Loss of ARHGAP15 affects the directional control of migrating interneurons in the embryonic cortex and increases susceptibility to epilepsy.

GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.

Genetic absence of the vesicular inhibitory amino acid transporter differentially regulates respiratory and locomotor motor neuron development.

During mid to late embryonic development (E13 to birth in mice), the neuromotor system is refined by reducing motor neuron (MN) numbers and establishing nascent synaptic connections onto and by MNs. Concurrently, the response to GABAergic and glycinergic synaptic activity switches from postsynaptic excitation to inhibition. Our previous studies on mutant mice lacking glycinergic transmission or deficient in GABA suggests that altered MN activity levels during this developmental period differentially regulates MN survival and muscle innervation for respiratory and non-respiratory motor pools. To determine if combined loss of GABAergic and glycinergic transmission plays a similar or exaggerated role, we quantified MN number and muscle innervation in two respiratory (hypoglossal and phrenic) and two locomotor (brachial and lumbar) motor pools, in mice lacking vesicular inhibitory amino acid transporter, which display absent or severely impaired GABAergic and glycinergic neurotransmission. For respiratory MNs, we observed significant decreases in MN number (-20 % hypoglossal and -36 % phrenic) and diaphragm axonal branching (-60 %). By contrast, for non-respiratory brachial and lumbar MNs, we observed increases in MN number (+62 % brachial and +84 % lumbar) and axonal branching for innervated muscles (+123 % latissimus dorsi for brachial and +61 % gluteal for lumbar). These results show that combined absence of GABAergic and glycinergic neurotransmission causes distinct regional changes in MN number and muscle innervation, which are dependent upon the motor function of the specific motor pool.

mGluR1-mediated excitation of cerebellar GABAergic interneurons requires both G protein-dependent and Src-ERK1/2-dependent signaling pathways.

Stimulation of type I metabotropic glutamate receptors (mGluR1/5) in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC) channels. GABAergic cerebellar molecular layer interneurons (MLIs) modulate firing patterns of Purkinje cells (PCs), which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABAB receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABAB receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src-ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.

GAD65 haplodeficiency conveys resilience in animal models of stress-induced psychopathology.

GABAergic mechanisms are critically involved in the control of fear and anxiety, but their role in the development of stress-induced psychopathologies, including post-traumatic stress disorder (PTSD) and mood disorders is not sufficiently understood. We studied these functions in two established mouse models of risk factors for stress-induced psychopathologies employing variable juvenile stress and/or social isolation. A battery of emotional tests in adulthood revealed the induction of contextually generalized fear, anxiety, hyperarousal and depression-like symptoms in these paradigms. These reflect the multitude and complexity of stress effects in human PTSD patients. With factor analysis we were able to identify parameters that reflect these different behavioral domains in stressed animals and thus provide a basis for an integrated scoring of affectedness more closely resembling the clinical situation than isolated parameters. To test the applicability of these models to genetic approaches we further tested the role of GABA using heterozygous mice with targeted mutation of the GABA synthesizing enzyme GAD65 [GAD65(+/-) mice], which show a delayed postnatal increase in tissue GABA content in limbic and cortical brain areas. Unexpectedly, GAD65(+/-) mice did not show changes in exploratory activity regardless of the stressor type and were after the variable juvenile stress procedure protected from the development of contextual generalization in an auditory fear conditioning experiment. Our data demonstrate the complex nature of behavioral alterations in rodent models of stress-related psychopathologies and suggest that GAD65 haplodeficiency, likely through its effect on the postnatal maturation of GABAergic transmission, conveys resilience to some of these stress-induced effects.

Morpho-physiological criteria divide dentate gyrus interneurons into classes.

