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.

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.

The apamin-sensitive Ca2+-dependent K+ channel molecular properties, differentiation and endogenous ligands in mammalian brain.

Apamin is a bee venom neurotoxin of 10 amino acids containing two disulphide bridges. Current-clamp and voltage-clamp experiments have shown that apamin externally applied blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylamonium-insensitive. It is distinct from the high conductance Ca+-dependent K+ channel revealed by patch-clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low: between 1 and 40 fmol/mg of protein. Radiation-inactivation analysis indicates a molecular mass for the apamin receptor of 250 000 Da whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular mass of about 30 000 Da. Autoradiography of 125I-apamin binding sites reveals the presence of Ca2+-activated K+ channels in many regions of the brain. There is an all-or-none control of the expression of the apamin-sensitive Ca2+-dependent K+ channel by innervation in mammalian skeletal muscle. There exists an endogenous equivalent of apamin in rat brain.

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.

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.

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.

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.

Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain.

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.

Cerebral glucose utilization after administration of apamin, a toxin active on Ca2+-dependent K+ channels.

The quantitative 2-[14C]deoxyglucose autoradiographic technique was used to examine the effects of acute intravenous administration of apamin, a bee venom toxin specific for one class of Ca2+-dependent K+ channels on brain energy metabolism. With doses of 0.5 mg/kg, the effects of apamin on local cerebral glucose utilization (LCGU) were limited to the habenulo-interpeduncular tract and the interpeduncular nucleus. After a 1.0 mg/kg dose, significant increases in rates of energy metabolism were additionally seen in two other limbic structures, the medial habenula and the lacunosum molecular of Ammon's horn as well as in the auditory cortex. Thirty minutes after the injection of 2 mg/kg apamin, LCGU was significantly decreased in the frontoparietal motor area, globus pallidus and accumbens nucleus. Ninety minutes after 2 mg/kg of the toxin, the average glucose utilization of the brain as a whole was enhanced by 35%, and rates of energy metabolism were significantly increased in 50 out of the 75 areas examined. The effects of apamin on cerebral glucose utilization are not totally related to the distribution of apamin binding sites. However, high densities of apamin binding sites are found in the habenulo-interpeduncular tract and the interpeduncular nucleus, the limbic areas that are highly affected by the toxin at the LCGU level.

Quantitative autoradiographic mapping in rat brain of the receptor of apamin, a polypeptide toxin specific for one class of Ca2+-dependent K+ channels.

The localization of the receptor for apamin, a specific toxin for one class of sensitive Ca2+-dependent K+ channel, was studied in rat brain using an in vitro autoradiographic technique. Radiolabeled monoiodoapamin binds specifically to rat brain sections with a high affinity (Kd = 25 pM) to a single class of sites. Autoradiograms demonstrated a very heterogeneous distribution of the apamin receptor throughout the brain. Very high grain densities were localized on the habenula, lateral septum, supraoptic and suprachiasmatic nuclei. Areas containing high levels of apamin binding sites included anterior olfactory nucleus, stratum oriens of hippocampus, pontine nuclei and granular layer of the cerebellar cortex and inferior olive. The thalamus, some nuclei of hypothalamus, hippocampus, tegmental area, red and oculomotor nuclei, vestibular nuclei and superior olive, among others, presented intermediate grain densities. In the other main areas, in particular basal ganglia, raphe, low to very low levels of apamin binding sites have been observed.

Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na+ channels, Ca2+-dependent K+ channels and slow Ca2+ channels identified as receptors for tetrodotoxin, apamin and (-)-desmethoxyverapamil.

