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The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

Brain abscesses most frequently occur because of bacterial dissemination from a primary lesion at a distant site or direct contiguous invasion from an adjacent site of infection. We examined serum and saliva cytokine levels in a brain abscess patient with severe periodontitis. A 66-year-old man with high-grade fever and right-sided paresis was hospitalized. Brain magnetic resonance imaging (MRI) revealed several nodules in his right parietal and occipital lobes. We also found increased leukocyte (10,532/ml) and cerebrospinal fluid white blood cell (235–7860 mm3) counts. Bacteriological examination of sputum showed Fusobacterium nucleatum and Prevotella. No ear, nose, throat, and gastrointestinal infections were observed. Severe periodontitis was noted. Bacteriological examination of the right maxilla showed Porphyromonas gingivalis, and we detected a high level of serum antibody to P. gingivalis. Broad-spectrum antimicrobial therapy, dental calculus removal, and intraoral remediation improved the patient’s general condition. Brain MRI at the time of discharge showed a decrease in the size of the nodules, and after three months, the level of serum antibody to P. gingivalis decreased. Elevated pro-inflammatory cytokines such as interleukin (IL)-1α and IL-1β and chemokines such as IL-8, macrophage chemoattractant protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) levels in the saliva significantly decreased after oral treatment. Similarly, elevated serum IL-1α, IL-1β, IL-8, MCP-1, eotaxin, regulated on activation, normal T-cell expressed and secreted (RANTES), VEGF, and tumor necrosis factor-alpha (TNF)-α levels decreased after oral treatment. These findings supported our hypothesis that periodontopathic bacteria produce inflammation cytokines, pro-inflammatory cytokines migrate from systemic circulation to the brain, and their expression increases, and transition of that response into an adaptive form might have been the etiology of brain abscess in our patient.

Background Research has clearly demonstrated that vacuolar H + -ATPase (V-ATPase) is involved in the acidification of intracellular organelles such as vacuoles, lysosomes, synaptic vesicles, endosomes, secretory granules, and the Golgi apparatus in all eukaryotic cells. V-ATPase is also involved in mediating proton transport across the plasma membrane of epithelial cells. Furthermore, V-ATPase senses and responds to acidic pH by mobilizing vesicle coat components that are critical for vesicle trafficking from the cytosol to the endosomal membrane. In the salivary gland, V-ATPase mRNA and protein were detected by RT-PCR, western blotting, and immunohistochemistry, although the function of V-ATPase remains unknown. Highlight The present review aims to clarify our current understanding of the localization and functional roles of V-ATPase, and to discuss how this enzyme is regulated in the salivary glands. Conclusion An increasing number of studies reveal that the immunoreactivity of V-ATPase is restricted to ductal cells with species- and gland-specific distribution, and that different subunit isoforms of V-ATPase could be involved in targeting it to various intracellular locations. Considering that the major function of salivary gland duct epithelial cells is the reabsorption of Na + and Cl − , and secretion of K + and HCO 3 − , V-ATPase may primarily function in transporting protons out of cells across the plasma membrane, or acidifying compartments, resulting in modifying fluid and electrolyte transport.

Neurons in the medial nucleus of the amygdala (MeA) play a key role in the innate maternal, reproductive, defensive, and social behaviors. However, it is unclear how activation of the vomeronasal system leads to the behavioral outputs that are associated with pheromones. Here, we characterized the electrophysiological and morphological properties of MeA neurons using whole-cell recordings in mice slice preparations. Biocytin labeling revealed that MeA neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. In 70% of recorded MeA neurons, monosynaptic excitatory postsynaptic currents (EPSCs) were evoked from the accessory olfactory bulb afferent in which the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate component was dominant and was rarely followed by the N-methyl-d-aspartic acid component. Norepinephrine increased the frequency of spontaneous inhibitory postsynaptic currents in some neurons, whereas α-methyl-5-hydroxytryptamine increased spontaneous EPSCs in other neurons. Morphologically and physiologically, heterogeneous MeA neurons appear likely to produce multiplex outputs of instinctive behaviors.

Functional magnetic resonance imaging (fMRI) has considerably advanced the understanding of peripheral and central neural mechanisms underlying orofacial movements (e.g., chewing, swallowing, digestion, and speech). The principle advantages of fMRI lie in its noninvasive nature, relatively high spatiotemporal resolution, and ability to identify the entire network of brain areas involved in particular tasks. However, there remain substantial and valid criticisms of fMRI based on its spatial and temporal limits. Although further improvements in the existing technology will enhance the scientific value of fMRI, the use of fMRI is in the early stages of translation from the research laboratory to clinical practice. In order to make clinically meaningful contributions, challenging questions must be answered regarding therapeutic applications of fMRI to the dental and craniofacial field.

