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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.

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.

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.

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.

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.

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.

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.