We investigated the properties of functional connections in the primary (S1) and secondary (S2) somatosensory cortex in rat cortical slices. S1 and S2 were identified according to the myeloarchitecture, which could be visualized in the translucent images of fresh slices illuminated by near vertical light. Neural activities were elicited by repetitive electrical stimulation, and visualized by green (500-550 nm) autofluorescence of flavoproteins in blue (450-490 nm) light. The stimulation applied in S1 produced neural responses spreading into S2 beyond the areal boundary between S1 and S2. However, the neural activities were not invaded into S1, when S2 was stimulated. This anisotropic information flow of neural activities probably reflects the physiological information flow from S1 to S2 in vivo. We also investigated activity-dependent changes in the neural activities in the barrel cortex. One or two weeks after trimming whiskers, slices were prepared from the contralateral barrel cortex. In these sensory-deprived slices, supragranular neural activities after stimulation of infragranular layers were clearly suppressed. These results suggest that flavoprotein autofluorescence imaging in the slice preparations are useful for investigating not only the properties of functional connections between S1 and S2 but also activity-dependent modification in somatosensory neural circuits. [Jpn J Physiol 54 Suppl:S145 (2004)]

View full abstract

Sensory information is processed in multiple cortical areas, which are connected with the pathways in the white matter. However, adjacent areas are also connected with direct pathways via the cortical boundary between them. To elucidate the roles of latter pathways, we investigated the functional connections via the boundary between the primary auditory cortex (area 41) and the surrounding areas in rat cortical slices. Neural activities were elicited by repetitive stimulation at 20 Hz for 1 s applied at layer V, and the propagating activities via the cortical boundaries were visualized using flavoprotein autofluorescence imaging method. The neural activities propagating in a short range (~0.5 mm) between area 41 and anterior area 39, or area 41 and dorsal area 18a spread around the boundary almost symmetrically. But the activities from area 41 into the secondary auditory cortices (ventral area 20 and posterior area 36) were more prominent than those in the opposite direction. This asymmetry was also observed in the activities propagating in a long range (~1.5 mm) between area 20 and the middle part of area 41. The application of 10 μM CNQX, an antagonist of non-NMDA glutamate receptor, suppressed the long-range propagation of neural activities, while cutting the white matter around the cortical boundary had no effect. Now we are investigating the possibility that these functional connections between areas might be modified by sensory deprivation in the slices obtained from deafened rats. [Jpn J Physiol 54 Suppl:S145 (2004)]

View full abstract

All animal care was performed in accordance with the University guidelines. The rodent primary somatosensory cortex (SI) contains a detailed representation of the whiskers on the face. The sensory information is transferred to SI from whiskers via the ventroposteromedial nucleus (VPM) of the thalamus. To investigate detailed synaptic activities of cortical neurons receiving input from thalamus, we have applied the in vivo whole-cell patch-clamp technique to rat SI. Male SD rats were anesthetized with urethane. Recording pipettes were filled with standard solution containing 0.2% neurobiotin. Rats were then perfused for the morphological studies. Most recorded neurons exhibited spontaneous EPSCs (V_H=-70mV) and spontaneous IPSCs (V_H=0mV). Deflection of contralateral whiskers elicited increased EPSCs in both frequency and amplitude. Some neurons exhibited membrane current fluctuation (oscillation). Application of CNQX through the perfusion line abolished the oscillatory current, indicating that the oscillation consisted of the summated EPSCs. Whisker stimulation distorted the regularity of the oscillation indicating that oscillation could be related to neural processing in stationary phase. A single electrical stimulation on VPM by using bipolar electrode induced an EPSC with short latency (2ms) as well as EPSCs with long latency (120ms) in cortical neurons and this stimulation also suppressed the oscillation suggesting that thalamic activities could regulate the oscillation. [Jpn J Physiol 54 Suppl:S145 (2004)]

View full abstract

Focal cortical dysplasia is one of the important causes of intractable epilepsies and characterized histologically by disorganized cortical lamination and cytomegalic dysplastic neurons. GABA is a major inhibitory transmitter in the brain. However, in immature neurons, GABA has an excitatory action because of a high intracellular Cl⁻ concentration. The change in GABAergic functions regulated by an alteration in Cl⁻ homeostasis, plays an important role in neocortical development by modulating such events as laminar organization and synaptogenesis. Cation-Cl⁻ cotransporters play a critical role in the regulation of [Cl⁻]_i, and of these NKCC1 promotes accumulation of Cl⁻ into, whereas KCC2 extrudes it out of the cell. Functional alterations in Cl⁻ homeostasis are known to be induced by neuronal insults such as trauma or axotomy. To investigate alterations in Cl⁻ homeostasis in human cortical dysplasia, NKCC1 and KCC2 expressions were examined by in situ hybridization histochemistry and immunohistochemistry. KCC2 mRNA and protein are expressed in both normal and displasic neurons. KCC2 mRNA and protein expressions were downregulated in the neurons around dysplastic neurons. NKCC1 expression was not changed. These results suggested that decrease of KCC2 expression might be changed GABAergic response to excitatory and be involved in the pathogenesis of dysplastic cortex. [Jpn J Physiol 54 Suppl:S145 (2004)]

View full abstract

Midazolam (MDZ), a benzodiazepine (BZD) analogue, is clinically used to induce sedation, relieve anxiety, and to impair memory of preoperative events. BZD act at mainly postsynaptic GABA_A receptors and enhance the action of inhibitory transmitter GABA. However, effects of MDZ on presynaptic GABA release have not been studied. Thus we examined the effects of MDZ on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in layer V pyramidal neurons in somatosensory cortex by using whole-cell patch-clamp technique for 2-3 weeks-old rat brain slices. The mIPSCs were significantly increased in frequency by 0.1-1 μM MDZ. However, other BZD analogues such as diazepam (0.5 μM) and zolpidem (0.5 μM) did not change the mIPSCs frequency, suggesting that MDZ might enhance the presynaptic GABA release via some mechanism other than GABA_A receptor activation. The MDZ-induced incremental effect of mIPSCs frequency was blocked by α7 nicotinic ACh receptor (nAChR) blocker methyllycaconitine, but not by α4β2 nAChR blocker dihydro-β-erythtroidine. Although nicotine alone fail to increase the mIPSCs frequency, pretreatment with MDZ enhanced the nicotine-induced GABA release. These results indicate that MDZ enhanced translocation of nAChR over the membrane surface, hence endogenous ACh increased the release of GABA via α7 nAChR on presynaptic nerve terminal. [Jpn J Physiol 54 Suppl:S146 (2004)]