Introduction Intracellular Ca 2+ is essential to many signal transduction pathways, and its level is tightly regulated by the Ca 2+ extrusion system in the plasma membrane, which includes the Na + -Ca 2+ exchanger (NCX). Although expression of NCX1 isoforms has been demonstrated in odontoblasts, the detailed properties of NCX remain to be clarified. In this study, we investigated localization and ion-transporting/pharmacologic properties of NCX isoforms in rat odontoblasts. Methods We characterized both the reverse and forward modes of NCX activity in odontoblasts in a dental pulp slice preparation. Ca 2+ influx by reverse NCX activity was measured by fura-2 fluorescence. Ca 2+ efflux by forward NCX activity elicited inward Na + current as measured by perforated-patch clamp recording. For immunohistochemical analysis, cryostat sections of incisors were incubated with antibodies against NCX. Results Immunohistochemical observation revealed localization of NCX1 and NCX3 in the distal membrane of odontoblasts. Inward currents by forward NCX activity showed dependence on external Na + . Fura-2 fluorescence measurement revealed that Ca 2+ influx by reverse NCX activity depended on extracellular Ca 2+ concentration, and that this influx was blocked by NCX inhibitor KB-R7943 in a concentration-dependent manner. However, Ca 2+ influx by NCX showed a slight sensitivity to SEA0400 (a potent NCX1 inhibitor), indicating that expression potencies in odontoblasts were NCX3 > NCX1. Conclusions These results suggest that odontoblasts express NCX1 and NCX3 at the distal membrane, and that these isoforms play an important role in the Ca 2+ extrusion system as well as in the directional Ca 2+ transport pathway from the circulation to the dentin-mineralizing front.

We examined the firing properties of vestibular ganglion cells (VGCs) acutely isolated from wild or heterozygous brain-derived neurotrophic factor null mice, using the patch-clamp technique. VGCs obtained from wild-type mice showed diverse firing properties during sustained membrane depolarization; approximately half of the neurons exhibited strong adaptation, generating just a single spike or a few spikes (phasic type), whereas approximately one-fourth of the neurons showed moderate adaptation or tonic firing (tonic type). In heterozygous mice, the majority of VGCs belonged to the tonic type, the rate of which was significantly different from that of wild-type. These results suggest that brain-derived neurotrophic factor not only contributes to the survival of the VGCs but also affects their firing properties.

GRIN1 (Gprin 1) is a signaling molecule coexpression of which with constitutively active form of Gαo can stimulate neurite extensions in Neuro2a cells, yet its in vivo roles remain elusive. Here, we examine expression profiles of GRIN1 during mouse development by in situ hybridization (ISH) and immunohistochemistry. ISH analysis revealed that GRIN1 expression was limited to the nervous system at all developmental stages tested: in the central nervous system, GRIN1 expression occurred within the entire embryonic mantle zones, while it became restricted to sets of nuclei at postnatal to adult stages. Immunohistochemistry using a GRIN1-specific antibody demonstrated that GRIN1 colocalized with Gαo at neuronal dendrites and axons, but it was not detected in glial cells. These results suggest that Gαo-GRIN1 pathway could mediate significant roles in neuronal migration and differentiation at embryonic stages and exert functions in wiring and/or maintenance of specific neural circuitries at postnatal to adult stages. Developmental Dynamics 237:2415–2429, 2008. © 2008 Wiley-Liss, Inc.

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp4e/- . During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process.

By altering their morphology, astrocytes, including those involved in the maintenance and plasticity of neurons and in clearance of transmitter, play important roles in synaptic transmission; however, the mechanism that regulates the morphological plasticity of astrocytes remains unclear. Recently, we reported that T1, a subtype of TrkB (a family of BDNF-specific receptors), altered astrocytic morphology through the control of Rho GTPases in primary astrocyte cultures. In this study, we extended this observation to investigate acute neocortical slices from adult rats. T1 siRNA-expression vectors were electroporated into astrocytes in neocortical layer I of living rats. In both normal slices and control vector-electroporated slices, BDNF induced the elongation of the astrocytic processes and increased the branching of processes in slices after 1 h incubation. In contrast, in T1 siRNA-electroporated slices, no such significant morphological changes were observed in the astrocytes. In addition, the number of synaptophysin + sites in contact with GFAP + processes increased in a BDNF-T1 -dependent manner without the increase in the total synaptophysin + sites. Therefore, the present study provides evidence of the regulation of layer I astrocytic morphology by the BDNF-T1 signal in adult rat neocortical slices.

Recently, ODA evaluation is recognized as important and necessary for securing accountability and improving the quality of ODA. This study is aimed at proposing an evaluation method for ODA projects in the water supply sector in terms of water quality. Achievement Grade (AG) and Risk Level of contamination were proposed for indicators of evaluating impact as potability. To demonstrate the evaluation results, field surveys were conducted in Nepal, Philippines, Vietnam and Indonesia. In addition to water quality analysis, the following surveys were conducted for multiple understanding of the actual situation: interview with those concerned with the projects, facility study and questionnaire survey to the beneficiaries.

