Increase in polysialyltransferase gene expression following LTP in adult rat dentate gyrus.

Neural cell adhesion molecule (NCAM) is frequently associated with polysialic acid (PSA), and its function is highly dependent on this polysialylation. PSA-NCAM plays an important role in synaptic plasticity in the hippocampus. STX and PST are the enzymes responsible for NCAM polysialylation. We investigated whether unilateral long-term potentiation (LTP) induction in vivo, in adult rat dentate gyrus (DG), triggered NCAM polysialylation by STX and PST produced in the hippocampus. We found that levels of STX and PST mRNA increased strongly since the early stage of hippocampal LTP and remained high during the maintenance of DG-LTP for 4 h. This rapid increase in polysialyltransferase gene expression occurred in both the hippocampi, probably resulting from bilateral LTP induction by strong unilateral HFS. Thus, LTP triggers interhemispheric molecular changes in the hippocampal network. This study is the first to describe the effects of LTP induction and maintenance on polysialyl-transferases in vivo. Our findings suggest that hippocampal synaptic remodeling requires NCAM polysialylation.

Characterization of mapacalcine-sensitive Ca(2+) channels in rat kidney.

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.

A new class of scorpion toxin binding sites related to an A-type K+ channel: pharmacological characterization and localization in rat brain.

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A-type current of striatum neurons in culture completely disappeared when 1 microM sBmTX3 was applied (Kd=54 nM), whereas the sustained K+ current was unaffected. 125I-sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol x mg(-1) of protein, Kd=0.21 nM). A panel of toxins yet described as specific ligands for K+ channels were unable to compete with 125I-sBmTX3. A high density of 125I-sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.

Apamin improves reference memory but not procedural memory in rats by blocking small conductance Ca(2+)-activated K(+) channels in an olfactory discrimination task.

Apamin blocks SK channels responsible for long-lasting hyperpolarization following the action potential. Using an olfactory associative task, the effect of an intracerebroventricular 0.3 ng apamin injection was tested on learning and memory. Apamin did not modify the learning of the procedure side of the task or the learning of the odor-reward association. To test reference memory specifically, the rats were trained on a new odor-association problem using the same procedure (acquisition session), and they were tested for retention 24 h later. Apamin injected before or after the acquisition session improved retention of the valence of a new odor pair. Apamin injected before the retention session did not affect the retrieval of the new valence. Thus, the results indicate that the blockage of apamin-sensitive SK channels facilitate consolidation on new-odor-reward association.

Kaliotoxin, a Kv1.1 and Kv1.3 channel blocker, improves associative learning in rats.

Olfactory associative learning was used to investigate the involvement of Kv channels containing Kv1.1 and Kv1.3 alpha-subunits in learning and memory. Kaliotoxin (KTX), a specific inhibitor of these Kv channels, was injected intracerebroventricularly in the rat brain, at a dose of 10 ng that did not disturb the rats' locomotor activity or drinking behaviour. In the first paradigm (odour-reward training), KTX improved learning but not information consolidation. Moreover, KTX increased the long-term retrieval of an odour-reward association tested by a reversal test 1 month after the odour-reward training. The second paradigm (successive odour-pair training) tested reference memory. The first session was an acquisition session where the rats learned a new odour-discrimination problem with the same procedure. The second was a retention session held 24 h later to test retrieval of the learned information. KTX injected before the acquisition or retention session improved performance, but no effect was found when KTX was injected immediately after acquisition. We showed that these effects were not due to the action of KTX on attention processes. Thus, these results suggest that the blockage of Kv1.1 or Kv1.3 channels by KTX facilitates cognitive processes as learning, in particular in a reference representation.

Distribution of mapacalcine receptors in the central nervous system of rat using the [125I]-labeled mapacalcine derivative.

Mapacalcine is a dimeric protein of Mr 19041 extracted from the marine sponge Cliona vastifica. Electrophysiological and pharmacological approaches have demonstrated that mapacalcine was blocking a calcium channel different from N-, L-, P-, T- or Q-type calcium channels on mouse intestinal smooth muscle. Recently a [125I]-labeled derivative of mapacalcine has been synthesized and characterized as a tool usable as a probe to investigate mapacalcine receptors. On rat brain membranes, it binds to its receptor with a K(d)=0.35 nM and a maximal binding capacity of 706 fmol/mg protein. We use here [125I]-mapacalcine to study the mapping of its receptors in the rat brain. Data obtained show a practically homogeneous labeling of the brain. Our experiments suggest that mapacalcine receptors are present on neuronal and glial cells. Interestingly, choroid plexus demonstrates a high density of mapacalcine receptors. These data would suggest that mapacalcine sensitive calcium channels could be involved in the control of calcium homeostasis of the cerebrospinal fluid.

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.

Behaviors and neurodegeneration induced by two blockers of K+ channels, the mast cell degranulating peptide and Dendrotoxin I.

