Alteration of Mesopontine Cholinergic Function by the Lack of KCNQ4 Subunit.

The pedunculopontine nucleus (PPN), a structure known as a cholinergic member of the reticular activating system (RAS), is source and target of cholinergic neuromodulation and contributes to the regulation of the sleep-wakefulness cycle. The M-current is a voltage-gated potassium current modulated mainly by cholinergic signaling. KCNQ subunits ensemble into ion channels responsible for the M-current. In the central nervous system, KCNQ4 expression is restricted to certain brainstem structures such as the RAS nuclei. Here, we investigated the presence and functional significance of KCNQ4 in the PPN by behavioral studies and the gene and protein expressions and slice electrophysiology using a mouse model lacking KCNQ4 expression. We found that this mouse has alterations in the adaptation to changes in light-darkness cycles, representing the potential role of KCNQ4 in the regulation of the sleep-wakefulness cycle. As cholinergic neurons from the PPN participate in the regulation of this cycle, we investigated whether the cholinergic PPN might also possess functional KCNQ4 subunits. Although the M-current is an electrophysiological hallmark of cholinergic neurons, only a subpopulation of them had KCNQ4-dependent M-current. Interestingly, the absence of the KCNQ4 subunit altered the expression patterns of the other KCNQ subunits in the PPN. We also determined that, in wild-type animals, the cholinergic inputs of the PPN modulated the M-current, and these in turn can modulate the level of synchronization between neighboring PPN neurons. Taken together, the KCNQ4 subunit is present in a subpopulation of PPN cholinergic neurons, and it may contribute to the regulation of the sleep-wakefulness cycle.

Alterations of the perivascular dystrophin-dystroglycan complex following brain lesions: an immunohistochemical study in rats.

Dystroglycan is a laminin receptor, which with dystrophins and other components forms the dystrophin-dystroglycan complex. It has an important role in the formation of gliovascular connections, cerebral vascularisation and blood-brain barrier. Dystroglycan consists of two sub-units, α and ß. Previous studies demonstrated that the ß-dystroglycan immunoreactivity of cerebral vessels temporarily disappeared in the area adjacent to the lesion, whereas the vascular laminin which is not immunoreactive in the intact brain became detectable. The present study extends these investigations over other components of the complex: utrophin, α1-syntrophin and α1-dystrobrevin. The experiments were performed on adult rats. The lesions were stab wounds or cryogenic lesions in deep ketamine-xylasine narcosis. Following survival periods 2 to 30 days, the animals were perfused and floating brain sections were processed for fluorescent immunohistochemistry. The α1-dystrobrevin, like ß-dystroglycan, vanished temporarily around the lesion. The immunoreactivity of utrophin changed in a similar way to that of laminin. In intact brains they were confined to the entering segments of the vessels and to the circumventricular organs. Following lesions their immunoreactivity manifested in the vessels around the lesions. However, utrophin followed laminin with a delay: their peaks were about POD (postoperative days) 21 and 7, respectively. Only immunoreactivity of α1-syntrophin appeared in the reactive astrocytes, peaking at POD 14. Double-labeling proved its co-localization with GFAP. Cryogenic lesions had similar immunohistochemical effects, but provided more suitable samples for Western blot analysis, which proved the altered levels of α1-dystrobrevin and α1-syntrophin. The phenomena may help to monitor the post-lesion vascular processes and the alterations of the gliovascular connections.

Targets, receptors and effects of muscarinic neuromodulation on giant neurones of the rat dorsal cochlear nucleus.

Although cholinergic modulation of the cochlear nucleus (CN) is functionally important, neither its cellular consequences nor the types of receptors conveying it are precisely known. The aim of this work was to characterise the cholinergic effects on giant cells of the CN, using electrophysiology and quantitative polymerase chain reaction. Application of the cholinergic agonist carbachol increased the spontaneous activity of the giant cells; which was partly the consequence of the reduction in a K(+) conductance. This effect was mediated via M4 and M3 receptors. Cholinergic modulation also affected the synaptic transmission targeting the giant cells. Excitatory synaptic currents evoked by the stimulation of the superficial and deep regions of the CN were sensitive to cholinergic modulation: the amplitude of the first postsynaptic current was reduced, and the short-term depression was also altered. These changes were mediated via M3 receptors alone and via the combination of M4, M2 and M3 receptors, when the superficial and deep layers, respectively, were activated. Inhibitory synaptic currents evoked from the superficial layer showed short-term depression, but they were unaffected by carbachol. In contrast, inhibitory currents triggered by the activation of the deep parts exhibited no significant short-term depression, but they were highly sensitive to cholinergic activation, which was mediated via M3 receptors. Our results indicate that pre- and postsynaptic muscarinic receptors mediate cholinergic modulation on giant cells. The present findings shed light on the cellular mechanisms of a tonic cholinergic modulation in the CN, which may become particularly important in evoking contralateral excitatory responses under certain pathological conditions.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

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.