Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.     

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca²⁺-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca²⁺-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.     

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II–III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.     

Caspase-3 immunoreactivity in different cortical areas of young and aging macaque (Macaca mulatta) monkeys

It has been shown that cytochrome-c-dependent caspase-3 activation is significantly elevated in the aging macaque brain. To assess the underlying age-related changes in the cellular distribution of caspase-3, we have examined the motor cortex, cerebellum and hippocampus of young (4-year-old, n = 4) and old (20-year-old, n = 4)rhesus monkeys by immunohistochemistry. Western blot analyses of brain homogenate showed that the antibody reacted only with inactive 32-kDa procaspase and its active 20- and 17-kDa subunits, formed after granzyme B exposure. In the motor cortex, pyramidal cells of layers III and V were moderately labeled; the underlying white matter contained weakly stained astrocytes. In the hippocampus, hilar neurons and pyramidal cells in CA3 showed the strongest immunoreaction, pyramidal cells in CA1 and granule cells of the dentate gyrus were also strongly labeled. In contrast, CA2 pyramidal cells were only weakly stained, and neurons of the molecular layer were unlabeled. Weak caspase-3 immunoreaction of CA2 neurons parallels known decreased susceptibility to apoptosis. In the cerebellar cortex, clusters of strongly labeled Purkinje cells were observed next to groups of weakly and unstained cells; granule cells were generally unstained. The brains of aging monkeys displayed a similar pattern of caspase-3 immunoreactivity. In neocortical layer V, however, scattered very strongly labeled pyramidal cells were regularly detected, which were not observed in younger animals. This clustering of caspase-3 indicates increased vulnerability of a subset of pyramidal cells in the aging brain.     

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.     

Comparison of VILIP-1 and VILIP-3 Binding to Phospholipid Monolayers

The neuronal calcium sensor proteins Visinin-like Proteins 1 (VILIP-1) and 3 (VILIP-3) are effectors of guanylyl cyclase and acetyl choline receptors, and transduce calcium signals in the brain. The “calcium-myristoyl” switch, which involves a post-translationally added myristoyl moiety and calcium binding, is thought to regulate their membrane binding capacity and therefore, play a critical role in their mechanism of action. In the present study, we investigated the effect of membrane composition and solvent conditions on the membrane binding mechanisms of both VILIPs using lipid monolayers at the air/buffer interface. Results based on comparison of the adsorption kinetics of the myristoylated and non-myristoylated proteins confirm the pivotal role of calcium and the exposed myristol moiety for sustaining the membrane-bound state of both VILIPs. However, we also observed binding of both VILIP proteins in the absence of calcium and/or myristoyl conjugation. We propose a two-stage membrane binding mechanism for VILIP-1 and VILIP-3 whereby the proteins are initially attracted to the membrane surface by electrostatic interactions and possibly by specific interactions with highly negatively charged lipids head groups. The extrusion of the conjugated myristoyl group, and the subsequent anchoring in the membrane constitutes the second stage of the binding mechanism, and ensures the sustained membrane-bound form of these proteins.     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

Structure of the fetal sheep brain in experimental growth retardation

A quantitative morphometric study of brain development has been made in growth-retarded fetal sheep. Intrauterine growth retardation was induced by removal of endometrial caruncles in the ewe prior to conception thereby reducing the size of the placenta in a subsequent pregnancy. Total brain and cerebellar weights were reduced by 21% (P less than 0.002) and the cerebrum by 20% (P less than 0.05) in the growth-retarded fetuses at 139 +/- 1 day (term = 146 days) compared with age matched control fetuses. Measurements of mean neuronal diameters were made on Purkinje cells, cerebellar granule cells, cortical cells in the motor and visual areas and hippocampal pyramidal cells; none were significantly different from control values. In growth-retarded fetuses compared with controls, there was a significant reduction in the thickness of the motor and visual cortices and the numerical density of neurones was significantly higher in these areas. In the cerebellar vermis, the number of Purkinje cells per unit surface area of Purkinje cell layer was higher, the numerical density of granule cells was significantly higher concomitant with a reduction in the area of the inner granular layer, and the area of the molecular layer was also reduced. In the hippocampal formation, the numerical density of pyramidal neurones was higher and the width of the stratum moleculare (dentate gyrus) was reduced. Migration of pyramidal neurones from the germinal layer to stratum pyramidale was not affected. These findings indicate that intrauterine growth retardation does not markedly affect cell size or neuronal migration (in the hippocampus) but does cause a significant reduction in the growth of the neuropil in the cerebellum, motor and visual cortices and the hippocampal formation.     

Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

Chronic prenatal ethanol exposure-induced decrease of guinea pig hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density

The brain is a key target of ethanol teratogenicity, in which ethanol can produce neurodegeneration in selected areas, including the hippocampus and cerebellum. The research objective was to test the hypothesis that chronic prenatal ethanol exposure, via maternal ethanol administration, produces differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells. Timed pregnant guinea pigs received chronic oral administration of ethanol, isocaloric-sucrose/pair-feeding, or water throughout gestation (term, about gestational day (GD) 68), and the offspring were studied at GD 62 (near-term fetus), postnatal day (PD) 1 (neonate), PD 5, and PD 12 (early postnatal life). Ethanol treatment, compared with isocaloric-sucrose/pair-feeding and water treatments, decreased brain, hippocampal, and cerebellar weights at GD 62, PD 1, PD 5, and PD 12. Hippocampal CA1 pyramidal cell linear density and cerebellar Purkinje cell linear density were unaffected at GD 62. Ethanol treatment produced 25, 30, and 30% decreases in linear density of hippocampal CA1 pyramidal cells at PD 1, PD 5, and PD 12, respectively, and a 30% decrease in linear density of cerebellar Purkinje cells at PD 12 only. At PD 5, Purkinje cell profile linear density remained unaffected; however, ethanol treatment appeared to increase linear density of apoptotic Purkinje cell nuclei, as determined by a modified TUNEL method. The data demonstrate that chronic prenatal ethanol exposure produces apparent differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells in the developing guinea pig.     

