Yoshinori Sahara

Defining Affinity with the GABAA Receptor

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 GABAA receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.

Microscopic Kinetics and Energetics Distinguish GABAA Receptor Agonists from Antagonists

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 GABA(A) 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 GABA(A) 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.

A Truncated Tropo-Myosine-Related Kinase B Receptor, T1, Regulates Glial Cell Morphology via Rho GDP Dissociation Inhibitor 1

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.

Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites

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.

Cellular localization of metabotropic glutamate receptors mGluR1, 2/3, 5 and 7 in the main and accessory olfactory bulb of the rat

The cellular localization of metabotropic glutamate receptors (mGluRs) (mGluR1alpha, 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. mGluR1alpha 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.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of α‐pompilidotoxin (α‐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 α‐PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of α‐PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and α‐scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of α‐PMTX on Na+ channels by whole‐cell recordings from rat trigeminal neurons. We found that α‐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 α‐PMTX had voltage‐dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that α‐PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of α‐PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro

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-phosphonovaleric 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.

Low-Voltage-Activated Potassium Channels Underlie the Regulation of Intrinsic Firing Properties of Rat Vestibular Ganglion Cells

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 approximately 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 alpha-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.

Truncated TrkB-T1 regulates the morphology of neocortical layer I astrocytes in adult rat brain slices

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.

TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium–calcium exchangers in rat odontoblasts

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.