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABAA and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABAA receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX slowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

At many central excitatory synapses, AMPA receptors relay the electrical signal, whereas activation of NMDA receptors is conditional and serves a modulatory function. We show here quite a different role for NMDA receptors at dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. In whole-cell patch-clamp recordings in bulb slices, stimulation of mitral cells elicited slowly decaying, GABAA receptor-mediated reciprocal IPSCs that reflected prolonged GABA release from granule cells. Although granule cells had a normal complement of AMPA and NMDA receptors, the IPSC was completely blocked by the NMDA receptor antagonist D,L-AP-5, suggesting that NMDA receptor activation is an absolute requirement for dendrodendritic inhibition. The AMPA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect on IPSCs in the absence of extracellular magnesium but modestly reduced IPSCs in 1 mM magnesium, indicating that the primary effect of the AMPA receptor-mediated depolarization was to facilitate the unblocking of NMDA receptors. Granule cell voltage recordings indicated that effective spike stimulation in granule cells depended on the slow NMDA receptor kinetics. Granule cells also showed a pronounced delay between synaptic stimulation and action potential generation, suggesting that their intrinsic membrane properties underlie the ineffectiveness of brief AMPA receptor-mediated EPSPs. NMDA receptors also seem to have a central role in dendrodendritic inhibition in vivo, because intraperitoneal dizocilpine maleate (MK-801) injection in young adult rats resulted in disinhibition of mitral cells as measured by the generation of c-fos mRNA. The unique dependence of dendrodendritic inhibition on slow EPSPs generated by NMDA receptors suggests that olfactory information processing depends on long-lasting reciprocal and lateral inhibition.

Premotor neurons projecting to the hypoglossal (XII) nucleus and participating in cortically-induced rhythmical tongue movements were defined by extracellular recording in the cat. Two thirds (37/57) of antidromically identified XII premotor neurons sampled in the rostral medullary parvocellular reticular formation showed changes in their firing pattern during cortically-induced rhythmical activity of XII motoneurons. Fifteen of the 37 neurons showed a firing in phase with rhythmical activity of either the medial or lateral branch of the XII nerve (phasic-type). The remaining 22 neurons showed an increase in discharge with no apparent correlation with cortically-induced rhythmical activity of the XII nerve (non-phasic-type). Among the phasic- and non-phasic-type neurons, 30 neurons received inputs from the cortical masticatory area, and 14 neurons received further excitatory inputs from the inferior alveolar nerve. By systematic mapping of the stimulation sites effective for antidromic activation, four phasic-type neurons were confirmed to project to either tongue-protruding or -retracting XII motoneuron pools in accordance with their burst firing, suggesting that the phasic-type premotor neurons contribute to excitation of XII motoneurons during cortically-induced rhythmical activity. It is concluded that there are the XII premotor neurons driving cortically-induced rhythmical activity of XII motoneurons in the rostral medullary parvocellular reticular formation.

Odontoblasts are involved in the transduction of stimuli applied to exposed dentin. Although expression of thermo/mechano/osmo-sensitive transient receptor potential (TRP) channels has been demonstrated, the properties of TRP vanilloid 1 (TRPV1)-mediated signaling remain to be clarified. We investigated physiological and pharmacological properties of TRPV1 and its functional coupling with cannabinoid (CB) receptors and Na(+)-Ca(2+) exchangers (NCXs) in odontoblasts. Anandamide (AEA), capsaicin (CAP), resiniferatoxin (RF) or low-pH evoked Ca(2+) influx. This influx was inhibited by capsazepine (CPZ). Delay in time-to-activation of TRPV1 channels was observed between application of AEA or CAP and increase in [Ca(2+)](i). In the absence of extracellular Ca(2+), however, an immediate increase in [Ca(2+)](i) was observed on administration of extracellular Ca(2+), followed by activation of TRPV1 channels. Intracellular application of CAP elicited inward current via opening of TRPV1 channels faster than extracellular application. With extracellular RF application, no time delay was observed in either increase in [Ca(2+)](i) or inward current, indicating that agonist binding sites are located on both extra- and intracellular domains. KB-R7943, an NCX inhibitor, yielded an increase in the decay time constant during TRPV1-mediated Ca(2+) entry. Increase in [Ca(2+)](i) by CB receptor agonist, 2-arachidonylglycerol, was inhibited by CB1 receptor antagonist or CPZ, as well as by adenylyl cyclase inhibitor. These results showed that TRPV1-mediated Ca(2+) entry functionally couples with CB1 receptor activation via cAMP signaling. Increased [Ca(2+)](i) by TRPV1 activation was extruded by NCXs. Taken together, this suggests that cAMP-mediated CB1-TRPV1 crosstalk and TRPV1-NCX coupling play an important role in driving cellular functions following transduction of external stimuli to odontoblasts.

