Phox2b-expressing neurons contribute to breathing problems in Kcnq2 loss- and gain-of-function encephalopathy models.

Loss- and gain-of-function variants in the gene encoding KCNQ2 channels are a common cause of developmental and epileptic encephalopathy, a condition characterized by seizures, developmental delays, breathing problems, and early mortality. To understand how KCNQ2 dysfunction impacts behavior in a mouse model, we focus on the control of breathing by neurons expressing the transcription factor Phox2b which includes respiratory neurons in the ventral parafacial region. We find Phox2b-expressing ventral parafacial neurons express Kcnq2 in the absence of other Kcnq isoforms, thus clarifying why disruption of Kcnq2 but not other channel isoforms results in breathing problems. We also find that Kcnq2 deletion or expression of a recurrent gain-of-function variant R201C in Phox2b-expressing neurons increases baseline breathing or decreases the central chemoreflex, respectively, in mice during the light/inactive state. These results uncover mechanisms underlying breathing abnormalities in KCNQ2 encephalopathy and highlight an unappreciated vulnerability of Phox2b-expressing ventral parafacial neurons to KCNQ2 pathogenic variants.

Functional downregulation of GluR2 in piriform cortex of kindled animals.

We have previously shown kindling-induced downregulation of the AMPA receptor GluR2 subunit in piriform cortex, as measured by Western blotting. In the present studies, we performed whole-cell patch clamp analysis of AMPA receptor-mediated currents from kindled and control animals to determine if the downregulation observed previously had any functional significance. These experiments were done in the absence and presence of N-hydroxyphenylpropanoyl spermine (HPPS), a polyamine that blocks currents through AMPA receptors lacking GluR2. We report that AMPA receptor-mediated currents recorded from piriform cortex layer II pyramidal cells in slices from animals kindled to 10 fully generalized seizures were blocked by HPPS. In contrast, application of HPPS had no effect on current amplitude in control animals, or in animals that had not been fully kindled. Western blotting revealed that decreases in GluR2 were seen in animals that had experienced at least one fully generalized seizure, but were not observed at earlier stages of kindling development. The increased polyamine sensitivity of AMPA receptor-mediated currents in kindled animals is consistent with the hypothesis that kindling induces formation of AMPA receptors that lack GluR2 in piriform cortex pyramidal cells. It has been demonstrated that polyamine sensitivity is directly correlated with the calcium permeability of the AMPA receptor, suggesting that kindling results in the formation of AMPA receptors that are calcium-permeable. Increases in intracellular calcium through these receptors could act as a second messenger and play a role in the initiation of long-term changes that contribute to the pathogenesis of kindling-induced epilepsy.

Molecular cloning and expression of the rat EAAT4 glutamate transporter subtype.

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. Here we report the cloning of rat EAAT4 (rEAAT4) cDNA from rat cerebellum. The nucleotide sequence of rEAAT4 was 88% identical to the human sequence, and the predicted peptide was 89% identical to the human protein. The transport activity encoded by rEAAT4 has high affinity for L-glutamate. In Xenopus laevis oocytes expressing rEAAT4, L-glutamate and other transporter substrates elicited a current predominantly carried by chloride ions. Like human EAAT4, the rEAAT4 mRNA was largely restricted to cerebellar Purkinje cells; the rEAAT4 protein was localized to Purkinje cell somas and dendrites.

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4.

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.

Alterations in glutamate transporter protein levels in kindling-induced epilepsy.

There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with Seizures. To date, three distinct Na(+)-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. NO changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.