Faculty Bibliography

Histamine-containing neurons of the tuberomammilary nucleus project to the hippocampal formation to innervate H1 and H2 receptors on both principal and inhibitory interneurons. Here we show that H2 receptor activation negatively modulates outward currents through Kv3.2-containing potassium channels by a mechanism involving PKA phosphorylation in inhibitory interneurons. PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons, which in turn negatively modulated high-frequency population oscillations recorded in principal cell layers. All these effects were absent in a Kv3.2 knockout mouse. These data reveal a novel pathway for histamine-dependent regulation of high-frequency oscillations within the hippocampal formation

M-currents are believed to play critical roles in the subthreshold behavior, and response to synaptic inputs of many CNS neurons. M-currents were first described in peripheral sympathetic neurons and have since been identified in neurons of the hippocampus, neocortex and olfactory bulb. Characteristic features of the classical M-current include a relatively low activation voltage, no measurable inactivation, and a low sensitivity to commonly used K+ channel blockers (4-AP and TEA). Another defining feature of the M-current is its inhibition by Ach via muscarinic receptors. It has been suggested that Kcnq2 and Kcnq3 subunits, when found in heteromultimers, are responsible for the classical M-current in sympathetic ganglia. However it is still unclear whether these genes are the molecular components for all M-like currents. Some members of the Eag family (eag,erg,elk) express voltage dependant currents with properties that resemble in some respects those of the M-current. These properties include a low voltage of activation and the ability to carry steady state currents in sub-threshold ranges. Eag K+ channels could thus play similar roles in neuronal excitability as members of the Kcnq family. To explore the role of these various genes in neuronal excitability, we used non-radioactive in situ hybridization to study the expression pattern of the Kcnq and Eag families of K+ channels in mouse and rat brain with particular emphasis on the thalamocortical system. These studies were then used to select cell types for electrophysiological recordings of M-like currents in vitro

Histamine-containing neurons of the hypothalamic tuberomammilary nucleus project to the hippocampal formation where they innervate both principal and inhibitory interneurons (INs) via H1 and H2 receptors. H2 receptor activation is positively linked to adenylyl cyclase formation although the downstream targets of these receptors are unclear. Using whole-cell recording of K+ currents from Dentate Gyrus INs in mouse hippocampal slices we now show that H2 receptor activation decreases K+ currents through Kv3.2-containing channels by a mechanism involving PKA phosphorylation. Currents through Kv3.2 channels activate rapidly, are essentially non-inactivating and endow INs with a 'fast spiking' phenotype. Histamine (10 uM) or dimaprit (H2 agonist, 10 uM) application reduced the K+-current by 40+-4% (n=13), and 28+-5% (n=10) respectively. Incubation in either the H2 antagonist cimetidine or PKA inhibitors prevented the reduction of Kv3.2 current by H2 agonists. H2 receptor-dependent-PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons (24+-3% decrease), which in turn decreased the high frequency (>70Hz) component of extracellular oscillations recorded in principal cell layers. All of these effects were reproduced by application of the PKA activator dibutyryl-cAMP (2mM) + IBMX (100 uM). H2 receptor modulation of outward currents and firing properties was absent in a Kv3.2 knockout mouse. Our data show that Kv3.2 containing channels are a target for histaminergic modulation and reveal a novel pathway for histamine-dependent regulation of high frequency oscillations within the hippocampal formation

Several two pore K+ channels (KT) have been identified in mammals. Amino acid sequence and phylogenetic analysis suggest the existence of 8 distinct subfamilies, with 3 subfamilies containing at least two members. Eight KT genes are expressed in the CNS. In heterologous expression systems KT genes produce currents at resting membrane potentials and therefore, are likely to play important roles in neuronal subthreshold properties. The diversity of these channels may be associated with a high degree of cellular or subcellular specificity. The diversity also provides multiple ways to regulate the subthreshold behavior of neurons. In situ hybridization experiments revealed that TASK-1 and KT3.2 are co-expressed in cerebellar granule cell layer, the olfactory bulb and in several brainstem nuclei like the ambigual, motor trigeminal, facial, vagal and hypoglossal nuclei. These two channels belong to the same subfamily (75% identity and 86% similarity in the core region) but differ in their pH dependence, and modulation by second messengers. TASK-1 is very sensitive to changes in pH in the physiological range while KT3.2 is almost insensitive to pH changes in the pH 7.0 to 8.0 range. KT3.2 is modulated by the phorbol ester PMA, a potent protein kinase C stimulator, but TASK-1 is insensitive to this treatment. If these channels are expressed in the same cells they may form heteromultimeric channels. Therefore the ability to form heteromeric complexes in vitro was investigated