The modulation of high-threshold Ca2+ currents by the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1- aminocyclopentane-1,3-dicarboxylic acid (ACPD), was investigated in cultured hippocampal neurons using whole-cell voltage-clamp recording. ACPD reduced high-threshold Ca2+ currents carried by Ba2+ with an EC50 of 15.5 microM. The inhibition was reversible, voltage dependent, and blocked by L-2-amino-3-phosphonopropionic acid (1 mM) or by pretreatment with pertussis toxin. Inhibition by ACPD was greatly enhanced, and became irreversible, when the nonhydrolyzable GTP analog GTP gamma S was included in the whole-cell pipette. In some neurons, the Ba2+ current was inhibited by L(+)-2-amino-4-phosphonobutanoic acid (L-AP4) as well as ACPD while most cells were insensitive to L-AP4, suggesting that these agonists activate distinct receptors. The inhibition of Ca2+ currents was reduced but not eliminated in the presence of either omega-conotoxin GVIA or nifedipine, suggesting that both N- and L-type Ca2+ currents were affected. The degree and kinetics of inhibition were dependent on intracellular calcium. With [Ca]i < 1 nM, inhibition had a fast onset (t approximately 1–2 sec) and a rapid recovery, consistent with a membrane-delimited pathway. However, a slow component of inhibition appeared when the steady state [Ca]i was increased to 100 nM (t onset approximately 3 min). The slow component did not require transient Ca2+ influx or release of intracellular Ca2+. We suggest that Ca2+ channel modulation by ACPD involves either two mGluR subtypes with separate coupling mechanisms or a single mGluR that couples to both mechanisms.

The expression of purinergic receptors (P2X) on rat vestibular ganglion neurons (VGNs) was examined using whole-cell patch-clamp recordings. An application of adenosine 5'-triphosphate (ATP; 100microM) evoked inward currents in VGNs at a holding potential of -60mV. The decay time constant of the ATP-evoked currents was 2-4s, which is in between the values for rapidly desensitizing subgroups (P2X1 and P2X3) and slowly desensitizing subgroups (P2X2, P2X4, etc.), suggesting the heterogeneous expression of P2X receptors. A dose-response experiment showed an EC(50) of 11.0microM and a Hill's coefficient of 0.82. Suramin (100microM) reversibly inhibited the ATP-evoked inward currents. Alpha, beta-methylene ATP (100microM), a P2X-specific agonist, also evoked inward currents but less extensively than ATP. An application of adenosine 5'-dihosphate (ADP; 100microM) evoked similar, but much smaller, currents. The current-voltage relationship of the ATP-evoked conductance showed pronounced inward rectification with a reversal potential more positive than 0mV, suggesting non-selective cation conductance. However, the channel was not permeable to a large cation (N-methyl-d-glucamine) and acidification (pH 6.3) had little effect on the ATP-evoked conductance. RT-PCR confirmed the expression of five subtypes (P2X2-P2X6) in VGNs. The physiological role of P2X receptors includes the modulation of excitability at the synapses between hair cells and dendrites and/or trophic support (or also neuromodulation) from supporting cells surrounding the VGNs.

Using the single-electrode voltage-clamp technique, we have examined the effects of a non -N- methyl- d -aspartate (NMDA) antagonist, Joro spider toxin (JSTX), and of an NMDA antagonist, zinc, on excitatory postsynaptic currents (EPSCs) evoked by stimulation of stratum radiatum in CA1 pyramidal cells of the guinea-pig hippocampal slice. Pressure application of a synthesized JSTX (JSTX-3) at 10–200 μM greatly reduced the EPSCs ( 14 19 cells). The block by JSTX-3 was observed in pyramidal cells where the EPSCs showed linear peak current-voltage (I–V) relations in the control. EPSCs remaining after JSTX-3 application showed non-linear peak I–V relationships ( 10 14 cells), and were blocked by puff application of the selective NMDA receptor antagonist dl -2-amino-5-phosphonovalerate (APV) at 200 μM ( 6 10 cells). In the presence of JSTX-3, the decay time constant of the EPSC was increased and was less affected by membrane potential. JSTX-3 had no detectable effects on EPSCs apparently mediated solely by NMDA receptor. These observations suggest that JSTX-3 blocks excitatory synaptic transmission mainly by suppressing non-NMDA-receptor-mediated EPSCs, and that the JSTX-3-insensitive component is mediated at least in part by NMDA receptors in the hippocampal slice. Zinc (100–200 μM) reversibly attenuated EPSCs ( 6 9 cells) and appeared to block a slower component of the EPSCs, suggesting that mainly NMDA receptor-mediated currents were affected.