View full abstract

Cajal-Retzius cells locating in marginal zone (MZ) play important roles in the construction of the cerebral cortex. In cortical plate, Ca²⁺ influx induced by GABA_A receptor- and/or glycine receptor-mediated depolarization is associated with cell migration and synapse formation. However, the roles of GABA_A receptor- and glycine receptor-mediated responses in synaptic transmission of MZ are yet to be elucidated. Tangential slices were made from MZ of postnatal 0-3 rats. Spread of excitation was recorded by voltage imaging using voltage-sensitive dye, JPW1114. Action potentials evoked by single electric stimuli radially spreaded over surrounding MZ, and those spread of excitation were completely blocked by tetrodotoxin or Ca²⁺-free medium. Although they were hardly affected by glutamate receptor blockers, AP5 and CNQX, GABA_A receptor antagonists, bicuculline and picrotoxin, and glycine receptor antagonist, strychnine largely inhibited them. Furthermore, Na⁺,K⁺-2Cl⁻ cotransporter inhibitor, bumetanide, also reduced spread of excitation in the MZ. Our data suggest that the high intracellular Cl⁻ concentration maintained mainly by NKCC1 is attributable to the excitatory roles of GABA and glycine receptor agonists, in the synaptic transmission of the MZ. Either synaptic or no-synaptic release of those ^"excitatory^" amino acids were further analysed by using microdyalysis technique. [Jpn J Physiol 54 Suppl:S146 (2004)]

View full abstract

The presence of columnar organization is most apparent in the rodent primary somatosensory cortex receiving inputs arising from whiskers, as revealed by a barrel structure in layer IV. By contrast, it is unclear whether the columnar organization exists or not in the insular cortex, the primary gustatory area. In the present study, we have performed optical imaging to investigate spatiotemporal patterns of excitation spread in the insular cortex in comparison with the barrel cortex. Slice preparations were incubated with a voltage-sensitive dye (RH414) for 1 hr. Following electrical stimulation applied to layer IV, excitation spread vertically from layer IV to layers II/III in the insular cortex, similar to the pattern seen in the barrel cortex. Then, the excitation spread horizontally in layers II/III in the both cortices. However, the extent of horizontal spread and the intensity of excitation in layers II/III were smaller and lower, respectively, in the insular than in the barrel cortex. An application of bicuculline (5-20 μM), a GABA_A receptor antagonist, enlarged the extent and prolonged the time course of excitation spread in the insular cortex as well as in the barrel cortex. These results suggest that although there is a columnar organization in the insular cortex, horizontal excitatory connections are less abundant in the insular cortex than in the barrel cortex while there is no prominent difference in GABA_A receptor-mediated lateral inhibition in layers II/III. [Jpn J Physiol 54 Suppl:S146 (2004)]

View full abstract

Thalamocortical connections undergo remarkable plasticity during critical period, and accumulating evidence demonstrated that an activation of silent synapses at postsynaptic site is an important underlying mechanism in this process. In contrast, relatively little is known about the nature of presynaptic property at this connection, especially in relation to the development and plasticity. We studied developmental changes of release probability at thalamocortical synapses on a layer IV neuron in the developing mouse barrel cortex using progressive block of EPSCs by MK-801, an open channel blocker of N-methyl-D-aspartate type of glutamate receptor, in combination with the conventional paired pulse ratio method. We found that there were two classes of terminals with high and low probability of release throughout the development, and only the higher one was further higher during the critical period. Furthermore, our analysis indicated a possibility that such higher probability was found selectively at terminals synapsing onto postsynaptic silent synapses. Our results suggest that presynaptic sites may also have an active role in plasticity, by working concomitantly with postsynaptic silent synapses during critical period. [Jpn J Physiol 54 Suppl:S146 (2004)]

View full abstract

In some nerve cells, synaptically released GABA acting on postsynaptic GABA_A receptor produce not only phasic inhibition, but also tonic inhibition by persistent activation of extrasynaptic receptors. However, functional consequences of the tonic GABA_A receptor activation are not well understood. We performed patch clamp experiments, using a CsCl based pipette solution, in the neocortical layer V pyramidal cells of brain slices taken from P28-40 rats. GABA_A receptor mediated currents were isolated by application of CNQX, APV, CGP55845 and TTX at 32 °C. Bath application of GABA_A receptor antagonist bicuculline blocked miniature IPSCs (mIPSCs) and outwardly shifted baseline holding current (Ihold). SR95531, a GABA_A receptor antagonist selective for phasic inhibition, similarly abolished mIPSCs but had no significant effect on Ihold. Thus, this tonic current was not a summation of mIPSCs attributable to the GABA_A receptor activation. Bath application of benzodiazepines such as midazolam produced inward tonic currents. These currents are also abolished by bicuculline but not by SR95531, indicating benzodiazepine augmentation of the tonic GABA_A receptor mediated current. These tonic currents are also abolished by bicuculline but not by SR95531, indicating benzodiazepine augmentation of the tonic GABA_A receptor mediated current. Therefore, the tonic GABA_A receptor mediated inhibition also might be regulating neocortical excitability, allowing a future studies to investigate its specific role in neuronal function. [Jpn J Physiol 54 Suppl:S147 (2004)]

View full abstract

The adenomatous polyposis coli (APC) tumor suppressor gene is linked to the development of familial adenomatous polyposis and sporadic colorectal tumors. Though APC is highly expressed in the central nervous system (CNS) and directly binds to PSD-95 through the extreme C terminus of APC, the significance of the interaction between APC and PSD-95 in CNS remains unknown. In this study, we investigated whether APC associates neural signal transduction via clustering of PSD-95 in neurons. We performed the calcium imaging and the patch clamp recording in 9-11 days cultured hippocampal neurons. Bath application of 1 μM AMPA increased intracellular calcium concentration in control cells. However, this response was inhibited in the dominant negative (APC-C72) cells, which were overexpressed a dominant negative molecule (APC-C72) that could disrupt the interaction between APC and PSD-95. Under patch clamp condition, no interactions were observed in the neighboring APC-C72 cells. These results suggest that APC may affect AMPA signal transduction via clustering of PSD-95. [Jpn J Physiol 54 Suppl:S147 (2004)]