GABAergic inhibitory interneurons control fundamental aspects of neuronal network function. Their functional roles are assumed to be defined by the identity of their input synapses, the architecture of their dendritic tree, the passive and active membrane properties and finally the nature of their postsynaptic targets. Indeed, interneurons display a high degree of morphological and physiological heterogeneity. However, whether their morphological and physiological characteristics are correlated and whether interneuron diversity can be described by a continuum of GABAergic cell types or by distinct classes has remained unclear. Here we perform a detailed morphological and physiological characterization of GABAergic cells in the dentate gyrus, the input region of the hippocampus. To achieve an unbiased and efficient sampling and classification we used knock-in mice expressing the enhanced green fluorescent protein (eGFP) in glutamate decarboxylase 67 (GAD67)-positive neurons and performed cluster analysis. We identified five interneuron classes, each of them characterized by a distinct set of anatomical and physiological parameters. Cross-correlation analysis further revealed a direct relation between morphological and physiological properties indicating that dentate gyrus interneurons fall into functionally distinct classes which may differentially control neuronal network activity.

Synaptic inhibition and γ-aminobutyric acid in the mammalian central nervous system.

Signal transmission through synapses connecting two neurons is mediated by release of neurotransmitter from the presynaptic axon terminals and activation of its receptor at the postsynaptic neurons. γ-Aminobutyric acid (GABA), non-protein amino acid formed by decarboxylation of glutamic acid, is a principal neurotransmitter at inhibitory synapses of vertebrate and invertebrate nervous system. On one hand glutamic acid serves as a principal excitatory neurotransmitter. This article reviews GABA researches on; (1) synaptic inhibition by membrane hyperpolarization, (2) exclusive localization in inhibitory neurons, (3) release from inhibitory neurons, (4) excitatory action at developmental stage, (5) phenotype of GABA-deficient mouse produced by gene-targeting, (6) developmental adjustment of neural network and (7) neurological/psychiatric disorder. In the end, GABA functions in simple nervous system and plants, and non-amino acid neurotransmitters were supplemented.

Genetic deficiency of GABA differentially regulates respiratory and non-respiratory motor neuron development.

Central nervous system GABAergic and glycinergic synaptic activity switches from postsynaptic excitation to inhibition during the stage when motor neuron numbers are being reduced, and when synaptic connections are being established onto and by motor neurons. In mice this occurs between embryonic (E) day 13 and birth (postnatal day 0). Our previous work on mice lacking glycinergic transmission suggested that altered motor neuron activity levels correspondingly regulated motor neuron survival and muscle innervation for all respiratory and non respiratory motor neuron pools, during this period of development [1]. To determine if GABAergic transmission plays a similar role, we quantified motor neuron number and the extent of muscle innervation in four distinct regions of the brain stem and spinal cord; hypoglossal, phrenic, brachial and lumbar motor pools, in mice lacking the enzyme GAD67. These mice display a 90% drop in CNS GABA levels ( [2]; this study). For respiratory-based motor neurons (hypoglossal and phrenic motor pools), we have observed significant drops in motor neuron number (17% decline for hypoglossal and 23% decline for phrenic) and muscle innervations (55% decrease). By contrast for non-respiratory motor neurons of the brachial lateral motor column, we have observed an increase in motor neuron number (43% increase) and muscle innervations (99% increase); however for more caudally located motor neurons within the lumbar lateral motor column, we observed no change in either neuron number or muscle innervation. These results show in mice lacking physiological levels of GABA, there are distinct regional changes in motor neuron number and muscle innervation, which appear to be linked to their physiological function and to their rostral-caudal position within the developing spinal cord. Our results also suggest that for more caudal (lumbar) regions of the spinal cord, the effect of GABA is less influential on motor neuron development compared to that of glycine.

Regulation of ERK1/2 mitogen-activated protein kinase by NMDA-receptor-induced seizure activity in cortical slices.