Duchenne muscular dystrophy (DMD) is accompanied by cognitive deficits and psychiatric symptoms. In the brain, dystrophin, the protein responsible for DMD, is localized to a subset of GABAergic synapses, but its role in brain function has not fully been addressed. Here, we report that defensive behaviour, a response to danger or a threat, is enhanced in dystrophin-deficient mdx mice. Mdx mice consistently showed potent defensive freezing responses to a brief restraint that never induced such responses in wild-type mice. Unconditioned and conditioned defensive responses to electrical footshock were also enhanced in mdx mice. No outstanding abnormality was evident in the performances of mdx mice in the elevated plus maze test, suggesting that the anxiety state is not altered in mdx mice. We found that, in mdx mice, dystrophin is expressed in the amygdala, and that, in the basolateral nucleus (BLA), the numbers of GABA(A) receptor alpha2 subunit clusters are reduced. In BLA pyramidal neurons, the frequency of norepinephrine-induced GABAergic inhibitory synaptic currents was reduced markedly in mdx mice. Morpholino oligonucleotide-induced expression of truncated dystrophin in the brains of mdx mice, but not in the muscle, ameliorated the abnormal freezing response to restraint. These results suggest that a deficit of brain dystrophin induces an alteration of amygdala local inhibitory neuronal circuits and enhancement of fear-motivated defensive behaviours in mice.

Individual primary vestibular afferents exhibit spontaneous activity the regularity of which can vary from regular to irregular. Different aspects of vestibular responsiveness have been associated with this dimension of regularity of resting discharge. Isolated rat vestibular ganglion cells (VGCs) showed heterogeneous intrinsic firing properties during sustained membrane depolarization: some neurons exhibited a strong adaptation generating just a single or a few spikes (phasic type), whereas other neurons showed moderate adaptation or tonic firing (tonic type). Tonic discharging VGCs were rare at postnatal days 5–7 and increased up to ∼60% of neurons during postnatal 2–3 wk. To explore the major factors responsible for the discharge regularity of primary vestibular afferents, we investigated the contribution of K+channels to the firing properties of isolated rat VGCs. Phasic firing became tonic firing in the presence of 4-aminopyridine or α-dendrotoxin, indicating that Kv1 potassium channels control the firing pattern of the phasic VGCs. Tetraethylammonium decreased the number of spikes during step current stimuli in all types. Blockade of Ca2+-activated K+channels decreased the number of spikes in tonic VGCs. Our results suggest that Kv1 channels are critical both in determining the pattern of spike discharge in rat vestibular ganglion neurons and in their proportional change during maturation.

It is known that painful tissue injury evokes an increase in dynorphin in spinal neurons. It is not known, however, whether dynorphinergic systems respond similarly to the pain that accompanies peripheral neuropathy. Radioimmunoassays and immunocytochemistry were used to evaluate changes in dynorphin A(1-8) in the spinal cord of rats with a painful peripheral neuropathy. The neuropathy is the result of a constriction injury that is created by tying loose ligatures around the common sciatic nerve. Signs of abnormal pain sensations, hyperalgesia, allodynia (pain after normally innocuous stimuli), and spontaneous pain (or dysesthesia), are first detected 2-5 days after injury, reach peak severity in about 10 days, and persist for 2-3 months (Bennett, G. J.; Xie, Y.-K. Pain 33:87-107; 1988). Dynorphin increased by 5 days in cells in laminae I-II and V-VII in the lumbar spinal cord ipsilateral to the injury. This increase, maximal at 10 days (262%), was still present 20 days after the injury but was now seen only in neurons in the deep laminae (V-VII). Thus, the spinal dynorphinergic system appears to respond to neuropathic pain. Furthermore, our results suggest that dynorphinergic cells in the superficial and deep laminae may have different roles in nociception.

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca2+-dependent exocytosis, which was abolished by the Ca2+-channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A.

Through tropo-myosine-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

The cellular localization of metabotropic glutamate receptors (mGluRs) (mGluR1α, 2/3, 5a and 7) in the main and accessory olfactory bulb (MOB and AOB) of adult rats was compared by using affinity purified polyclonal antibodies directed to their C-termini. mGluR1α and mGluR5a immunoreactivities were located in comparable structures of the MOB and AOB with different levels of intensity. mGluR5a reactivity was high in the AOB. mGluR2/3 showed a different pattern of expression in the MOB compared to that observed in the AOB; the periglomerular region of the MOB was strongly stained, but in the AOB it was the mitral/tufted cell layer that was intense. The mitral cell bodies in the MOB were strongly immunoreactive for mGluR7. These differences in the distribution of mGluRs in the MOB and AOB may reflect differences in synaptic transmission and sensitivity to neuromodulation in the two systems.

