Ágnes Pór

Presence and distribution of three calcium binding proteins in projection neurons of the adult rat cochlear nucleus

The presence and distribution of three cytoplasmic calcium binding proteins, calbindin, calretinin, and parvalbumin, have been investigated in the projection neurons of the cochlear nucleus complex in adult rats by using immunohistochemistry in free-floating slices. Identification of the individual cell types was carried out on the basis of their intranuclear localization, morphological characteristics, and (in the cases of pyramidal and bushy neurons) by retrograde labeling with rhodamine-dextran. The most important findings were confirmed by using confocal microscopy. The data obtained in these experiments are the first to demonstrate the presence of parvalbumin in pyramidal neurons and globular and spherical bushy cells of rat cochlear nucleus, whereas octopus and giant cells did not show positivity for parvalbumin. Calretinin was not present in either Purkinje-like cells or giant neurons. According to the double immunolabeling co-localization experiments, the pyramidal neurons, Purkinje-like cells, globular bushy cells, and octopus cells express two different calcium binding proteins in their cytoplasm (although in different combinations) whereas giant cells and spherical bushy cells contain solely calbindin and parvalbumin, respectively. The presence of calretinin in globular bushy cells provides a tool for distinguishing them from spherical bushy cells. The immunolabeling of the fibers and axonal endings of the acoustic nerve in the ventral part of the cochlear nucleus indicated that these structures are also parvalbumin positive. It is concluded that the heterogenous cell composition of the cochlear nucleus is accompanied by a rather complex expression pattern of the cytoplasmic calcium binding proteins.

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-μm-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 μM 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.

Voltage-gated and background K+ channel subunits expressed by the bushy cells of the rat cochlear nucleus.

Bushy cells of the ventral cochlear nucleus produce a single, short latency action potential at the beginning of long depolarisations. In the present work an immunochemical survey was performed to detect the presence of K+ channel subunits which may contribute to the specific membrane properties of the bushy cells. The immunocytochemical experiments conducted on enzymatically isolated bushy cells indicated positive immunolabelling for several subunits known to be responsible for the genesis of rapidly inactivating K+ currents. Bushy cells showed strong expression of Kv3.4, 4.2 and 4.3 subunits, with the lack of Kv1.4 specific immunoreaction. The Kv3.4-specific immunoreaction had a specific, patchy appearance. Bushy cells also expressed various members of the Kv1 subunit family, most notably Kv1.1, 1.2, 1.3 and 1.6. Weak positivity could be observed for Kv3.2 subunits. The positive immunolabelling for Kv3.4, Kv4.2 and Kv4.3 was confirmed in free-floating tissue slices. Voltage-clamp experiments performed on positively identified bushy cells in brain slices corroborated the presence and activity of Kv3.4 and Kv4.2/4.3 containing K+ channels. Bushy cell showed strong immunopositivity for TASK-1 channels too. The results presented in this work indicate that bushy cells possess several types of voltage-gated K+ channel subunits whose activity may contribute to the membrane properties and firing characteristics of these neurones.

Voltage-gated Potassium Channel (Kv) Subunits Expressed in the Rat Cochlear Nucleus

Because the neuronal membrane properties and firing characteristics are crucially affected by the depolarization-activated K+ channel (Kv) subunits, data about the Kv distribution may provide useful information regarding the functionality of the neurons situated in the cochlear nucleus (CN). Using immunohistochemistry in free-floating slices, the distribution of seven Kv subunits was described in the rat CN. Positive labeling was observed for Kv1.1, 1.2, 1.6, 3.1, 3.4, 4.2, and 4.3 subunits. Giant and octopus neurons showed particularly strong immunopositivity for Kv3.1; octopus neurons showed intense Kv1.1- and 1.2-specific reactions also. In the latter case, an age-dependent change of the expression pattern was also documented; although both young and older animals produced definite labeling for Kv1.2, the intensity of the reaction increased in older animals and was accompanied with the translocation of the Kv1.2 subunits to the cell surface membrane. The granule cell layer exhibited strong Kv4.2-specific immunopositivity, and markedly Kv4.2-positive glomerular synapses were also seen. It was found that neither giant nor pyramidal cells were uniform in terms of their Kv expression patterns. Our data provide new information about the Kv expression of the CN and also suggest potential functional heterogeneity of the giant and pyramidal cells.