Both the Mast Cell Degranulating (MCD) peptide and Dendrotoxin I (DTX(I)), two blockers of fast activation and slowly inactivating K+ channels, induced epileptiform seizures and brain damage after intracerebroventricular injection (200 pmol) in Sprague-Dawley rats. A considerable variation in the response of the rats was observed for each toxin. The neurodegeneration included the hippocampal formation, subiculum, septum, amygdala, and the cerebellum for both toxins, and the neocortex and anterior thalamic nuclei exclusively for DTX(I) treatment.

Apamin, a blocker of the calcium-activated potassium channel, induces neurodegeneration of Purkinje cells exclusively.

Following acute intracerebroventricular injections of 1 ng of apamin and chronic apamin infusion (0.4 ng/microl, 0.5 microl/h, 14 days), the rat brains exhibited bilateral damage only in the cerebellum. The argyrophilic cells were Purkinje cells in copula pyramis, flocculus, paraflocculus, and paramedian lobules. These data demonstrate that the inactivation of small conductance Ca2+-activated K+ channels by apamin induces a non-limbic neurodegeneration.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.

Scyllatoxin, a blocker of Ca(2+)-activated K+ channels: structure-function relationships and brain localization of the binding sites.

Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.

Increase of sodium channels in demyelinated lesions of multiple sclerosis.

Redistribution of sodium channels along demyelinated pathways in multiple sclerosis (MS) could be an important event in restoring conduction prior to other reparative mechanisms such as remyelination. Sodium channels in human multiple sclerosis lesions were identified by quantitative light microscopic autoradiography using tritiated saxitoxin (STX), a highly specific sodium channel ligand. Demyelinated areas in various central nervous system regions containing denuded but vital axons exhibited a high increase of STX-binding sites by up to a factor of 4 as compared to normal human white matter. This important finding could explain aspects of fast clinical remissions and 'silent' MS lesions on functional and morphological properties. Demyelinated axons may functionally reorganize their membranes and adapt properties similar to those of slow conducting unmyelinated nerve fibres which have a higher amount and a more diffuse distribution of STX binding sites. This report is the first description of an altered distribution of voltage-sensitive sodium channels in human multiple sclerosis lesions.

Effect of apamin, a toxin that inhibits Ca(2+)-dependent K+ channels, on learning and memory processes.

Apamin, a neurotoxin extracted from bee venom, specifically binds to a particular class of Ca(2+)-activated K+ channels which are involved in the slow afterhyperpolarization (S-AHP) that follows action potentials in many excitable cells. We tested in mice the effects of apamin on learning and memory processes. The results showed that pre-training injection of apamin accelerated the acquisition of a bar-pressing response but also increased the bar-pressing rates of the animals. This latter result suggests that apamin accelerated acquisition because it increased behavioral activity in general and the number of bar-presses in particular. Post-training apamin injection retroactively and non-contingently facilitated memory processes taking place shortly after training in a bar-pressing task. The lack of an effect of the delayed apamin injection showed that apamin did not act proactively on memory retrieval processes. These results suggest that apamin-sensitive KCa channels may contribute to memory processes.

Specific hippocampal lesions indicate the presence of sulfonylurea binding sites associated to ATP-sensitive K+ channels both post-synaptically and on mossy fibers.

The density of glibenclamide (GB) binding sites decreases considerably in the dentate gyrus (greater than 80%), the CA1 field (approximately 75%) and the stratum lucidum of CA3 field (approximately 70%) after intradentate colchicine injections in rat hippocampus. The density of GB receptors is unchanged in kainic acid damaged hippocampus. These data show that GB binding sites associated to adenosine triphosphate-sensitive K+ channels are mainly located in granular cells in both pre- and post-synaptic positions. They are present in mossy fibers.

Saxitoxin-sensitive Na+ channels: presynaptic localization in cerebellum and hippocampus of neurological mutant mice.

The autoradiographic distribution of saxitoxin (STX) binding sites associated with voltage-sensitive Na+ channel was studied in the cerebellum of neurological weaver (wv/wv), Purkinje cell degeneration (pcd/pcd), nervous (nr/nr) and reeler (rl/rl) mutant mice. The Purkinje cell layer contains the highest density of STX binding sites in normal mice. High densities were observed in the molecular layer. Intermediate and very low densities were present in the granular layer and the white matter respectively. There was an important decrease of grain density in the molecular layer and Purkinje cell layer of wv/wv cerebellum, where a large majority of granular cells had disappeared. In pcd/pcd and nr/nr mutants, a small decrease was observed in Purkinje cell layer where the Purkinje cells had almost all degenerated. In rl/rl mutants where all neuronal cells were malpositioned, the compacted molecular layer contained an increased STX binding sites density. Conversely the labelling of Purkinje cells areas was decreased. The hippocampal formation of rl/rl mutants presents an homogeneous repartition of the Na+ channel protein in contrast with the laminated distribution observed in normal mice. Our autoradiographic data suggest that a major proportion of STX-sensitive Na+ channels are localized in parallel fibers of granular cells and in axons of basket cells in a presynaptic position. In Purkinje cells, the dendritic arborization seems to be devoid of STX binding sites conversely to somata.