View full abstract

Aiming at an improved form of the evanescence microscope, we constructed a system scanning with a slit beam thinner than 1 μm in width for imaging a single exocytosis in neuronal cells stained with fluorescent dyes and proteins. The slit-scanning fluorescence (SSF) microscope consisted of an objective lens (x100 apo, NA=1.65), adjustable slits (0.1 mm width), and a 473 nm DPSS laser. The illuminated area in the focal region was approximately 1 x 10 μm/square, and about 1 μm thick when the angle of the incident light was set at the critical angle for total internal reflection. By shifting the angle below 50 degree, the beam passed through the specimen on the high refractive index glass (n=1.78). The image of glass-attached fluorescent beads of 3 μm in diameter appeared as a spot or a ring of 1 μm in diameter. This result indicates that the slit beam was indeed 1 μm or less in width and was able to form the optically-sliced images thinner than 1 μm thick, a better performance than confocal microscopy. In cultured neurons stained with FM1-43, many vesicle like structures (smaller than 0.3 μm) and their axonal movements were visible. A high resolution analysis of the synaptic function may be facilitated not only by the evanescence microscopy but also by the SSF microscopy proposed above. [Jpn J Physiol 54 Suppl:S147 (2004)]

View full abstract

A novel protein for cytomatrix at the active zone (CAZ), named CAST, directly binds RIM1 and indirectly binds to Munc13 through RIM1. The interaction of Munc13-1 with RIM1 is implicated in priming of synaptic vesicles. All the CAZ proteins, thus far known, including CAST, RIM1, Munc13-1, Bassoon and Piccolo, from a large molecular complex in the brain. RIM1 and Bassoon bind directly to the C-terminus and central region of CAST, forming a ternary complex. To examine the functional role for CAST in neurotransmitter release, we monitored changes in synaptic transmission produced by perturbing the interaction of CAST with the other CAZ proteins in rat superior cervical ganglion neurons in culture. These neurons express CAST, RIM1 and Bassoon at presynaptic terminals, as shown by co-localization of these proteins with synaptiphysin, a synaptic vesicle associated protein. The synaptic transmission is reduced by microinjection of RIM1- or Bassoon-binding region of CAST. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission. These results indicate that CAST serves as a key component for the CAZ structure and is involved in neurotransmitter release by directly binding these CAZ proteins. [Jpn J Physiol 54 Suppl:S147 (2004)]

View full abstract

Degradation of GTP by GTPase is thought to be essential for synaptic vesicle recycling. For example, in the endocytic process, the non-hydrolyzable GTP analogue GTPγS blocks the dynamin-dependent fission of synaptic vesicles. At the calyx of Held synapse, GTPγS loaded into the nerve terminal blocks recovery of synaptic responses from the use-dependent depression. To address whether these phenomena are co-related, we made capacitance measurements from the calyx of Held presynaptic terminal visually identified in brainstem slices of juvenile rats. A depolarizing command pulse (from –80 mV to 0 mV, 10ms) induced a rapid increase in capacitance followed by a slow recovery, suggesting that they represent vesicular exo- and endocytosis, respectively. When the presynaptic pipette contained GTP (0.3 mM) or GDPβS (3 mM), the shape and peak amplitude of the capacitance change remained stable at least for 24 min (12 stimuli). However, in the presence of GTPγS (0.2 mM) in the patch pipette, the capacitance change no longer recovered, and its peak underwent a use-dependent decline. The proline-rich domain peptide of dynamin included in the presynaptic pipette (together with GTP) mimicked the effects of GTPγS. We conclude that the dynamin-dependent GTP hydrolysis plays an essential role in vesicle endocytosis, thereby contributing to the recovery from use-dependent synaptic depression. [Jpn J Physiol 54 Suppl:S148 (2004)]

View full abstract

Co-release of glycine and GABA is established in which both are co-accumulated into the same synaptic vesicles in the spinal network. Here we show that the pattern of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and focal evoked IPSCs (eIPSCs) eliciting from a single synaptic terminal depends on the preferential uptake of glycine and GABA into the synaptic vesicles. Long time exposure with bafilomycin A1, a vacuolar-type H⁺/ATPase inhibitor, decreased in the mIPSC frequency and amplitude. During washing out bafilomycin A1, recovery of GABAergic mIPSCs was slower than for glycinergic mIPSCs as well as that of GABAergic components in the mixed (glycinergic/GABAergic) mIPSCs. Focal stimulation of a single inhibitory synaptic terminal revealed that glycine and GABA are randomly co-released from the synaptic terminals. This suggests that the uptake of glycine and GABA may provide selectivity to the synaptic vesicles. [Jpn J Physiol 54 Suppl:S148 (2004)]

View full abstract

G-protein-coupled adenosine receptors (ARs) play pivotal roles in synaptic modulation throughout the brain and thereby regulate arousal, cognition, memory, emotion, and neuronal survival. Here we show a novel action of G_i/o-coupled type-1 AR (A1R) on excitatory postsynaptic signaling. In cultured mouse cerebellar Purkinje cells, A1R activation led to acute depression of type-1 metabotropic glutamate receptor (mGluR1)-mediated inward currents and intracellular Ca²⁺ mobilization. By contrast, activation of B-type γ-aminobutyric acid receptor, another G_i/o-coupled receptor, augmented the mGluR1-mediated inward currents. The action of A1R on the inward currents was resistant to a G_i/o-protein inhibitor and was not mimicked by stimulation of the G_s-protein signaling cascade. A1R did not show functional coupling to G_q-protein. These findings demonstrate that A1R reduces postsynaptic mGluR1 signaling independently of the major G-proteins unlike its classical functions. [Jpn J Physiol 54 Suppl:S148 (2004)]

