Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits.

The distribution of the binding sites for kaliotoxin (KTX), a blocker of voltage-dependent K(+) channels, was studied with quantitative autoradiography in adult rat brain and during postnatal brain maturation. Iodinated KTX bound specifically to tissue sections with a high affinity (K(d) = 82 pM) and a maximal binding capacity of 13.4 fmol/mg protein. The distribution of KTX binding sites within the central nervous system was heterogeneous. The highest densities were found in the neocortex, hypothalamus, dentate gyrus, bed nucleus of the stria terminalis, and parabrachial nuclei. The lowest level was observed in the white matter. From postnatal day 5 onward, KTX binding sites were detectable only in the hindbrain. The density of KTX binding sites in whole brain drastically increased after postnatal day 15 to achieve adult levels at postnatal day 60 in the whole brain. Bath application of KTX to Xenopus laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels potently and Kv1.2 channels less potently, with respective K(d) values of 0.1, 1.5, and 25 nM. KTX affinities for each of these channels expressed in mammalian cells were about 10-fold lower. A comparison of the distribution of KTX binding sites with that of Kv1 channel polypeptides, together with the pharmacology of KTX block, suggests that the principal targets for KTX in rat brain are K(+) channels containing Kv1.1 and Kv1.3 alpha-subunits.

Single-channel and whole-cell recordings from non-dissociated sympathetic neurones in rabbit coeliac ganglia.

A procedure is described for performing patch-clamp recordings on mammalian sympathetic neurones within intact ganglia. The plasma membrane of superficial neurones was cleaned by blowing (1.5-3 h) a gentle stream of Ringer saline onto ganglia, the connective sheath of which was previously softened by a short protease treatment. This procedure preserved the intraganglionic connectivity so that the neurones could be activated either synaptically or antidromically by stimulating the appropriate nerves. Depending on the duration of the mechanical cleaning step, recordings were performed on either the neurones or the satellite glial cells covering the neuronal cell bodies. The applicability of the various configurations of the patch-clamp technique to studying sympathetic neurones is illustrated by recordings of whole-cell voltage, whole-cell currents and single-channel currents in cell-attached and excised patches. With these techniques, the resolution of the membrane current recordings is higher than with conventional microelectrodes. The results obtained show that mammalian sympathetic neurones have a very high input resistance (0.5 G omega), are electronically compact and may display pacemaker activity. These techniques provide a useful tool for studying the synaptic transmission and neuromodulation mechanisms operating within the sympathetic ganglia.

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus. An ultrastructural and stereological study.

Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Group of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus under-goes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.

Autoradiographic detection of indolamine and catecholamine neurons in the nervous system of Owenia fusiformis (Polychaeta, Annelida).

Light- and electron-microscopic autoradiographic studies were carried out on Owenia to detect selectively catecholaminergic and indolaminergic neurons at two appropriate body levels according to the regenerative properties of this annelid, i.e. in the 1st and the 4th abdominal segments. Autoradiographically, intense 3H-5-hydroxytryptamine accumulation is seen in the 1st abdominal segment, and a less intense autoradiographic reaction in the 4th segment. Roughly, a similar difference in the distribution of the label is seen at both levels following 3H-noradrenaline administration. These observations, confirmed by a quantitative uptake analysis, suggest the presence of catecholaminergic and indolaminergic neurons in Owenia.

Radioautographic identification of serotonergic neurones in Aplysia.

Radioautography shows that 3H 5-HT specifically labels metacerebral and 2 other serotonergic cell bodies in Aplysia cerebroid ganglia as well as 5-HT terminals in close contact with giant neurone membrane in buccal ganglia.