We recorded intracellularly with sharp electrodes from layers 2 to 6 of S1 barrel cortex, in Ketamine-xylazine anesthetized mice in vivo. The bipolar EEG (pial surface - depth) was obtained simultaneously in the vicinity (usually apprx 1mm) of the intracellular pipette. Cells were identified as regular spiking, intrinsically bursting, fast spiking or regular fast bursting, according to the firing patterns in response to current injection through the micropipette and during synaptic activity evoked by thalamic or cortical stimulation. In addition corticothalamic projection cells were identified by their antidromic responses to thalamic stimulation. Cells oscillated in synchrony with the local EEG during the slow oscillation as described elsewhere (Steriade et al., 1993 J Neurosci. 13:3252-65; Contreras D, Steriade M 1995 J Neurosci 15: 604-22). In addition, most cells showed spontaneous spindle oscillations in synchrony with the local bipolar EEG. Cells responded with an epsp-ipsp sequence to weak electrical stimulation of the ventrobasal nucleus of the thalamus. These responses were very similar to stimulation of individual or multiple whiskers. Whiskers were stimulated with either a small hand held probe or with a piezoelectric device described by Simons, (Simons, D.J. 1983 Brain Res. 276: 178-182). In order to measure the contribution of ipsps to the responses to whisker stimulation we displaced cell's membrane potential with direct current and constructed current vs. voltage plots. In addition some cells (n=6) were recorded with pipettes filled with KCl in order to reverse chloride dependent ipsps

Inhibitory interneurons have critical functions in neocortical circuits. There are several types of inhibitory interneurons in the neocortex which differ in morphology, axonal connectivity, and firing patterns. Two major groups of inhibitory interneurons are the parvalbumin-containing fast spiking (FS) and the somatostatin-containing low-threshold spiking (LTS) cells. Both types are known to function in independent neuronal networks through reciprocal synaptic connections and gap junctions. Both types of neurons are known to contain Kv3 potassium channels. Kv3.2 and Kv3.1 channels are thought to be involved in the high frequency firing characteristic behavior of these cells due to their unusual channel properties. Kv3.2-Kv3.1 channels do not show significant activation until -10 mV and have the fastest deactivation rates of all cloned voltage-gated K+ channels. Using thalamocortical brain slices we studied the firing characteristics of FS and LTS cells in wild-type and Kv3.2 knockout (KO) mice. Electrophysiological properties of FS and LTS cells in deep neocortical layers were altered in the KO both in their repetitive firing properties and action potential waveforms. Steady state firing frequency of FS cells was lower in the KO than WT. In addition, KO FS cells were impaired in their ability to sustain high frequency firing. The cellular alterations of these interneurons may disrupt the synchrony of the inhibitory neuronal networks as suggested by in vivo data from Kv3.2 mutants

The role of a given potassium channel in neuronal excitability depends on its subcellular localization within the neuron. Several K+ channel genes encode multiple alternatively spliced proteins which differ only in their C-terminal sequence and produce channels with similar electrophysiological properties. We have previously shown that alternatively spliced products of the Kv3.2 and Kv3.1 genes are differentially localized when expressed in a polarized system such as MDCK cells, and that Kv3.1 isoforms show a differential expression in mouse brain, suggesting that the intracellular C-terminal domain is responsible for the differential targeting. We have studied the distribution of the different isoforms in infected 10-12 d.i.v. hippocampal neurons obtained from Kv3.2 knockout mice. By using the ecdysone inducible expression system combined with adenoviral vectors we show that, as expected, Kv3.2a was in axons in few cases (10% stained axons, n=21) while Kv3.2b infected neurons showed 77% stained axons, n=36, at 3 hours after induction. At longer times, both isoforms showed a similar distribution (Kv3.2a: 60%, n=29; Kv3.2b: 56%, n=28 at 9h after induction). These results support the conclusion that Kv3 C-terminal domains dictate the differential targeting behavior. However, it appears that protein overexpression can 'overload' the targeting machinery resulting in ectopic localization

Potassium channels that are open at very negative membrane potentials govern the subthreshold behavior of neurons. These channels contribute to the resting potential and help regulate the degree of excitability of a neuron by affecting the impact of synaptic inputs and the threshold for action potential generation. They can have large influences on cell behavior even when present at low concentrations because few conductances are active at these voltages. We report the identification of a new K(+) channel pore-forming subunit of the ether-a-go-go (Eag) family, named Eag2, that expresses voltage-gated K(+) channels that have significant activation at voltages around -100 mV. Eag2 expresses outward-rectifying, non-inactivating voltage-dependent K(+) currents resembling those of Eag1, including a strong dependence of activation kinetics on prepulse potential. However, Eag2 currents start activating at subthreshold potentials that are 40-50 mV more negative than those reported for Eag1. Because they activate at such negative voltages and do not inactivate, Eag2 channels will contribute sustained outward currents down to the most negative membrane potentials known in neurons. Although Eag2 mRNA levels in whole brain appear to be low, they are highly concentrated in a few neuronal populations, most prominently in layer IV of the cerebral cortex. This highly restricted pattern of cortical expression is unlike that of any other potassium channel cloned to date and may indicate specific roles for this channel in cortical processing. Layer IV neurons are the main recipient of the thalamocortical input. Given their functional properties and specific distribution, Eag2 channels may play roles in the regulation of the behavioral state-dependent entry of sensory information to the cerebral cortex