View full abstract

Application of serotonin (5-HT) induces a slow Na⁺-current response in the identified neurons of Aplysia ganglia under voltage clamp. We previously reported that this type of response is produced by the activation of cholera toxin-sensitive G-protein. The Na⁺-current response to 5-HT was gradually and irreversibly depressed after intracellular injection of Clostridium difficile toxin B, which is known to mono-glucosylate and inactivate all Rho family proteins RhoA-C, Rac and Cdc42. Furthermore, intracellular injection of Clostridium botulinum exoenzyme C3, which is known to specifically ADP-ribosylate the monomeric G-protein, RhoA-C, and uncouple its effectors, irreversibly depressed the 5-HT-induced response. In contrast, intracellular injection of L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane. Application of active form of Rac, or Cdc42 in the Rho-family G-protein did not have any effect either on the 5-HT-induced current response or resting membrane. These results suggest that the 5-HT-induced Na⁺-current response may be facilitated by the activation of monomeric G-protein RhoA. [Jpn J Physiol 54 Suppl:S148 (2004)]

View full abstract

We used high-speed optical recordings to study the effect of PREGS, one of the most important neurosteroids, on glutamate releases in rat hippocampal slices stained with the voltage-sensitive dye RH-155. PREGS acutely and dose-dependently enhanced the propagation of neuronal excitation in the dentate gyrus by augmenting the EPSP in granule cells. PREGS enhanced paired-pulse facilitation (PPF), a parameter reflecting changes in the properties of presynaptic terminals, suggesting that the augmented EPSP arises from the increased glutamate releases. To test this hypothesis we measured synaptically-induced glial depolarization (SIGD) that is an indicative of the glutamate transporter activity representing the amount of presynaptic glutamate releases. PREGS increased the amplitude and duration of SIGD, strongly supporting the idea that PREGS enhanced glutamate releases. The PREGS-effect on SIGD was prevented by the selective α7nAChR antagonists, α-BGT and MLA. Moreover GTS-21, a selective α7nAChR agonist, mimicked the PREGS-effect on SIGD and antagonized the effect of PREGS. The magnitude of the PREGS effect depended on external Ca²⁺ concentration and was attenuated by the blockade of L-type Ca channel. Taken together we propose a novel mechanism of the PREGS effect that the drug sensitizes presynaptic α7nAChR followed by an activation of L-type Ca channels to increase glutamate releases. [Jpn J Physiol 54 Suppl:S149 (2004)]

View full abstract

Dendritic spines, which are functional round-shape postsynaptic units of most excitatory synapses, are organized by glutamate receptors, actin-cytoskeletal proteins and postsynaptic density (PSD) scaffold proteins. Further, spines represent the activity-dependently pleomorphic structures based on actin cytoskeleton. We previously demonstrated that spine morphogenesis in neuronal development requires synaptic clustering of actin components and the following synaptic clustering of PSD proteins in dendritic filopodia, which are precursors of spines. (J. Neurosci.23(16):6586-95. Takahashi et. al.) However, it is unknown how synaptic activity regulates synaptic clustering of these postsynaptic molecules in neuronal development. In this study, we investigate how AMPA receptor (AMPAR) and NMDA receptor (NMDAR) activities regulate synaptic clustering of filamentous actin (F-actin) and drebrin during development. Chronic blockage of AMPAR activity inhibits their synaptic clustering, and also inhibits the morphological spine maturation from filopodia. In contrast, chronic blockage of NMDAR activity slightly promotes their clustering and the spine maturation. Our data suggest that AMPAR and NMDAR activities bidirectionally regulate the postsynaptic actin organization in developing dendritic spines. [Jpn J Physiol 54 Suppl:S149 (2004)]

View full abstract

In order to explore the facilitatory GABAergic control of central noradrenergic neurons, we investigated the effect of muscimol, a GABA_A receptor agonist, on spontaneous excitatory postsynaptic currents in mechanically dissociated rat locus coeruleus neurons attached with native glutamtatergic nerve terminals. The activation of presynaptic GABA_A receptors by muscimol facililtates spontaneous glutamate release by activating tetrodotoxin-sensitive Na⁺ channels and high-threshold Ca²⁺ channels. This depolarizng action of muscimol was caused by an outwardly directed Cl⁻ driving force for GABA_A receptors; that is, intracellular Cl⁻ concentration of glutamatergic nerve terminals was higher than that predicted for a passive distribution. The higher Cl⁻ concentration was generated by bumetanide-sensitive Na-K-Cl cotransporters and was responsible for the GABA_A receptor-mediated presynaptic depolarization. Thus, GABA_A receptor-mediated modulation of spontaneous glutamatergic transmission may contribute to the regulation of activity of locus coeruleus noradrenergic neurons. [Jpn J Physiol 54 Suppl:S149 (2004)]

View full abstract

Recently, neuropsychological and pharmacological studies provide compelling supports for a central noradrenergic hypothesis in the pathophysiology of attention deficit / hyperactivity disorder (AD/HD) that is a development disorder of children. Therefore, the nucleus locus coeruleus (LC) where is the most major source of noradrenergic tone in a central nervous system is explored to examine the effect of methylphenidate (MPH), since MPH is the most commonly used agent for a treatment of AD/HD. In the present study, the effect of MPH on neural activity of the LC of juvenile rats was examined by using a whole-cell patch clamp technique. MPH caused an outward current associated with an increase in inward rectifier K⁺ channels. MPH significantly potentiated the amplitude and time course of slow IPSCs in LC neurons, although MPH did not affect EPSCs. MPH increased the amplitude of slow IPSCs in a dose dependent manner (0.3< MPH< 3 μM), however higher dose of MPH (> 10 μM) contrary inhibited slow IPSCs. MPH enhanced the amplitude of miniature IPSCs in presence of TTX. These results were the same as the data from adult rats. Further more, noradrenalin (NA) content in a brain slice preparation was measured with and without MPH treatment by HPLC. On the LC of juvenile rats as well as adult rats, MPH may enhance the concentration of NA in the synapscleft as NA re-uptake blocker. These results suggest that noradrenergic agents play the important role in the therapy of AD/HD. [Jpn J Physiol 54 Suppl:S149 (2004)]