Extracellular signal-regulated kinase 1/2 (ERK1/2) that belongs to a subfamily of mitogen-activated protein kinases (MAPKs) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro, as well as in NMDA-receptor (NMDA-R)-dependent long-term potentiation in the hippocampus. On the other hand, recent studies in cultured neurons have shown that NMDA-R stimulation could result in either ERK1/2 activation or non-activation, depending on the pharmacological manipulations. To assess NMDA-R-dependent regulation of ERK1/2 activity in vivo, here we examined the effect of NMDA-R-induced seizure activity on ERK1/2 activation by using rat cortical slice preparations. NMDA-R-dependent seizure activity introduced by Mg2+ -free condition did not cause ERK1/2 activation. On the other hand, when picrotoxin was added to concurrently suppress GABAA-receptor-mediated inhibition, profound ERK1/2 activation occurred, which was accompanied by strong phospho-ERK1/2-staining in the superficial and deep cortical layer neurons. In this case, prolonged membrane depolarization and enhanced burst action potential firings, both of which were much greater than those in Mg2+ -free condition alone, were observed. Differential ERK1/2 activation was supported by the concurrent selective increase in phosphorylation of a substrate protein, phospho-site 4/5 of synapsin I. These results indicate that NMDA-R activation through a release from Mg2+ -blockade, which accompanies enhancement of both excitatory and inhibitory synaptic transmission, was not enough, but concurrent suppression of GABAergic inhibition, which leads to a selective increase in excitatory synaptic transmission, was necessary for robust ERK1/2 activation to occur within the cortical network.

GABAergic inhibition regulates developmental synapse elimination in the cerebellum.

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.

Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells.

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.

The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice.

BACKGROUND: The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. RESULTS: VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. CONCLUSION: VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.

Ontogeny-recapitulating generation and tissue integration of ES cell-derived Purkinje cells.

Purkinje cells are the sole output neurons of the cerebellar cortex and their dysfunction causes severe ataxia. We found that Purkinje cells could be robustly generated from mouse embryonic stem (ES) cells by recapitulating the self-inductive signaling microenvironments of the isthmic organizer. The cell-surface marker Neph3 enabled us to carry out timed prospective selection of Purkinje cell progenitors, which generated morphologically characteristic neurons with highly arborized dendrites that expressed mature Purkinje cell-specific markers such as the glutamate receptor subunit GluRδ2. Similar to mature Purkinje cells, these neurons also showed characteristic spontaneous and repeated action potentials and their postsynaptic excitatory potentials were generated exclusively through nonNMDA glutamate receptors. Fetal transplantation of precursors isolated by fluorescence-activated cell sorting showed orthotopic integration of the grafted neurons into the Purkinje cell layer with their axons extending to the deep cerebellar nuclei and dendrites receiving climbing and parallel fibers. This selective preparation of bona fide Purkinje cells should aid future investigation of this important neuron.

Action potential-enhanced ATP release from taste cells through hemichannels.

Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the alpha-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels.

Fast activation of feedforward inhibitory neurons from thalamic input and its relevance to the regulation of spike sequences in the barrel cortex.

Thalamocortical afferents innervate both excitatory and inhibitory cells, the latter in turn producing disynaptic feedforward inhibition, thus creating fast excitation-inhibition sequences in the cortical cells. Since this inhibition is disynaptic, the time lag of the excitation-inhibition sequence could be approximately 2-3 ms, while it is often as short as only slightly above 1 ms; the mechanism and function of such fast IPSPs are not fully understood. Here we show that thalamic activation of inhibitory neurons precedes that of excitatory neurons, due to increased conduction velocity of thalamic axons innervating inhibitory cells. Developmentally, such latency differences were seen only after the end of the second postnatal week, prior to the completion of myelination of the thalamocortical afferent. Furthermore, destroying myelination failed to extinguish the latency difference. Instead, axons innervating inhibitory cells had consistently lower threshold, indicating they had larger diameter, which is likely to underlie the differential conduction velocity. Since faster activation of GABAergic neurons from the thalamus can not only curtail monosynaptic EPSPs but also make disynaptic ISPSs precede disynaptic EPSPs, such suppression theoretically enables a temporal separation of thalamically driven mono- and disynaptic EPSPs, resulting in spike sequences of 'L4 leading L2/3'. By recording L4 and L2/3 cells simultaneously, we found that suppression of IPSPs could lead to deterioration of spike sequences. Thus, from the end of the second postnatal week, by activating GABAergic neurons prior to excitatory neurons from the thalamus, fast feedforward disynaptic suppression on postsynaptic cells may play a role in establishing the spike sequences of 'L4 leading L2/3 cells'.

