A pilot study to assess the origin of the spectral power increases of heart rate variability associated with transient changes in the R-R interval.

Spectral analysis of heart rate variability (HRV) is used to assess cardiovascular autonomic function. In the power density spectrum calculated from a time series of the R-R interval (RRI), three main components are distinguished: very-low-frequency (VLF; 0.003-0.04 Hz), low-frequency (LF; 0.04-0.15 Hz), and high-frequency (HF; 0.15-0.4 Hz) components. However, the physiological correlates of these frequency components have yet to be determined. In this study, we conducted spectral analysis of data segments of various lengths (5, 30, 100, and 200 s) of the RRI time series during active standing. Because of the trade-off relationship between time and frequency resolution, the analysis of the RRI data segment shorter than 30 s was needed to identify the temporal relationships between individual transient increases in RRI and the resulting spectral power changes. In contrast, the segment of 200 s was needed to properly evaluate the magnitude of the increase in the VLF power. The results showed that a transient increase in the RRI was tightly associated with simultaneous increases in the powers of the VLF, LF, and HF components. We further found that the simultaneous power increases in these three components were caused by the arterial baroreceptor reflex responding to rapid blood pressure rise.

Temporal relationships among changes in the RR-interval and the powers of the low- and high-frequency components of heart rate variability in normal subjects.

Spectral analysis of heart rate variability (HRV) is widely used as a non-invasive method to assess the cardiovascular autonomic function. Of the two main frequency components of HRV, namely low-frequency (LF, 0.04-0.15 Hz) and high-frequency (HF, 0.15-0.4 Hz) components, it is generally accepted that the HF power reflects modulation of heart rate which is mediated by cardiac parasympathetic (vagal) nerve activity. In contrast, the origin and functional correlates of the LF component are still controversial. Although several lines of evidence have indicated a close correlation between LF power and the baroreflex modulation of autonomic outflows, the detailed mechanisms underlying the genesis of the LF component remain unclarified. In this study, we conducted an ultra-short-term (UST) spectral analysis of R-R interval (RRI) time series using Fast Fourier Transform (FFT) with 5- and 25-s windows to clarify the temporal relationships among transient changes in the RRI and, LF and HF powers in healthy subjects. We found that during active standing, transient RRI increases occurred sporadically. The UST spectral analysis revealed that this RRI increase was associated with a simultaneous increase in HF power which was closely linked to the prominent LF power increase. These results indicate that during active standing, increases in LF and HF powers occur simultaneously, and they may reflect enhanced cardiac vagal activity which generates transient bradycardia.

Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

Differences in exercise-induced blood pressure changes between young trained and untrained individuals.

There are no studies assessing short-term blood pressure (BP) changes induced by daily exercise load in young trained individuals. The authors enrolled 25 healthy, trained (mean age 19.7 ± 0.1 years, 36% female) and 26 healthy, untrained (mean age 20.4 ± 0.3 years, 50% female) individuals and measured BP after the Master two-step test. Among them, 42 individuals underwent echocardiography after BP measurements to assess left ventricular mass index (LVMI). The baseline systolic BP (SBP) levels of trained and untrained individuals were 122.7 ± 2.9 versus 117.4 ± 1.5 mmHg, respectively (p = .016). Trained individuals showed a significant suppression of the SBP increase soon after exercise loads and lower SBP levels at 1, 2, and 3 min after exercise loads compared with untrained individuals. The peak SBP level over the study period was also significantly lower in trained individuals than in untrained individuals: 156.4 ± 3.3 versus 183.7 ± 5.2 mmHg (p < .001). Trained individuals showed significantly higher LVMI compared with untrained individuals: 129.4 versus 101.6 g/m2 (p < .001). These findings demonstrated that trained individuals showed significant suppression of short-term BP variability in response to by daily exercise loads and prompt SBP recovery from acute exercise loads compared with untrained individuals. Our results would be useful to understand short-term BPV and LV hypertrophy induced by adaptive responses of the heart to regular exercise loads.

An increase in the deoxygenated hemoglobin concentration induced by a working memory task during the refractory period in the hemodynamic response in the human cerebral cortex.