View full abstract

The nucleus locus coeruleus (LC) contains cell bodies of noradrenergic neurons and has widespread projection of noradrenergic neurons to cerebrum, thalamus, limbic system, hypothalamus, brain stem and spinal cord. Milnacipran is a recently released antidepressant as a class of a selective serotonin and noradrenaline re-uptake inhibitor (SNRI). In this study, we examined the effect of milnacipran on activity of LC neurons by using an intracellular recording and a whole-cell patch clamp technique. Bath application of milnacipran (0.3-100 μM) produced a hyperpolarizing response in LC neurons. Yohimbine, α₂ receptor antagonist, inhibited milnacipran induced hyperpolarization. Milnacipran (0.3-10 μM) increased the amplitude and the time course of the inhibitory postsynaptic current (IPSC) mediated by NA in LC neurons. Milnacipran (3-10 μM) significantly inhibited the amplitude of an excitatory postsynaptic current (EPSC) and such an inhibitory effect on EPSCs was attenuated by WAY100635, an 5-HT_1A receptor antagonist. We discuss the effects of milnacipran on membrane potential and synaptic transmission on LC neurons and compare milnacipran with methylphenidate that is the most common therapeutic agent to improve major symptoms of attention deficit/hyperactivity disorder (AD/HD). [Jpn J Physiol 54 Suppl:S150 (2004)]

View full abstract

We examined the role of dopamine in neurons of the rat dorsolateral septal nucleus(DLSN) by intracellular recording methods. Dopamine(DA,1-100μM) was administrated by the bath-application. DA(100μM) induced a hyperpolarization(4.9±0.6mV;n=17). In some neurons, the DA-induced hyperpolarization was gradually decreased during the application of DA. The DA-induced hyperpolarization was associated with a decrease in input membrane resistance and reversed polarity at –90mV, suggesting an increase in the potassium conductance. This hyperpolarization was not affected by TTX(1μM). We investigated the effects of DA on the EPSP and the slow IPSP in the presence of bicuculline(15µM). As a result, DA(100µM) reversibly inhibited slow IPSP in all the cells tested(63±8％;n=6), while DA(100µM) inhibited the EPSP in some neurons(26±7％;5 of 6cells). This inhibition was observed in the neurons which did not show any change in input membrane resistance. During a long-term administration(1hour), DA(1µM) progressively facilitated EPSP (~30％) in half of the neurons tested. D1 type receptor agonist SKF38393(50µM) inhibited slow IPSP in the DLSN(21±4％;n=11), while D2 type receptor agonist quinpirole(50µM) produced facilitation of the EPSP in majority of the neurons(20±3％;10 of 15 cells). However, neither D1 nor D2 type receptor agonists produced membrane hyperpolarization. These results suggest that DA causes multiple effects on the efficacy of synaptic transmission in the DLSN. [Jpn J Physiol 54 Suppl:S150 (2004)]

View full abstract

Involvement of P2X and P2Y receptors in various brain functions is now established. Yet unclear is the molecular mechanism of ATP release, especially in native tissues. To address this issue, we visualized changes in [ATP]_o in acute brain slices. The slices of the hippocampus (CA1 and CA3), cortex and the nucleus of the solitary tract (NTS) were submerged in artificial cerebrospinal fluid containing luciferin and luciferase. The bioluminescence in a 320 μm X 240 μm field was detected with an image intensifier-coupled VIM camera with a 2.5- to 30-s exposure time. Continuous electrical stimulation delivered with a co-centric bipolar electrode (0.5 mA, 10 Hz) for 10-30 s markedly increased the light intensity (LI) around the electrode (in a radius of ~150 μm), which lasted during the stimuli and faded within 15-30 s in all three structures. This LI increase was not reproduced when the same locus was stimulated but observed similarly in the same slice when a distinct region was stimulated. Stimulation of the afferent fibers increased LI at the site of stimulation but not at the postsynaptic regions. The LI increase persisted in the presence of tetrodotoxin. Application of high [K⁺]_o solution did not increase LI. It is concluded that electrical stimulation evokes ATP release at the site of stimulation, which was independent of action potential generation. Supported by Grants-in-Aid from the MECSST, Japan (No. 15650071). [Jpn J Physiol 54 Suppl:S150 (2004)]

View full abstract

Muscarinic acetylcholine receptors are believed to play important roles in higher brain functions. Here we report that muscarinic activation suppresses inhibitory transmission through two distinct mechanisms, namely cannabinoid-dependent and -independent mechanisms. When cannabinoid CB1 receptor was blocked, oxotremorine M (oxo-M), a muscarinic agonist, suppressed IPSCs in a subset of hippocampal neuron pairs. This suppression was blocked by the M2-prefering antagonist gallamine, and totally absent in neuron pairs from M2-KO mice. When CB1 was not blocked, oxo-M suppressed IPSCs in a gallamine-resistant manner in cannabinoid-sensitive pairs. This suppression was blocked by the CB1 antagonist AM281, and completely eliminated in neuron pairs from M1/M3-KO mice. Our immunohistochemical examination showed that M2 and CB1 were present at different inhibitory presynaptic terminals. These results indicate that two distinct mechanisms mediate the muscarinic suppression. In a subset of synapses, activation of presynaptic M2 suppresses GABA release directly. In contrast, in a different subset of synapses, activation of postsynaptic M1/M3 induces the release of endocannabinoids that act on presynaptic CB1 receptors and suppress the GABA release. [Jpn J Physiol 54 Suppl:S150 (2004)]

View full abstract

Muscarinic acetylcholine (ACh) receptor activation presynaptically inhibited the putative striato-nigral GABAergic IPSC at substantia nigra pars reticulata (SNr) GABA neurons from young rats. This IPSC inhibition by muscarine was abolished by the application of M₁- and M₃-muscarinic ACh receptor antagonists, 4-DAMP and pirenzepine. On this inhibition, 4-DAMP was 100 times more potent than pirenzepine. The values of IC₅₀ were 19 nM (4-DAMP) and 1.7 μM (pirenzepine), respectively. This result clearly indicated that muscarine activated the M₃-receptors in this inhibition. Since it was reported that muscarine had its presynaptic effects through the production of cannabinoid (CB) at postsynaptic neurons in other parts of brain and that CB presynaptically inhibited the IPSC at SNr GABA neurons, the effect of a CB₁-receptor antagonist, AM251, was observed. AM251 (5 μM) had no significant effect on the IPSC inhibition by muscarine. The mean values of the relative inhibition ratio were 0.529 ± 0.091 in control and 0.509 ± 0.085 in AM251 (mean ± s.e.m.; n=4, p=0.362). The activation of PKC by an activator (phorbol 12, 13-dibutyrate; PDBu, pretreatment for 15 min at 1 μM) did not mimic the effect of muscarine but significantly reduced the IPSC inhibition by muscarine. The mean values of the relative inhibition ratio were 0.449 ± 0.053 in control and 0.809 ± 0.102 in PDBu (n=4, p=0.008). This result might suggest the novel possibility that the M₃-muscarinic ACh receptor activation negatively links the activation of PKC. [Jpn J Physiol 54 Suppl:S151 (2004)]