The postnatal development of the distribution of 3 different ionic channel proteins in rat brain was studied using light microscopic autoradiography. [3H]Ethylenediaminetetrodotoxin, [125I]apamin and (-)-[3H]desmethoxyverapamil were used to label one class of voltage-dependent Na+ channel proteins, one class of Ca2+-dependent K+ channel proteins, and the slow Ca2+ channel protein, respectively. Ca2+-dependent K+ channel proteins are detected very early in the germinative zone. They are associated to neuronal somas during their migration and their maturation. In hippocampus and cerebral cortex, apamin binding sites are already present at birth and their density increases to day 20 postnatal when the adult localization is established. Slow Ca2+ channel protein development occurs later in CNS ontogenesis. The development of slow Ca2+ channels seems to follow the development of dendrites. Density of these channel proteins increases regularly until adult age. At the resolution level of this analysis, Na+ channel proteins are absent in diencephalon at birth. Their appearance and their increase in density are strictly correlated to the synaptogenesis in particular in cerebral and cerebellar cortex and hippocampus. Although cerebellum, neocortex and hippocampus have been particularly analyzed, other brain structures have also been examined.

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.

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.

Distribution of voltage-dependent Na+ channels identified by high-affinity receptors for tetrodotoxin and saxitoxin in rat and human brains: quantitative autoradiographic analysis.

The localization of a putative voltage-dependent Na+ channel in adult rat and human brain was studied by light microscopic quantitative autoradiography using a tritiated derivative of tetrodotoxin ([3H]enTTX) and tritiated saxitoxin [( 3H]STX). Equilibrium binding experiments in the whole rat brain gave dissociation constants of 7.0 nM ([3H]enTTX) and 5.0 nM ([3H]STX). The dissociation constant for the binding of [3H]STX in the different human brain regions was near 1.5 nM. Autoradiograms demonstrated a heterogeneous distribution of toxin binding sites in the brain with a very good correlation of the mapping of tetrodotoxin and saxitoxin receptors. With the exception of a few regions, the same type of cartography was observed for human and rat brain structures. If toxin receptors were present in all brain regions, their density was particularly important in cerebral cortex, hippocampus, lateral septum and molecular layer of cerebellar cortex. Conversely, the medulla oblongata contained only low amounts of binding sites.

High affinity receptors for the bee venom MCD peptide. Quantitative autoradiographic localization at different stages of brain development and relationship with MCD neurotoxicity.

High densities of MCD receptors were found in the stratum radiatum of Ammon's horn, the neocortex, the molecular layer of the cerebellum, colliculi and pons. Conversely areas such as the stratum lacunosum moleculare of Ammon's horn contained only low levels of MCD binding sites. The density of MCD receptors is low during the perinatal period and increases rapidly by postnatal day 10 with a decrease of the receptor affinity for MCD. The adult distribution of MCD receptors was reached at postnatal day 30. Increases in density of MCD receptors are discussed in relation with increased neurotoxicity of MCD during brain development. Effects of MCD during the perinatal period are very weak. However, the threshold MCD dose to induce seizures drastically decreased after the first postnatal week. The efficient dose corresponding to adult stage is reached after postnatal day 40.

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.

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.

The brain response to the bee venom peptide MCD. Activation and desensitization of a hippocampal target.

The mast cell-degranulating peptide (MCD) isolated from bee venom has been found previously to have receptor sites in rat brain. Behavioral and electrocorticographic responses following intracerebroventricular injections of various doses of MCD have been analyzed. MCD produced a quasi-permanent hippocampal theta rhythm in the motionless animal alternating with epileptiform spike waves and paroxystic seizures. At a dose of 70 pmol seizures occurred for half of the treated rats. At a dose of 100 pmol generalized paroxystic crises were observed for all the rats. These effects were not antagonized by naloxone, morphine, diazepam and progabide. Rats recovered 24 h after a 100 pmol injection of MCD. A second ipsilateral injection to these rats showed the occurrence of a desensitization phenomenon. Desensitization was not observed when the second injection was contralateral. These physiological responses were studied in relation with a biochemical approach on membrane sites of action of MCD using [125I]MCD and their behavior in the desensitization process. The target of [125I]MCD is the ipsilateral hippocampus. Recovery from MCD effects was not due to MCD degradation. Desensitization was not due to down-regulation of the MCD receptor level.

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.