In activated brain regions, the deoxygenated hemoglobin (deoxy-Hb) concentration decreases despite an increase in oxygen consumption. This is attributed to the fact that the cerebral blood flow (CBF) induced by neuronal activation exceeds the accompanying increase in the cerebral metabolic rate of oxygen (CMRO2). The discrepancy between large CBF and disproportionately small CMRO2 responses provides the basis for detecting the hemodynamic correlates of neuronal activities by functional magnetic resonance imaging (fMRI). However, this implies that if the supply of oxygen is made smaller than the oxygen consumed by the suppression of stimulus-induced CBF, the polarity of signals would be reversed. We used near-infrared spectroscopy (NIRS) to search for a condition wherein a marked decrease in the stimulus-evoked oxygenated Hb (oxy-Hb) concentration change was accompanied by an increase in the deoxy-Hb concentration in the human brain. We found that when a specific brain region was activated by two working memory (WM) task blocks in rapid succession, the local change in the deoxy-Hb concentration evoked by the second task block was reversed to an increase due to the refractory effect in the hemodynamic response. The result suggests that the polarity of the blood oxygenation level-dependent (BOLD) signal could change during repetitive neuronal activation, and thus caution must be taken in the interpretation of the BOLD signal under such situations.

Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation.

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

Relationship between afterhyperpolarization profiles and the regularity of spontaneous firings in rat medial vestibular nucleus neurons.

Our previous in vivo and in vitro whole-cell patch-clamp recording studies demonstrated that neurons in the medial vestibular nucleus (MVN) could be characterized on the basis of three electrophysiological properties: afterhyperpolarization (AHP) profile; firing pattern; and response pattern to hyperpolarizing current pulses. In the present study, to clarify which types of the classified MVN neurons correspond to neurons with regular or irregular firing, we investigated their spike discharge patterns using whole-cell patch-clamp recording in both in vivo and in vitro preparations. The discharge regularity was related to AHP profiles, and we found that: (i) the coefficient of variation (CV) of interspike intervals during spike discharges was smaller in neurons exhibiting AHP with a slow component [AHP(s+)] than in those without a slow component [AHP(s-)], or with a slow AHP component preceded by afterdepolarization (ADP) [AHP(s+) with ADP]; (ii) the blockade of Ca(2+)-dependent K(+) channels by 100 nm apamin abolished the slow component and increased the CV in neurons exhibiting AHP(s+); and (iii) the modulation of firing (firing gain) in response to ramp current was larger in neurons exhibiting AHP(s-) than in the other two neuronal types. These results suggest that neurons exhibiting AHP(s+) are regularly discharging neurons with small firing gains to stimulus, neurons exhibiting AHP(s+) with ADP are irregularly discharging neurons with small firing gains, and neurons exhibiting AHP(s-) are irregularly discharging neurons with large firing gains. The regular firing of neurons exhibiting AHP(s+) is attributed to the activation of apamin-sensitive Ca(2+)-dependent K(+) channels.

Membrane properties of excitatory and inhibitory neurons in the rat prepositus hypoglossi nucleus.

The prepositus hypoglossi nucleus (PHN) is thought to be a neural structure involved in transforming eye or head velocity signals into eye position signals for horizontal eye movements. In this study, we investigated the relationship between electrophysiological membrane properties and expression patterns of cellular markers for excitatory and inhibitory neurons by whole-cell patch clamp recordings followed by reverse transcription polymerase chain reaction (RT-PCR) analysis in rat brainstem slices. Three types of voltage response properties, namely afterhyperpolarization (AHP), firing pattern, and response to hyperpolarizing current pulses, were characterized in each neuron. Following RT-PCR analysis, we identified PHN neurons as either glutamatergic (n = 22) or GABAergic (n = 43), although a small number of cholinergic (n = 2) and glycinergic neurons (n = 1) were also identified. Both glutamatergic and GABAergic neurons showed a wide variety of membrane properties; however, we found several characteristic relationships between neuronal type and membrane properties. Most neurons exhibiting (i) AHP without a slow component, (ii) a firing pattern with a delay in the generation of the first spike, (iii) a firing pattern with a transient burst and (iv) a firing pattern with a prolonged initial interspike interval were GABAergic. On the other hand, glutamatergic neurons were primarily characterized by a low firing rate. These results indicate that there is a close relationship between specific electrophysiological membrane properties and expression of chemical markers in some types of glutamatergic and GABAergic PHN neurons.

Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover.

A glial glutamate transporter, GLAST, is expressed abundantly in Bergmann glia and plays a major role in glutamate uptake at the excitatory synapses in cerebellar Purkinje cells (PCs). It has been reported that a higher percentage of PCs in GLAST-deficient mice are multiply innervated by climbing fibers (CFs) than in the wild-type (WT) mice, and that CF-mediated EPSCs with small amplitude and slow rise time, designated as atypical slow CF-EPSCs, are observed in these mice. To clarify the mechanism(s) underlying the generation of these atypical CF-EPSCs, we used (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA), an inhibitor of glial glutamate transporters. After the application of PMB-TBOA, slow-rising CF-EPSCs were newly detected in WT mice, and their rise and decay kinetics were different from those of conventional fast-rising CF-EPSCs but similar to those of atypical CF-EPSCs in GLAST-deficient mice. Furthermore, both slow-rising CF-EPSCs in the presence of PMB-TBOA in WT mice and atypical CF-EPSCs in GLAST-deficient mice showed much greater paired-pulse depression compared with fast-rising CF-EPSCs. In addition, both of them were more markedly inhibited by gamma-d-glutamyl-glycine, a low-affinity competitive antagonist of AMPA receptors. These results indicated that both of these types of EPSCs were mediated by a low concentration of glutamate released from neighboring CFs. Based on all of these findings, we suggest that glial transporters prevent glutamate released from a single CF from spilling over to neighboring PCs other than the synaptically connected PC, and play an essential role in the maintenance of the functional one-to-one relationship between CFs and PCs.

Membrane properties of rat medial vestibular nucleus neurons in vivo.

In our previous study using the whole-cell patch clamp technique combined with reverse transcription-polymerase chain reaction analysis in rat brainstem slices, we demonstrated that the classification of neurons in the medial vestibular nucleus (MVN) based on three membrane properties detected as voltage response properties to depolarizing and hyperpolarizing current pulses, namely afterhyperpolarization (AHP) profiles, firing patterns, and response patterns to hyperpolarizing current pulses, is useful for clarifying the relationship between membrane properties and cellular markers for excitatory and inhibitory neurons. These membrane properties characterized in vitro, however, have not been ascertained in vivo. To address this issue, we applied the whole-cell patch clamp recording method to in vivo preparations of young adult rats and investigated voltage responses to depolarizing and hyperpolarizing current pulses. We found three AHP profiles, three firing patterns, and three response patterns to hyperpolarizing current pulses in MVN neurons in vivo that were characterized in our previous in vitro study. The MVN neuronal populations classified on the basis of the three membrane properties in vivo were comparable to those obtained in vitro. This finding indicates that the classification of MVN neurons based on the three membrane properties is applicable to in vivo preparations.

Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice.

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.

Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses.

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate-aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses.

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.

Kainate receptor-dependent short-term plasticity of presynaptic Ca2+ influx at the hippocampal mossy fiber synapses.

Transmitter release at the hippocampal mossy fiber (MF)-CA3 synapse exhibits robust use-dependent short-term plasticity with an extremely wide dynamic range. Recent studies revealed that presynaptic kainate receptors (KARs), which specifically localized on the MF axons, mediate unusually large facilitation at this particular synapse in concert with the action of residual Ca2+. However, it is currently unclear how activation of kainate autoreceptors enhances transmitter release in an activity-dependent manner. Using fluorescence recordings of presynaptic Ca2+ and voltage in hippocampal slices, here we demonstrate that paired-pulse stimulation (with 20-200 msec intervals) resulted in facilitation of Ca2+ influx into the MF terminals, as opposed to other synapses, such as the Schaffer collateral-CA1 synapse. These observations deviate from typical residual Ca2+ hypothesis of facilitation, assuming an equal amount of Ca2+ influx per action potential. Pharmacological experiments reveal that the facilitation of presynaptic Ca2+ influx is mediated by activation of KARs. We also found that action potentials of MF axons are followed by prominent afterdepolarization, which is partly mediated by activation of KARs. Notably, the time course of the afterdepolarization approximates to that of the paired-pulse facilitation of Ca2+ influx, suggesting that these two processes are closely related to each other. These results suggest that the novel mechanism amplifying presynaptic Ca2+ influx may underlie the robust short-term synaptic plasticity at the MF-CA3 synapse in the hippocampus, and this process is mediated by KARs whose activation evokes prominent afterdepolarization of MF axons and thereby enhances action potential-driven Ca2+ influx into the presynaptic terminals.