View full abstract

We previously reported that transient application of PREGS, one of the most important neurosteroids, to hippocampal slices in rat (4 weeks) evoked a rapid and sustained potentiation of EPSP over 60 min at the dentate gyrus (DG), termed PREGS-inuced long-lasting potentiation (LLP_PREGS). LLP_PREGS was external Ca²⁺ dependent and prevented by NMDA receptor antagonist (AP5), GluRε2 subunit inhibitor (Ifenprodil) and open-channel blocker (MK-801), suggesting that a Ca²⁺ entry through NMDA receptors is involved in the LLP_PREGS. The present study aims to directly examine whether PREGS elicits elevation of intracellular Ca²⁺ in hippocampal granule cells. A Ca²⁺ indicator, Indo-1 AM (1 mM), was injected into the lateral part of the molecular layer of DG in horizontal slice. Indo-1 AM could be taken into the soma and dendrites of multiple granule cells within 15 min after injection. Changes in intrasomatic Ca²⁺ concentration ([Ca²⁺]_i) in several stained granule cells were measured using a two-photon laser scanning microscope (wave length 720nm). The result revealed that the pre-treatment of the slice with PREGS (50 μM) caused a potentiation of the rise in [Ca²⁺]_i induced by an application of 5 μM glutamate for 1-2 min. This finding strongly suggests that LLP_PREGS induction is due, in part, to an augmentation of the glutamate action to NMDA receptors. [Jpn J Physiol 54 Suppl:S151 (2004)]

View full abstract

Adenosine amine congener (ADAC), a selective A₁ agonist, has been reported to protect hippocampal neurons against ischemia without producing harmful cardiovascular side effects. We examined effects of ADAC on the neuronal activity of CA1 neurons in rat hippocampal slice preparations using intracellular and whole-cell patch-clamp recording methods. Bath-application of ADAC (0.1-2μM) produced a hyperpolarizing response and outward current (I_ADAC) in a concentration-dependent manner. At a concentration of 1μM, ADAC produced a hyperpolarizing response (4.4±0.3mV, n=9) and I_ADAC (173.3±27.5pA, n=6) with an increased membrane conductance to 140％ of the control at –65mV. The I_ADAC reversed its polarity at near –90mV, that is close to the equilibrium potential for K⁺ in the ACSF. The I_ADAC was not blocked by TTX (1μM) but partially depressed by BaCl₂(100μM). ADAC (2μM) depressed the amplitude of EPSC to 10％ of the control (n=4). ADAC did not affect the glutamate-induced inward currents. Furthermore, ADAC depressed the frequency of mEPSP without changing their amplitude. 8-CPT, a selective A₁ antagonist, blocked the I_ADAC and the depression of the EPSC. These results suggest that ADAC depresses the release of glutamate via A₁ receptors. [Jpn J Physiol 54 Suppl:S151 (2004)]

View full abstract

Neurodevelopmental abnormalities in the medial temporal lobe including hippocampas are implicated in the pathophysiology of schizophrenia. The dysfunction of the dopaminergic system is of particular interest in schizophrenia. Dopamine can enhance or inhibit synaptic transmission in various brain regions. In the hippocampus of early neonatal rat, however, the modulatory effects of dopamine on synaptic transmission has not yet been clarified. In this study we studied the effect of dopamine on glutamatergic and GABAergic transmission during the period of synaptogenesis in rat hippocampus. Whole-cell patch clamp recordings were obtained from CA1 pyramidal cells. Dopamine (100μM) reversively attenuated the amplitude of excitatory postsynaptic currents (EPSCs) to 31.6 ± 7.3% of the control and the amplitude of GABA_A receptor-mediated postsynaptic currents (GABA_A-PSCs) to 32.2 ± 5.4% of the control. SKF38393, an agonist of D1-like receptors, decreased the amplitude of EPSCs and GABA_A-PSCs, while the effect of dopamine was partially antagonized by SCH23390, an antagonist of D1-like receptors. Kainic acid (100μM)- or GABA (100μM)-induced inward currents were not changed by dopamine. Dopamine had no effect on the amplitude of miniature EPSCs or miniature GABA_A-PSCs. In conclusion, dopamine depresses glutamatergic and GABAergic transmission in the hippocampus at the stage of synaptogenesis and the depression is thought to be mediated through D1-like receptors by presynaptic mechanism. [Jpn J Physiol 54 Suppl:S151 (2004)]

View full abstract

Application of either AMPA or Quisqualic acid (QA), agonists for AMPA-receptor, induces inward current response in the CA1 pyramidal neuron in rat hippocampal slice under whole-cell patch clamp. The responses to AMPA and QA were almost completely blocked by 30 μM NBQX and GYKI52466, antagonists for AMPA-receptor, and the response reversed at +10 mV, showing opening of the cationic channel. When one of these agonists was applied repeatedly, the second response to AMPA or QA was occluded at the shorter interval of the agonist application. However, there was no sign of the desensitization in the response, although GABA-induced Cl^–-current response examined in the same type of cell exhibited strong desensitization. Furthermore, perfusion of cyclothiazide (CTZ), a reagent that relief the desensitization of AMPA-receptor, markedly augmented the AMPA- and QA-induced response without affecting the time course of the respopnse. Dose-response curve obtained in the presence of CTZ showed upward shift without the significant charge in K_D value. These results suggested that the AMPA-receptor of the pyramidal neuron in the rat hippocampal slice may be lacking a nature of the desensitization or may be under suppression of the desensitization. [Jpn J Physiol 54 Suppl:S152 (2004)]