Voltage-gated K+ channel (Kv) subunit expression of the guinea pig spiral ganglion cells studied in a newly developed cochlear free-floating preparation

The spiral ganglion accommodates the cell bodies of the acoustic nerve fibres connecting the hair cells to the central nervous system. As the ionic channels containing various voltage-gated K+ channel (Kv) subunits play pivotal roles in determining the functional properties and firing behaviour of the spiral ganglion cells (SGCs), every piece of information concerning the Kv expression of the SGCs is valuable. In the present work a comprehensive immunohistochemical analysis was performed to describe the expression of 9 Kv subunits in the guinea pig cochlea on traditional wax-embedded sections as well as employing a newly developed preparation that allowed confocal analysis, reconstruction of the three-dimensional appearance and precise morphological characterisation of the SGCs. Besides determining their Kv expression patterns, differences between type I and type II SGCs were sought. SGCs showed positivity for 8 out of the 9 Kv subunit-specific antibodies with varying intensity and proportion of the immunopositive cells; whereas no obvious Kv3.2 positivity could be noted. Type I and type II cells demonstrated similar expression patterns for all subunits tested, with the exception of Kv1.2, whose presence was confirmed in only 50% of the type II cells. Although the present findings suggest that type I and type II cells do not differ fundamentally in the Kv subunits they possess; they also imply that SGCs may not form a homogeneous cell population, and might provide explanation of the previously noted heterogeneity of the membrane properties of the SGCs.

Removal of Ca2+ following depolarization-evoked cytoplasmic Ca2+ transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca(2+)] ([Ca(2+)](i)) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca(2+) removal following K(+) depolarization-induced Ca(2+) transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca(2+)](i)) had monophasic time course (time constant of 6.43 +/- 0.48 s). In the cases of higher Ca(2+) transients biphasic decay was found. The early time constant decreased (from 3.09 +/- 0.26 to 1.46 +/- 0.11 s) as the peak intracellular [Ca(2+)] increased. The value of the late time constant was 18.15 +/- 1.60 s at the smallest transients, and showed less dependence on [Ca(2+)](i). Blockers of Ca(2+) uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca(2+) transients and slowed their decay. La(3+) (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca(2+) transients as a plateau developed and persisted until the La(3+) was present. When the other Ca(2+) removal mechanisms were available, reduction of the external [Na(+)] to inhibit the Na(+)/Ca(2+) exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca(2+) depends mainly on the activity of Ca(2+) pump mechanisms.

Endocannabinoid Tone Regulates Human Sebocyte Biology

We have previously shown that endocannabinoids (eCBs) (e.g., anandamide) are involved in the maintenance of homeostatic sebaceous lipid production in human sebaceous glands and that eCB treatment dramatically increases sebaceous lipid production. Here, we aimed to investigate the expression of the major eCB synthesizing and degrading enzymes and to study the effects of eCB uptake inhibitors on human SZ95 sebocytes, thus exploring the role of the putative eCB membrane transporter, which has been hypothesized to facilitate the cellular uptake and subsequent degradation of eCBs. We found that the major eCB synthesizing (N-acyl phosphatidylethanolamine-specific phospholipase D, and diacylglycerol lipase-α and -β) and degrading (fatty acid amide hydrolase, monoacylglycerol lipase) enzymes are expressed in SZ95 sebocytes and also in sebaceous glands (except for diacylglycerol lipase-α, the staining of which was dubious in histological preparations). eCB uptake-inhibition with VDM11 induced a moderate increase in sebaceous lipid production and also elevated the levels of various eCBs and related acylethanolamides. Finally, we found that VDM11 was able to interfere with the proinflammatory action of the TLR4 activator lipopolysaccharide. Collectively, our data suggest that inhibition of eCB uptake exerts anti-inflammatory actions and elevates both sebaceous lipid production and eCB levels; thus, these inhibitors might be beneficial in cutaneous inflammatory conditions accompanied by dry skin.