Muscarine-induced increase in frequency of spontaneous EPSCs in Purkinje cells in the vestibulo-cerebellum of the rat.

Cholinergic projections are relatively sparse in the cerebellum compared with other parts of the brain. However, some mossy fibers in the vestibulo-cerebellum are known to be cholinergic. To clarify the functional roles of cholinergic mossy fibers in the vestibulo-cerebellum, we investigated the effects of acetylcholine (ACh) on the membrane electrical properties of both granule cells and Purkinje cells in slices of the cerebellar vermis of the rat using whole-cell patch-clamp techniques. The bath application of ACh induced a marked increase in the frequency of spontaneous EPSCs (sEPSCs) in Purkinje cells specifically in the vestibulo-cerebellum. This effect of ACh was mimicked by muscarine but not by nicotine. It was abolished by application of either tetrodotoxin or the antagonist of AMPA receptors, indicating that the ACh-induced enhancement of sEPSCs occurred indirectly via the activation of neurons sending glutamatergic projections to Purkinje cells. In approximately 15% of granule cells tested in the vestibulo-cerebellum, muscarine elicited membrane depolarization accompanied by a decrease in membrane conductance and increased the neuronal excitability. The muscarine-induced depolarization of granule cells in the vestibulo-cerebellum was attributable to the inhibition of standing-outward K+ currents (IKSO) most likely via the activation of muscarinic M3 receptors. Taken together, these results indicate that ACh increases the firing frequency of granule cells by inhibiting IKSO, which in turn increases the frequency of sEPSCs in Purkinje cells in the rat vestibulo-cerebellum.

Presynaptic Ca2+ entry is unchanged during hippocampal mossy fiber long-term potentiation.

The hippocampal mossy fiber (MF)-CA3 synapse exhibits NMDA receptor-independent long-term potentiation (LTP), which is expressed by presynaptic mechanisms leading to persistent enhancement of transmitter release. Recent studies have identified several molecules that may play an important role in MF-LTP. These include Rab3A, RIM1alpha, kainate autoreceptor, and hyperpolarization-activated cation channel (I(h)). However, the precise cellular expression mechanism remains to be determined because some studies noticed essential roles of release machinery molecules, whereas others suggested modulation of the ionotropic processes affecting Ca2+ entry into the presynaptic terminals. Using fluorescence recordings of presynaptic Ca2+ in hippocampal slices, here we demonstrated that MF-LTP is not accompanied by an increase in presynaptic Ca2+ influx during an action potential. Whole-cell recordings from CA3 neurons revealed long-lasting increases in mean frequency, but not mean amplitude, of miniature EPSCs after the high-frequency stimulation of MFs. These data indicate that the presynaptic expression mechanisms responsible for enhanced transmitter release during MF-LTP involve persistent modification of presynaptic molecular targets residing downstream of Ca2+ entry.

