Hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective channel subunit expression pattern of guinea-pig spiral ganglion cells.

Although the hyperpolarization-activated non-specific cationic current (I(h)) plays important roles in determining the membrane characteristics of the spiral ganglion cells (SGCs), neither the exact types of the hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective channel (HCN) subunits contributing to the molecular assembly of the relevant channels, nor their distribution pattern presented by the SGCs is known. In the present work immunolabeling and Western blot analysis were performed to describe the presence and distribution of all four known HCN subunits in the guinea-pig spiral ganglion. Besides determining the expression of the HCN1-HCN4 subunits by both type I and type II SGCs, the presence of possible apico-basal gradients in the expression patterns was also sought. The results indicate that both type I and type II SGCs express all four HCN subunits. The intensity of the immunolabeling of the cell surface membrane was generally strong, but it showed pronounced cell-to-cell variability. The Western blot experiments in combination with densitometry revealed that the amount of the HCN1 and HCN3 proteins was more significant in the apical than in the basal third of the guinea-pig cochlea. These findings not only imply potential heteromeric HCN channel formation of the spiral ganglion neurons, but they also offer a possible explanation of the previously reported heterogeneity of I(h) recorded in functional studies.

Glutamate-induced cytoplasmic Ca2+ transients in neurones isolated from the rat dorsal cochlear nucleus.

Extracellular application of glutamate elicited cytoplasmic Ca2+ transients in freshly dissociated rat neurones of the dorsal cochlear nucleus (DCN) (identified as pyramidal cells) with half-maximal concentration of 513 micromol/l while saturating doses (5 mmol/l) of this neurotransmitter caused transients of 46.1 +/- 3.0 nmol/l on an average. The genesis of these glutamate-evoked Ca2+ transients required extracellular Ca2+. When [Mg2+]o was 1 mmol/l, the NMDA receptor antagonist AP5 (100 micromol/l) had no effects while 100 micromol/l CNQX and 10 micromol/l NBQX, inhibitors of the AMPA receptors, greatly decreased the glutamate-induced Ca2+ transients (a decrease of 92 and 57%, respectively). When facilitating the activation of the NMDA receptors (50 micromol/l glycine, 20 micromol/l [Mg2+]o) in the presence of 100 micromol/l CNQX, Ca2+ transients of 55.4 +/- 13.1 nmol/l could be produced. Block of the voltage-gated Ca2+ channels (200 micromol/l Cd2+) decreased the Ca2+ transients to approx. 50%. The data indicate that under our control experimental circumstances the glutamate-induced Ca2+ transients of the isolated DCN neurones are produced mainly by Ca2+ entry through voltage-gated Ca2+ channels and AMPA receptors. However, when the activation of the NMDA receptors may take place, these receptors also contribute significantly to the genesis of the glutamate-evoked cytoplasmic [Ca2+] elevations.

Differential distribution of TASK-1, TASK-2 and TASK-3 immunoreactivities in the rat and human cerebellum.

In this work, the distributions of some acid-sensitive two-pore-domain K+ channels (TASK-1, TASK-2 and TASK-3) were investigated in the rat and human cerebellum. Astrocytes situated in rat cerebellar tissue sections were positive for TASK-2 channels. Purkinje cells were strongly stained and granule cells and astrocytes were moderately positive for TASK-3. Astrocytes isolated from the hippocampus, cerebellum and cochlear nucleus expressed TASK channels in a primary tissue culture. Our results suggest that TASK channel expression may be significant in the endoplasmic reticulum of the astrocytes. The human cerebellum showed weak TASK-2 immunolabelling. The pia mater, astrocytes, Purkinje and granule cells demonstrated strong TASK-1 and TASK-3 positivities. The TASK-3 labelling was stronger in general, but it was particularly intense in the Purkinje cells and pia mater.

HCN channels contribute to the intrinsic activity of cochlear pyramidal cells.

A hyperpolarization-activated current recorded from the pyramidal cells of the dorsal cochlear nucleus was investigated in the present study by using 150- to 200-microm-thick brain slices prepared from 6- to 14-day-old Wistar rats. The pyramidal neurones exhibited a slowly activating inward current on hyperpolarization. The reversal potential of this component was -32 +/- 3 mV (mean +/- SE, n = 6), while its half-activation voltage was -99 +/- 1 mV with a slope factor of 10.9 +/- 0.4 mV (n = 27). This current was highly sensitive to the extracellular application of both 1 mM Cs+ and 10 microM ZD7288. The electrophysiological properties and the pharmacological sensitivity of this current indicated that it corresponded to a hyperpolarization-activated non-specific cationic current (Ih). Our experiments showed that there was a correlation between the availability of the h-current and the spontaneous activity of the pyramidal cells, suggesting that this conductance acts as a pacemaker current in these neurones. Immunocytochemical experiments were also conducted on freshly isolated pyramidal cells to demonstrate the possible subunit composition of the channels responsible for the genesis of the pyramidal h-current. These investigations indicated the presence of HCN1, HCN2 and HCN4 subunits in the pyramidal cells.