1. Paired whole-cell recordings were made from a glutamatergic giant nerve terminal, the calyx of Held, and its postsynaptic target cell in the medial nucleus of the trapezoid body (MNTB) in the brainstem slice of juvenile rat. Excitatory postsynaptic currents (EPSCs) were evoked by presynaptic action potentials triggered by brief (1 ms) depolarizing pulses. 2. In normal artificial cerebrospinal fluid (ACSF), EPSCs of several nanoamperes in amplitude were evoked at a relatively constant latency with no failure, whereas in low [Ca(2+)](o)-high [Mg(2+)](o) solutions, EPSCs fluctuated both in amplitude and latency, and stochastic failures of transmitter release were observed in response to presynaptic action potentials. 3. After blocking action potentials with tetrodotoxin (TTX), direct depolarization of the calyceal preterminal elicited asynchronous release of miniature EPSCs (mEPSCs). When the magnitude of depolarization was increased, mEPSCs increased in frequency. Being consistent with their quantal nature, their mean amplitude remained constant over a wide range of frequencies. The amplitude distribution of mEPSCs was slightly skewed (skewness = 1.06), with a mean conductance of 0.45 nS and a coefficient of variation (c.v.) of 0.43. 4. Single-channel conductance underlying mEPSCs was estimated using non-stationary fluctuation analysis. The weighted mean single channel conductance was 20.4 pS, suggesting that a single quantum opens 22 postsynaptic glutamate receptor channels on average. 5. After washing out TTX, EPSCs evoked by presynaptic action potentials were tested for quantal analysis based upon the mean amplitude of mEPSCs and their variance. In low [Ca(2+)](o)-high [Mg(2+)](o) solutions, quantal contents estimated from the EPSC/mEPSC ratio, rate of failures or c.v. assuming Poisson's statistics, coincided with each other. Evoked EPSCs could be fitted by integer multiples of mEPSCs with an assumption of incremental variance more adequately than the constant variance assumption. 6. It is concluded that the rat central auditory synaptic transmission is made in a quantal manner as at the frog neuromuscular junction.

At nicotinic and glutamatergic synapses, the duration of the postsynaptic response depends on the affinity of the receptor for transmitter (Colquhoun et al., 1977; Pan et al., 1993). Affinity is often thought to be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor selects for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested these assumptions for the GABAAreceptor by dissecting the contributions of microscopic binding and unbinding kinetics for agonists of equal efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed that agonist unbinding rates could not account for affinity if diffusion-limited binding was assumed. However, direct measurement of the instantaneous competition between agonists and a competitive antagonist revealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound and bound states determines GABAAreceptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, seeEdmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

In order to measure unitary properties of receptor channels at the postsynaptic site, the noise within the decay phases of inhibitory postsynaptic currents (IPSCs) and of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents (EPSCs) in rat hippocampal neurons was studied by nonstationary fluctuation analysis. Least squares scaling of the mean current was used to circumvent the wide variation in amplitude of postsynaptic currents. The variance of fluctuations around the expected current was analyzed to calculate single channel conductance, and fluctuation kinetics were studied with power spectra. The single channel conductance underlying the IPSC was measured as 14 pS, whereas that underlying the EPSC was 42 pS. Openings of the EPSC channel could also be resolved directly in low-noise whole-cell recordings, allowing verification of the accuracy of the fluctuation analysis. The results are the first measurements of the properties of single postsynaptic channels activated during synaptic currents, and suggest that the technique can be widely applicable in investigations of synaptic mechanism and plasticity.

A neurotoxin (JSTX) was isolated from the venom of spider (Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CA1 pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus. JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.