Postsynaptic expression of a new calcium pathway in hippocampal CA3 neurons and its influence on mossy fiber long-term potentiation.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is induced by postsynaptic Ca(2+) influx via NMDA receptors (NMDARs). However, this synaptic plasticity occurs independently of NMDARs when Ca(2+)-permeable AMPA receptors (AMPARs) are expressed at postsynaptic sites using various genetic techniques, indicating that an increase in Ca(2+) level at critical postsynaptic sites, regardless of its entry pathway, triggers the induction of LTP at CA1 synapses. In contrast, NMDARs are sparsely distributed on mossy fiber (MF) synapses in CA3 hippocampal neurons, and most evidence favors the presynaptic mechanism for LTP induction, although some reports suggested a postsynaptic mechanism. In this study, we examined whether Ca(2+) influx through the newly produced postsynaptic receptors during high-frequency stimulation affects the induction of MF LTP. For this purpose, we expressed Ca(2+)-permeable AMPARs in CA3 pyramidal neurons by Sindbis viral-mediated gene transfer of the unedited form of the glutamate receptor 2 (GluR2Q) subunit, as a new pathway for postsynaptic Ca(2+) entry, in rat hippocampal organotypic cultures. Virally expressed myc-tagged GluR2Q was detected at the complex spines known as the thorny excrescences, which serve as postsynaptic targets for MF synaptic input, on the proximal apical dendrites of CA3 pyramidal cells. Furthermore, endogenous Ca(2+)-impermeable AMPARs at MF synapses were converted into Ca(2+)-permeable receptors by GluR2Q expression. However, the postsynaptic expression of Ca(2+)-permeable AMPARs had no significant influence on the two types of MF LTP induced by different stimulus protocols. These results supported the notion that MF LTP is independent of postsynaptic Ca(2+).

Effects of a novel glutamate transporter blocker, (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), on activities of hippocampal neurons.

Glutamate transporters rapidly take up synaptically released glutamate and maintain the glutamate concentration in the synaptic cleft at a low level. (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) is a novel glutamate transporter blocker that potently suppresses the activity of glial transporters. TFB-TBOA inhibited synaptically activated transporter currents (STCs) in astrocytes in the stratum radiatum in rat hippocampal slices in a dose-dependent manner with an IC50 of 13 nM, and reduced them to approximately 10% of the control at 100 nM. We investigated the effects of TFB-TBOA on glutamatergic synaptic transmission and cell excitability in CA1 pyramidal cells. TFB-TBOA (100 nM) prolonged the decay of N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs), whereas it prolonged that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated EPSCs only when the desensitization of AMPARs was reduced by cyclothiazide (CTZ). Furthermore, long-term application of TFB-TBOA induced spontaneous epileptiform discharges with a continuous depolarization shift of membrane potential. These epileptiform activities were mainly attributed to NMDAR activation. Even after pharmacological block of NMDARs, however, TFB-TBOA induced similar changes by activating AMPARs in the presence of CTZ. Thus, the continuous uptake of synaptically released glutamate by glial transporters is indispensable for protecting hippocampal neurons from glutamate receptor-mediated hyperexcitabilities.

Identification and characterization of a novel Delphilin variant with an alternative N-terminus.

Delphilin is identified as a Glutamate receptor delta2 (GluRdelta2) subunit interacting protein, consisting of a PDZ domain and formin homology (FH) domains 1 and 2, in addition to a C-terminal coiled-coil structure. Delphilin has been shown to be selectively expressed in cerebellar Purkinje cells where it co-localizes with the GluRdelta2 subunit at the Purkinje cell-parallel fiber synapses. Although Delphilin specifically interacts with the GluRdelta2 C-terminus via its PDZ domain, the physiological role of the interaction is not yet understood. Here, we report that the Delphilin protein exhibits diversity at its N-terminus by variable usage of the first several exons. Interestingly, the two Delphilin mRNAs which correspond to the first one initially identified (now designated as Delphilin alpha) and the second that contains a newly identified first exon (designated as Delphilin beta), show different chronological expression profiles. Delphilin beta mRNA was not decreased throughout the cerebellar development in vivo and in vitro, while in vivo Delphilin alpha mRNA gradually decreases following the first postnatal week. Delphilins alpha and beta also revealed different subcellular distribution with some overlap. Specifically, the cerebellar synaptosomal membrane fraction contained the Delphilin beta protein. Both Delphilin alpha and beta localized at the dendritic spines with GluRdelta2; however, dendritic shafts in cultured Purkinje cells also included Delphilin beta. In MDCK cells upon becoming confluent, Delphilin alpha moved to the cell-cell junction area, whereas Delphilin beta maintained a diffuse distribution pattern throughout the cytoplasm. Taken as a whole, these two different Delphilins seemed to play functionally different roles in developing and matured cerebellar Purkinje cells.