Differential gene expression in migrating cortical interneurons during mouse forebrain development.

Gamma-aminobutyric acid (GABA)ergic interneurons play a vital role in modulating the activity of the cerebral cortex, and disruptions to their function have been linked to neurological disorders such as schizophrenia and epilepsy. These cells originate in the ganglionic eminences (GE) of the ventral telencephalon and undergo tangential migration to enter the cortex. Currently, little is known about the signaling mechanisms that regulate interneuron migration. We therefore performed a microarray analysis comparing the changes in gene expression between the GABAergic interneurons that are actively migrating into the cortex with those in the GE. We were able to isolate pure populations of GABAergic cells by fluorescence-activated cell sorting of cortex and GE from embryonic brains of glutamate decarboxylase 67 (GAD67)-green fluorescent protein (GFP) transgenic mice. Our microarray analysis identified a number of novel genes that were upregulated in migrating cortical interneurons at both E13.5 and E15.5. Many of these genes have previously been shown to play a role in cell migration of both neuronal and non-neuronal cell types. In addition, several of the genes identified are involved in the regulation of migratory processes, such as neurite outgrowth, cell adhesion, and remodeling of the actin cytoskeleton and microtubule network. Moreover, quantitative polymerase chain reaction and in situ hybridization analyses confirmed that the expression of some of these genes is restricted to cortical interneurons. These data therefore provide a framework for future studies aimed at elucidating the complexities of interneuron migration and, in turn, may reveal important genes that are related to the development of specific neurological disorders.

Molecular identity of periglomerular and short axon cells.

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic-GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65+ GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic-GABAergic cells have extensive interglomerular projections. DAergic-GABAergic SA cells comprise two subgroups. One subpopulation contacts 5-12 glomeruli and is referred to as "oligoglomerular." Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON-->ET-->SA circuit. The second population of DAergic-GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive "polyglomerular" network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Short Promoters in Viral Vectors Drive Selective Expression in Mammalian Inhibitory Neurons, but do not Restrict Activity to Specific Inhibitory Cell-Types.

Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown.

Visual receptive field structure of cortical inhibitory neurons revealed by two-photon imaging guided recording.

Synaptic inhibition plays an important role in shaping receptive field (RF) properties in the visual cortex. However, the underlying mechanisms remain not well understood, partly because of difficulties in systematically studying functional properties of cortical inhibitory neurons in vivo. Here, we established two-photon imaging guided cell-attached recordings from genetically labeled inhibitory neurons and nearby "shadowed" excitatory neurons in the primary visual cortex of adult mice. Our results revealed that in layer 2/3, the majority of excitatory neurons exhibited both On and Off spike subfields, with their spatial arrangement varying from being completely segregated to overlapped. In contrast, most layer 4 excitatory neurons exhibited only one discernable subfield. Interestingly, no RF structure with significantly segregated On and Off subfields was observed for layer 2/3 inhibitory neurons of either the fast-spike or regular-spike type. They predominantly possessed overlapped On and Off subfields with a significantly larger size than the excitatory neurons and exhibited much weaker orientation tuning. These results from the mouse visual cortex suggest that different from the push-pull model proposed for simple cells, layer 2/3 simple-type neurons with segregated spike On and Off subfields likely receive spatially overlapped inhibitory On and Off inputs. We propose that the phase-insensitive inhibition can enhance the spatial distinctiveness of On and Off subfields through a gain control mechanism.

Discrimination of taste qualities among mouse fungiform taste bud cells.

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

Kinase-dead knock-in mouse reveals an essential role of kinase activity of Ca2+/calmodulin-dependent protein kinase IIalpha in dendritic spine enlargement, long-term potentiation, and learning.

Ca2+/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIalpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIalpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIalpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIalpha (K42R) mice, kinase activity was reduced to the same level as in CaMKIIalpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIalpha was preserved. In addition, CaMKIIalpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIalpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.