1. The afferent-evoked synaptic input to lumbar spinal cord (L5-S1) neurons that were activated antidromically from the medial pontomedullary reticular formation (nucleus reticularis gigantocelluaris and vicinity) was investigated with the use of intracellular recordings in pentobarbital sodium-anesthetized cats. 2. Spinoreticular tract (SRT) neurons (n = 33) were categorized into three types (“deep-inhibited,” “deep-complex,” and “intermediate”) on the basis of their locations and of their responses to natural and electrical stimulation. 3. The deep-inhibited-type neurons, located in the medial part of the deeper laminae (approximately VI-VIII), comprised a large component of the sample (20/33). They had no demonstrable excitatory receptive field (RF). However, electrical stimulation of low-threshold cutaneous afferents of hindlimb nerves evoked inhibitory postsynaptic potentials (IPSPs) via an oligosynaptic linkage. High-threshold cutaneous and muscle afferents also evoked IPSPs. 4. In the deep-complex-type neurons (8/33), electrical stimulation of low-threshold cutaneous afferents evoked complex IPSP-excitatory postsynaptic potential (EPSP) sequences. With intense stimuli, long-latency C-fiber-like EPSPs were evoked. Two of these eight neurons were characterized as wide-dynamic-range (WDR) neurons with large, excitatory and inhibitory cutaneous RFs. 5. Intermediate-type neurons (5/33) were concentrated in the lateral spinal gray and relatively superficially (approximately lamina V). These neurons had convergent low- and high-threshold cutaneous inputs (WDR neurons). Electrical stimulation of low-threshold cutaneous afferent fibers from within the excitatory RF evoked mono- or disynaptic EPSPs followed by IPSPs. High-threshold muscle and cutaneous afferents also evoked EPSPs. 6. These results show that SRT neurons have a variety of response characteristics resulting from various degrees of spatial and temporal summation of primary afferent input. Neurons with widespread inhibitory responses but no excitatory drive from the periphery comprise a surprisingly large component of the SRT: the function of these cells is unknown. It is apparent that the spinoreticular projection has considerable functional heterogeneity.

Intracellular recordings were made from hypoglossal motoneurons during cortically-induced fictive mastication in paralyzed encephale isole cats. Repetitive stimulation of the masticatory area of the cerebral cortex induced rhythmical tongue movements coordinated with jaw movements. After the animal was immobilized, the cortical stimulation still induced rhythmical burst activities in the hypoglossal nerve and the digastric nerve. The burst activities in the medial and lateral branches of the hypoglossal nerve alternated rhythmically, and were in and out of phase with the burst activities of the digastric nerve, respectively. All hypoglossal motoneurons showed rhythmical intracellular potentials during repetitive cortical stimulation. The rhythmical depolarizing potentials superimposed by spike bursts appeared in phase with rhythmical bursts in either the lateral or medial branch of the hypoglossal nerve. No hyperpolarization was present between consecutive depolarizing potentials. Synaptic activation noise increased coincidentally with the depolarizing potential, indicating that EPSPs were involved in the generation of the depolarizing potential. No evidence was obtained for the existence of IPSPs during the inter-depolarizing phase by intracellular current injection. It was concluded that rhythmical bombardment of excitatory impulses to hypoglossal motoneurons was responsible for the rhythmical activity induced by repetitive stimulation of the cortical masticatory area.

During food ingestion in cats, the activity of single bulbar reticular neurons showed rhythmical spike bursts during the active jaw opening phase of mastication. By utilizing spike-triggered averaging techniques, certain reticular cells were strongly suggested to be inhibitory neurons projecting to jaw closer motoneurons. We propose that these bulbar reticular neurons participate in the central generation of masticatory jaw movements by rhythmically inhibiting jaw closer motoneurons during mastication.

We sought to identify those cells involved in the generation of atonia of the masseter muscles during active sleep. A neuronal population was examined in the medullary reticular formation which has been shown to project monosynaptically to trigeminal motoneurons and provide inhibitory input to them. These neurons exhibited a pattern of state-dependent discharge which was characterized by a tonic increase in firing frequency which paralleled the tonic decrease in somatomotor reflex activity (within the trigeminal system) in the continuum of wakefulness to quiet (NREM) sleep to active sleep. This population of cells discharged at extremely high rates during active sleep, especially during periods of rapid eye movements, when postsynaptic inhibitory control of motoneurons is most prominent. We therefore suggest that these medullary units are the inhibitory neurons which are responsible for the postsynaptic inhibition of trigeminal motoneurons during active sleep.

Synaptic basis of the rhythmical activity induced by repetitive stimulation of the masticatory area of the cerebral cortex was studied by intracellular recording from hypoglossal motoneurons in cats, with the following results: (1) Repetitive cortical stimulation induced rhythmical tongue movements coordinated with rhythmical jaw movements. After the animal was paralyzed, the cortical stimulation still induced a rhythmically alternating efferent burst activity in the medial and lateral branches of the hypoglossal nerve; the bursts in the medial and lateral branches were in and out of phase with that of the digastric nerve, respectively. (2) In all of the recorded 36 hypoglossal motoneurons, repetitive cortical stimulation induced a rhythmical depolarizing potential superimposed by spike bursts corresponding with the rhythmical efferent burst in the hypoglossal nerve. No hyperpolarizing potential was present between the consecutive depolarizing potential. (3) Synaptic activation noise was increased coincidentally with the depolarizing potential, indicating that EPSPs were involved in the generation of the depolarizing potential. By intracellular application of either DC current or Cl-, no evidence was obtained for existence of IPSPs during the inter-excitatory phase.It was concluded that rhythmical bombardment of excitatory impulses to hypoglossal motoneurons was responsible for their rhythmical activity induced by repetitive stimulation of the cortical masticatory area.