View full abstract

In CA1 neurons of guinea pig hippocampal slices, long-term depression (LTD) was induced in the field EPSP response in the absence of test synaptic inputs (1 stimulus every 20 sec) by application of the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD). This effect was blocked and long-term potentiation (LTP) was induced by co-application of N-methyl-D-aspartate (NMDA) during ACPD perfusion (ACPD/NMDA-induced LTD). These results indicate that the state of NMDA receptor activation during ACPD perfusion determines whether LTP or LTD is induced in hippocampal CA1 neurons. Co-application of an inositol 1, 4, 5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate, during ACPD application had no effect on the ACPD/NMDA-induced LTP, but increased the magnitude of the ACPD-induced LTD, suggesting that the ACPD/NMDA-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to the ACPD-induced LTD. [Jpn J Physiol 54 Suppl:S152 (2004)]

View full abstract

Comparing with the excitatory synaptic plasticity, the modulation of GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) that modify excitatory synaptic transmission has not been well characterized. Post tetanic depression of electrically-evoked IPSCs has been reported by several investigators. However, relatively few investigations were made on potentiation of the IPSCs. We examined changes of evoked and spontaneous GABA_Aergic synaptic transmission after tetanic stimulation, in acute slices of rat hippocampus. Using patch-clamp recording method, IPSCs were recorded from CA3 neurons. Excitatory glutamatergic currents were blocked by specific inhibitors during recording of spontaneous IPSCs. High frequency stimulation was applied by microelectrode. After intensive tetanus, transient and marked facilitation of spontaneous IPSCs were observed. Miniature IPSCs, recorded in the presence of tetrodotoxin, were also facilitated by the tetanic stimulation. Suppression, rather than facilitation, of evoked IPSC was observed after tetanization under similar conditions. The facilitation of the spontaneous IPSC was still detected in nominally Ca²⁺-free solution, though the control IPSC at rest was depressed by Ca²⁺ elimination. In addition, increment of intracellular calcium concentration after high frequency stimulation was also demonstrated in hippocampal slice preparation by fluorescent imaging. [Jpn J Physiol 54 Suppl:S152 (2004)]

View full abstract

Forskolin activates PKA through stimulation of adenylate cyclase and induces late phase LTP in neurons. We previously reported that repetitive exposure of cultured hippocampal slice to forskolin induced a synaptic enhancement developed slowly and lasted for several weeks (forskolin-RISE). The occurrence of the forskolin-RISE was dependent on the times and interval of forskolin application. The forskolin-RISE, originally detected as an enlarged field excitatory postsynaptic potential, was accompanied by an increase in immunohistochemically-identified synaptic structures. We made here an electron-microscopic observation of cultures under development of the RISE and confirmed that the number of ultrastructurally-identified synapses increased in parallel with the RISE. It is assumed that an ultrastructure called 'perforated synapse' (a synapse with interrupted postsynaptic density) is a transitional form of multiplying synapse. Confirming this assumption, the numbers of perforated synapses and of synapses with short postsynaptic density (presumably representing newly-formed synapses) increased in parallel with the RISE. We recently found that a similar synaptic enhancement occurred in cultured hippocampal slice after repeated exposure to glutamate (Glu-RISE). If perforated synapses and short synapses are indices of synaptogenesis, similar ultrastructural changes are expected to occur in the slices developing Glu-RISE. This expectation was also confirmed by the present study. [Jpn J Physiol 54 Suppl:S152 (2004)]

View full abstract

Synaptic plasticity is regulated in part by the complex modulations of the intracellular phosphorylation state. Protein tyrosine phosphataseζ(PTPζ) is a receptor-like tyrosine phosphatase expressed primarily in the brain. The extracellular domain of PTPζ binds various cell-recognition, cell-adhesion molecules and growth factors. However, little is known about the functional roles of PTPζ. To investigate the role of PTPζ in synaptic transmission and plasticity, we recorded excitatory synaptic responses in the CA1 region of hippocampal slices obtained from PTPζ-deficient mice. Although we found no difference in basic synaptic properties such as paired-pulse facilitation between the genotypes, long-term potentiation (LTP) in mutant mice was age-dependently enhanced. Since properties of the NMDA receptor-mediated synaptic response before and during tetanic stimulation were indistinguishable between the genotypes, enhanced LTP in mutant mice was unlikely due to NMDA receptor modification by the lack of PTPζ. However, because LTP enhancement in mutant mice was dependent on NMDA receptor activation, we examined possible involvement of the process downstream of NMDA receptor activation. We conclude that PTPζ regulates synaptic plasticity through modification of intracellular biochemical processes involved in LTP expression via the control of tyrosine phosphorylation. [Jpn J Physiol 54 Suppl:S153 (2004)]

View full abstract

The adenylyl cyclase activator forskolin or membrane permeable cAMP analogues enhance synaptic transmission presynaptically at many synapses, but exact mechanism underlying these effects is unknown. We have studied the intracellular mechanism of the cAMP action at the calyx of Held, where it is possible to make simultaneous pre- and postsynaptic recordings. Forskolin (0.5-50 μM) presynaptically potentiated EPSCs in 8-22 days old rats with the magnitude of potentiation being reduced with postnatal development. Loading cAMP (0.5 mM) into calyceal terminal in simultaneous pre- and postsynaptic whole-cell recordings also markedly potentiated EPSCs. The forskolin-induced synaptic potentiation was associated with an increase in release probability (p) and size of readily releasable pool (RRP) of synaptic vesicles deduced from experiments using tetanic stimulation or MK-801. Forskolin had no effect on the peak amplitude of presynaptic Ca2+ currents or K+ currents recorded from calyceal terminals, suggesting that it directly stimulates exocytotic machinery downstream of Ca2+ influx. Phorbol ester applied in addition to forskolin further potentiated EPSCs with no apparent occlusion, suggesting that the mechanism underlying synaptic potentiations by cAMP and PKC are distinct. Whereas neither a PKA inhibitor nor Ih channel blocker affected the forskolin-induced EPSC potentiation. Direct loading of the cAMP-GEF activator 8CPT-2Me-cAMP into calyceal terminal potentiated EPSCs. We conclude that cAMP-induced synaptic potentiation is caused by increasing both p and RRP size, possibly through cAMP-GEF pathway at the calyx of Held. [Jpn J Physiol 54 Suppl:S153 (2004)]