Brain ischemia alters the density of binding sites for glibenclamide, a specific blocker of ATP-sensitive K+ channels.

Transient ischemia in normoglycemic animals leads to delayed neuronal damage which is confined to selectively vulnerable regions. In at least one of these, the CA1 sector of the hippocampus, cell death is preceded by neuronal hyperactivity, presumed to be caused by loss of inhibitory control. Hyperglycemic subjects develop postischemic seizures, and show enhanced damage. The ATP-sensitive K+ channel, which may be important in inhibitory control, is the target of antidiabetic sulfonylureas. We determined densities of sulfonylurea binding sites in rat brain after forebrain ischemia. Normoglycemic animals showed a decrease of glibenclamide receptor binding in the CA3 field, hilus and dentate gyrus of the hippocampus after 1 day of recovery. After 4 days of recovery, levels of sulfonylurea binding sites decreased mainly in the CA1 field and in the hilus, as well as in the substantia nigra. After 1 day of recovery, hyperglycemic animals did not show any significant variations of densities of sites compared to control animals. It is proposed that reduction of inhibitory control by ATP-sensitive K+ channels may be associated with delayed neuronal death.

Sulfonylurea binding sites associated with ATP-regulated K+ channels in the central nervous system: autoradiographic analysis of their distribution and ontogenesis, and of their localization in mutant mice cerebellum.

The localization of a putative ATP-regulated K+ channel in normal rat and neurological mutant mice was studied by light microscopic quantitative autoradiography using a tritiated glibenclamide, an antidiabetic sulfonylurea. Glibenclamide binding sites presented a heterogeneous distribution in the rat central nervous system. Their density was particularly important in substantia nigra reticulata, septohippocampal nucleus, globus pallidus, neocortex, molecular layer of cerebellum, CA3 field and dentate gyrus of hippocampus. Conversely hypothalamic areas, medulla oblongata and spinal cord contained only low amounts of glibenclamide receptors. The ontogenesis of sulfonylurea binding sites was a postnatal phenomenon and seemed to correlate with the maturation of neuronal connectivity. In the cerebellum of neurological mutant mice, the autoradiographic patterns were different to that of wild-type cerebellum. In particular, in the molecular layer of weaver cerebellum, a decrease of 82% of binding site density suggested a presynaptic position of glibenclamide receptors in parallel fibers.

Analogies and differences in the mode of action and properties of binding sites (localization and mutual interactions) of two K+ channel toxins, MCD peptide and dendrotoxin I.

Both the bee venom toxin, mast cell degranulating (MCD) peptide, and the snake toxin, dendrotoxin 1 (DTX1) induce epileptiform activity and paroxystic seizures after intracerebroventricular (i.c.v.) injection to rats. Although many of the properties of the two toxins, which are blockers of the same K+ channel, appear to be very similar, a number of differences have been found. (1) Induced seizures have an hippocampal origin for MCD and two different origins, situated in the cortex and in the limbic system, for DTX1. (2) A first i.c.v. administration of DTXI desensitizes against a second ipsilateral injection of the same peptide as we had previously observed for MCD. However no cross-desensitization was observed between the two different toxins. (3) The number of high affinity (Kd = 41 pM) binding sites for 125I-DTXI in synaptic membranes is about 5 times higher than the number of high affinity (Kd = 158 pM) binding sites for 125I-MCD. (4) Autoradiographic analysis of the distribution of high affinity 125I-DTX1 binding sites has been compared to our previous analysis of high affinity 125I-MCD binding sites. High levels of high affinity binding sites for both toxins seem to be localized in synapse-rich areas. However high affinity binding sites for the two toxins are not always co-localized. Analysis of the mutual interactions between DTXI and MCD binding sites has revealed the presence of classes of low affinity binding sites for MCD. In most areas of the brain, a large proportion of high affinity binding sites for DTXI is allosterically related to low affinity binding for MCD.

Antidiabetic sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices.

The distribution of antidiabetic sulfonylurea [( 3H]glibenclamide) binding sites is heterogeneous in rat brain. Pyramidal and extrapyramidal motor system contain the highest densities of sites, particularly in the substantia nigra and in the globus pallidus. Only low levels are present in the hypothalamic nuclei and the main medulla oblongata regions. In hippocampal formation the stratum lucidum and the stratum lacunosum moleculare of CA3 show an important density of glibenclamide binding sites. Electrophysiological studies with hippocampal slices show that glibenclamide blocks hyperpolarization induced by anoxia, suggesting the involvement of adenosine triphosphate-sensitive K+ channel in this early hyperpolarization event.