Many of the neuropathic pain states that occur when human somatosensory nerves are damaged by disease or trauma are thought to involve pathological changes within the spinal segments innervated by the affected nerve. Investigations in animals that have had a peripheral nerve transected or crushed have revealed several neurochemical changes within the spinal cord that may be of pathophysiological significance. Both transection and crush injuries cause a complete peripheral denervation, but their behavioral consequences differ (Wall >et al, 1979a). Rats with a transection have a marked tendency to attack the denervated region (autotomy); animals with a crush injury display autotomy infrequently and when present it is mild. It has been hypothesized that autotomy is an animal’s response to a neuropathic pain state (Wall >et al, 1979a,b; Rodin and Kruger, 1984; Coderre >et al, 1986).

1. Single-unit activity was recorded from 215 neurons in the medial bulbar reticular formation during the masticatory sequence, from intake to deglutition, of 3 kinds of food (cat food pellets, canned fish, and milk) in 8 chronically prepared, unanesthetized, spontaneously respiring cats with their head fixed to a stereotaxic apparatus without pain or pressure. The firing patterns were compared to the simultaneously recorded EMGs of the jaw-closing and -opening muscles and to the jaw movement. 2. Fifty neurons changed their firing patterns during mastication. Nine neurons increased and one neuron decreased or stopped firing coincident with the masticatory sequence without an apparent rhythmical modulation of frequency corresponding with the masticatory rhythm (nonphasic group). The firing pattern of the remaining 40 neurons was modulated in phase with jaw movement (phasic group); 34 neurons either showed a spike burst or attained the highest firing frequency during the jaw-opening phase (opening type), while 6 neurons did so during the jaw-closing phase (closing type). The firing patterns of each neuron were essentially the same regardless of the kind of food ingested, except for 2 opening-type neurons that showed a rhythmical burst during mastication of solid food and tonic activity during lapping milk. 3. For 16 phasic neurons, there were significant correlations between some aspects of the firing pattern and a parameter of the movement during ingestion of solid food and/or milk. With one exception, these relationships did not appear to be due to sensory feedback. 4. We detected a monosynaptic excitatory projection from 3 opening-type neurons to the anterior digastric motoneurons, and monosynaptic inhibitory projections to the temporal or masseter motoneurons from 3 other opening-type neurons, by spike-triggered averaging of the full-wave rectified EMG of the jaw-closing and -opening muscles. No monosynaptic projections from the closing-type neurons or nonphasic group neurons to either jaw-opener or -closer motoneurons were detected. 5. The instantaneous firing frequency of all 3 inhibitor premotor neurons was positively correlated with the opening velocity, and the firing of 2 was also related to the jaw displacement.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of a toxin (JSTX) obtained from Nephila clavata (Joro spider) on the CA1 pyramidal neurons of the hippocampus was studied using slice preparations. JSTX blocked the excitatory postsynaptic potentials (EPSPs) in the pyramidal neuron evoked by Schaffer collateral stimulation but was without effect on the antidromic action potentials or on the resting conductance. Depolarization induced by ionophoretic application of glutamate was readily suppressed by JSTX but aspartate-induced depolarization was much less sensitive to the toxin. Among preferential agonists activating 3 receptor subtypes for excitatory amino acids, quisqualate responses were most effectively suppressed by JSTX. Kainate responses were similarly suppressed but in some cells higher concentration of the toxin was needed to block the responses. N-methyl- D -aspartate (NMDA) responses were the least sensitive to JSTX but they were suppressed by ± 2-amino-5-phonovaleric acid (APV). Long term potentiation (LTP) once it had taken place was not completely inhibited by APV. In the presence of JSTX, however, LTP was blocked and tetanic stimuli produced only a short-lived potentiation. In Mg2+ free solution, an orthodromic stimulation evoked repetitive spike responses which were superimposed on the depolarization following the initial spike. APV suppressed the depolarization and associated spikes leaving an orthodromic response which was sensitive to JSTX. The results suggest that JSTX blocks EPSPs in CA1 pyramidal neurons which are mediated by non-NMDA type receptors.

The digastric nerve reflex response to stimulation of the upper lip was studied in urethan-anesthetized rabbits paralysed with pancuronium bromide. Rhythmic bursts of masticatory activity were evoked in the nerve by repetitive electrical stimulation of the motor cortex. The amplitude and latency of the reflex responses during fictive mastication were compared with preceding control values. When stimuli close to threshold were given, the largest and earliest responses occurred during the digastric burst. When intense stimuli were employed, the largest responses were out of phase with the burst, although the latency was still shortest when the motoneurons were rhythmically active. Since the pattern is essentially the same as that seen during normal mastication, we conclude that the cyclical modulation of reflex amplitude and latency is not the result of sensory feedback generated by the movements themselves but is instead governed by the central motor program.