Permanent neuronal deficits in rats exposed to alcohol during the brain growth spurt

The purpose of this study was to determine whether developmental alcohol exposure could induce permanent neuronal deficits, whether the peak blood alcohol concentration (BAC) influences the severity of the effects, and whether the effects are gender related. Rat pups were reared artificially over postnatal days (PD) 4 through 11 (a period of rapid brain growth, comparable to part of the human third trimester). Alcohol treatments were administered on PD 4 through 9. Patterns of alcohol exposure that produce different peak BACs have been shown to affect differentially the amount of brain weight deficits and neuron loss shortly after the exposure period, so this study investigated whether the pattern of alcohol exposure was also effective in producing permanent deficits. Two groups received a daily alcohol dose of 4.5 g/kg, condensed into either four or two feedings. A third group received a higher daily alcohol dose of 6.6 g/kg administered in 12 uniformly spaced daily feedings. Pups were fostered back to dams on PD 11 and perfused on PD 90. Brain weights were measured, and Purkinje cells and granule cells were counted in each of the 10 lobules of the cerebellar vermis. In the hippocampal formation, cell counts were made of the pyramidal cells of fields CA1 and CA2/3, the multiple cell types of CA4 and the granule cells of the dentate gyrus. The groups receiving the lower daily dose (4.5 g/kg) condensed into either four or two feedings were exposed to higher peak BACs and suffered significant permanent brain weight deficits and neuronal losses, relative to controls. The group receiving the higher daily dose (6.6 g/kg) in continuous fractions had no significant brain weight reductions or neuronal loss. Vulnerability to alcohol-induced neuronal loss varied among regions and cell populations and as a function of peak BAC. In the hippocampus, only the CA1 pyramidal cells were significantly reduced in number and only in group receiving the most condensed alcohol treatment. In the cerebellum, the severity of Purkinje cell and granule cell losses varied among lobules, and Purkinje cell vulnerability appeared to depend on the maturational state of the neuron at the time of the alcohol exposure, with the more mature Purkinje cells being the more vulnerable.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Immunocytochemical Localization of TASK-3 Protein (K2P9.1) in the Rat Brain

Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

Visinin-like proteins (VSNLs): interaction partners and emerging functions in signal transduction of a subfamily of neuronal Ca<Superscript>2+</Superscript>-sensor proteins

The visinin-like protein (VSNL) subfamily, including VILIP-1 (the founder protein), VILIP-2, VILIP-3, hippocalcin, and neurocalcin δ, constitute a highly homologous subfamily of neuronal calcium sensor (NCS) proteins. Comparative studies have shown that VSNLs are expressed predominantly in the brain with restricted expression patterns in various subsets of neurons but are also found in peripheral organs. In addition, the proteins display differences in their calcium affinities, in their membrane-binding kinetics, and in the intracellular targets to which they associate after calcium binding. Even though the proteins use a similar calcium-myristoyl switch mechanism to translocate to cellular membranes, they show calcium-dependent localization to various subcellular compartments when expressed in the same neuron. These distinct calcium-myristoyl switch properties might be explained by specificity for defined phospholipids and membrane-bound targets; this enables VSNLs to modulate various cellular signal transduction pathways, including cyclic nucleotide and MAPK signaling. An emerging theme is the direct or indirect effect of VSNLs on gene expression and their interaction with components of membrane trafficking complexes, with a possible role in membrane trafficking of different receptors and ion channels, such as glutamate receptors of the kainate and AMPA subtype, nicotinic acetylcholine receptors, and Ca²⁺-channels. One hypothesis is that the highly homologous VSNLs have evolved to fulfil specialized functions in membrane trafficking and thereby affect neuronal signaling and differentiation in defined subsets of neurons. VSNLs are involved in differentiation processes showing a tumor-invasion-suppressor function in peripheral organs. Finally, VSNLs play neuroprotective and neurotoxic roles and have been implicated in neurodegenerative diseases. Work in the laboratories of K.H.B. has been supported by grants from DFG (Br1579/8–1 and Br1579/9–1, Priority Program of the German Research Foundation SPP1226), Deutsche Krebshilfe, Charité Berlin, and Kultusministerium des Landes Sachsen-Anhalt. Work in the laboratory A.J.K. has been supported by grants from the National Institutes of Health CA107257, CA06927, by an appropriation from the Commonwealth of Pennsylvania, and by a grant from the Pennsylvania Department of Health. An erratum to this article can be found at     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current

Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABA(A) channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocampal slices that insulin (1 nM) "turns on" new extrasynaptic GABA(A) channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC(50)) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABA(A) antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.