing, which was blocked by nimodipine. Because intrinsic firing may play a critical role in febrile seizures, we tested the effect of nimodipine in an in vivo model of febrile seizures and found that this drug dramatically reduces both the incidence and duration of febrile seizures in rat pups, suggesting new possibilities of intervention for this important pathological condition.
Commentary
Seizures that accompany fever (febrile seizures) occur in 2 to 4 percent of children in countries with advanced economies and represent the most prevalent form of seizure disorder (1). Febrile seizures typically occur in children between the ages of 6 months and 3 years (median age of first seizure: 18 months), are associated with a fever of 101°F (39°C) or higher, and occur in the absence of an infection of the central nervous system or previous seizure history (1). Febrile seizures can be classified into two somewhat broad clinical categories: simple and complex (2). A febrile seizure is classified as simple if the seizure is generalized, short in duration (<15 minutes), and does not reoccur within 24 hours. Febrile seizures that last longer than 15 minutes, have focal clinical features, and are recurrent, or that comprise a combination of these features, are considered to be complex. Although the vast majority of children (60–70%) who have a febrile seizure will have no further complications (1), repeated or prolonged febrile seizures are thought to be a risk factor for subsequent epilepsy. This is especially true for children who experience febrile status epilepticus, which is a complex seizure that lasts more than 30 minutes. At present, it remains unknown how febrile seizures might contribute to the development of epilepsy later in life, but data from animal models of prolonged febrile seizures have uncovered a number of long-lasting changes that alter neuronal function and plasticity (3). And although there have been a number of genes and proteins identified as being putatively involved (4), the biology that underlies the genesis of febrile seizures continues to be poorly understood.
Recently, it has been demonstrated that hippocampal slices in vitro can exhibit fairly dramatic increases in intrinsic excitability when exposed to hyperthermic conditions, suggesting that elevated temperature can directly mediate hyperexcitability (5). The present study by Radzicki and colleagues follows up on this earlier work and provides evidence that L-type voltage-gated calcium channels may modulate firing rates of pyramidal neurons in a temperature-dependent manner, suggesting that these channels may contribute to a state of hyperexcitability during febrile episodes. For these experiments, hippocampal slices were prepared from 18-day-old Long-Evans rat pups, and recordings were made in the CA1 region of the hippocampus using either loose-seal (extracellular) or whole-cell intracellular recordings. Slice temperature was manipulated during the recordings by simply varying the temperature of the bath perfusate (artificial cerebrospinal fluid [aCSF]). Under both recording conditions, the authors were able to convincingly demonstrate that a steady increase in aCSF temperature from 32 to 40°C resulted in depolarization and spontaneous firing in about 40% of recorded neurons in CA1. Similar results were obtained in the entorhinal cortex and, to a somewhat lesser extent, in the occipital cortex. It is important to note that this spontaneous firing is an intrinsic property, as the hyperexcitability persists even in the presence of kynurenic acid and picrotoxin, which were used to block excitatory and inhibitory synaptic transmission, respectively. Similarly, the temperature-dependent depolarization and firing appear to be independent of the internal solution of the recording pipette, as these phenomena were observed when the potassium gluconate internal was substituted with a potassium phosphate–based recording solution. In addition to the spontaneous firing (which typically started at ~38°C), depolarizing steps delivered to the soma via the patch pipette resulted in significantly higher firing rates at the elevated temperature. The authors next turned to investigating the currents that might underlie the temperature-dependent depolarization and firing in hopes of identifying a specific ion channel or channels that contribute to the temperature sensitivity. In light of previous reports that a class of transient receptor potential channels (TRPV) exhibited a temperature-dependent response, the authors’ initial efforts focused on using non-specific as well as subtype specific blockers of TRPV channels but found no indication of involvement. While blocking TRPV channels had no effect, the authors did identify a temperature-sensitive background current, which was active near the resting membrane potential (−65 mV). This current appears to be similar to sodium currents that are mediated by NALAN (sodium leak channel nonselective protein) channels, in that a large portion of the current at 39°C could be blocked by bath application of gadolinium and not cadmium.
In addition to the voltage-independent sodium background current, the authors discovered that elevated temperature also significantly altered the functional kinetics of voltage-gated calcium channels (VGCCs). Specifically, temperatures around 39°C induced a leftward shift in the activation range of a voltage-sensitive inward current that was completely blocked by cadmium. In addition, a more modest decrease in inactivation kinetics was observed. In subsequent experiments, Radzicki and colleagues demonstrated that the temperature-sensitive current was blocked by nimodipine, suggesting that L-type VGCCs (L-VGCCs) are likely involved. Of interest, similar experiments carried out using mice in which one of the major L-VGCC subtypes (CaV1.3) had been constitutively deleted revealed no significant differences in peak current kinetics or firing rates when compared with control animals. Although the authors did not examine CaV1.2 knock-out mice (the other major subtype expressed in the mammalian brain), it stands to reason by subtraction that the majority of the current that was blocked by nimodipine was in fact carried by CaV1.2.
Could dihydropyridines such as nimodipine be used as a therapy to acutely reduce febrile seizures? While the experiments will need to be replicated and extended, the authors present data demonstrating that nimodipine can reduce the temperature-induced in vivo ictal activity in a rodent model of febrile seizures. This reduced in vivo ictal activity mirrors the reduction in firing rates observed in vitro, suggesting that L-VGCC channels could be an attractive therapeutic target to slow the seizure progression or perhaps ameliorate some of the downstream consequences of prolonged febrile seizure. An additional advantage to this approach would be that there are currently a number of L-VGCC antagonists that are approved for a variety of uses in the United States and Europe, and these drugs by and large have reasonable safety margins. As the authors point out in the discussion, L-VGCC antagonists might be especially attractive as an alternate therapy in cases where sodium channel blockers (e.g., phenytoin, lamotrigine, carbamazepine) may worsen seizures, as is the case for patients with Dravet syndrome.