View full abstract

The brain stem contains neural circuits that mediate vital functions. Many studies have addressed the development of brainstem neurons using molecular genetic and anatomical techniques. Studies of the development of functional connections have lagged behind, partly because of the anatomical complexity of the brain stem and because electrophysiological techniques for assessing neuronal activity are limited in the number of cells that can be recorded simultaneously. Here we use an optical recording technique to visualize activity in emerging vestibular and auditory circuits in the intact brain stem of the chicken embryo. Dissected brain stems at embryonic days 5-9 were labeled with a voltage-sensitive dye, NK2761. Vestibular or cochlear afferents were stimulated with suction electrodes, and a 1020-element photodiode array recorded membrane potential changes in large areas of the brain stem. Signals related to synaptic potentials in first- and second-order vestibular and cochlear nuclei, and in ocular motoneurons involved in vestibulo-oculomotor reflexes, were visualized at appropriate latencies representative of conduction and synaptic delays. Inhibitory and excitatory potentials could be discriminated by pharmacological blockade of GABA and glutamate receptors. Activation of specific neuron groups emerged in a developmental sequence that matched the anatomical development of vestibular and auditory projections. [Jpn J Physiol 54 Suppl:S153 (2004)]

View full abstract

The multiple-site optical recording of transmembrane potential activity with a voltage-sensitive merocyanine-rhodanine dye, NK2761, was used to survey extensively the early functional organization of neural circuits related to the vagus nerve in the embryonic chick brainstem. In addition to the response area on the stimulated side, including the vagal sensory nucleus (the nucleus of the tractus solitarius (NTS)) and the motor nucleus (the dorsal motor nucleus of the vagus nerve (DMNV)), vagal stimulation induced an additional response area at the level of the cerebellum on the contralateral side. This response appeared to come through the second synaptic pathway from the NTS to the contralateral side. Blockade of non-NMDA receptors in the NTS abolished the responses on the contralateral side, while blockade of NMDA receptors did not abolish the responses. The responses on the contralateral side were first detected from the 7-day old embryonic stage, when the glutamatergic EPSP in the NTS was first observed. This result suggests that the second synaptic pathway from the NTS to the contralateral side is already generated when the primary vagal afferents make functional synapses on the NTS neurons. [Jpn J Physiol 54 Suppl:S153 (2004)]

View full abstract

Nucleus magnocellularis (NM) is a division of the avian cochlear nucleus and extracts the timing of auditory signals. We compared the neurons' membrane excitability and the synaptic transmissions along the tonotopic axis of NM. The high characteristic frequency (CF) neurons demonstrated a high level expression of Kv1.1 mRNA and protein and the expression decreased along the tonotopic axis, while the expression of Kv1.2 mRNA did not change. Neurons showed tonotopic differentiation in the following membrane properties: the resting potential, the threshold, the spike amplitude and the membrane rectification. All neurons were sensitive to 100 nM DTX, but the effects were most significant in the high CF neurons. The excitatory post-synaptic current (EPSC) recorded by the stimulation of auditory nerve fiber (ANF) was 17 times larger in the high CF neurons than in the low CF neurons. EPSCs were generated in an all-or-none manner in the high CF neurons when stimulus intensity was increased, while EPSCs were gradually developed in the low CF neurons, with many sub-steps being observed. Terminal morphologies of ANF were visualized by placing Di-I into the cochlea. ANF formed enfolding terminals around the cell body (end bulb of Held) in the high CF region; however, large enfolding terminals were not found in the low CF region. These differentiation may be suitable for extracting and preserving the timing information of auditory signals for a wide range of frequencies. [Jpn J Physiol 54 Suppl:S154 (2004)]

View full abstract

Interaural time difference (ITD) is a cue for localizing a sound origin along the azimuth. In birds, Neurons in nucleus laminaris (NL) act as coincidence detectors of binaural synaptic inputs, and ITD is detected as a place of NL neurons firing maximally within the nucleus. Accurate coincidence detection (CD) is essential for this ITD detection in the NL. Previously, we showed that precise CD is achieved by the rapid EPSP time course due to the activation of low-threshold K⁺ currents and hyperpolarization-activated cation currents. In the NL, neurons are tonotopically organized, and characteristic frequencies (CF) gradually decrease from rostro-medial to caudo-lateral direction in the nucleus. Along with this axis, NL neurons change their morphologies systematically; high CF neurons have a small soma with short dendrites, while low CF neurons have a large soma with long dendrites. Furthermore, resolution of localizing sound is dependent on the sound frequency, and each animal species has its optimal frequency for sound localization. However, it is not known how morphological variations among different CF neurons affect their electrophysiological properties and acuity of CD in the NL. In this study, we examined CD along the tonotopic axis in the chick (P3-10) NL, using the whole-cell slice-patch recordings. We found that the acuity was the sharpest in middle-low CF (1-2 kHz) neurons, and became broad in both high (around 3 kHz) and very low CF (around 0.5 kHz) neurons. We explored the cellular mechanisms underlying this difference in the CD. [Jpn J Physiol 54 Suppl:S154 (2004)]

View full abstract

In birds, neurons in nucleus laminaris (NL) act as coincidence detectors of binaural synaptic inputs to localize sound sources. Acuity of coincidence detection affects the resolution of sound localization, and is determined by the EPSP time course. NL is organized tonotopically; neurons in rostromedial region have a high characteristic frequency (CF) and those in caudolateral a low CF. Electrophysiologically, the input impedance was lower and the time course of voltage sag during hyperpolarization was faster in low CF neurons. Previously, we reported that properties and distribution of hyperpolarization-activated cation current (I_h) were different along the tonotopic axis, and contributes to those differences of membrane properties in the NL. We also reported that the existence of I_h modulation by elevation of cAMP via noradrenaline (NA) in the NL. In this study, we examined I_h contributions to the EPSP time course, using the whole-cell recordings in slices. In low CF neurons, the EPSP was faster and prolonged to a larger extent after block of I_h by ZD7288. These indicate that I_h is activated at the rest and accelerate the EPSP in low CF. Application of NA accelerated the EPSP much in high CF neurons, because of reducing the input impedance and depolarizing the membrane potential. This depolarization would activate the low-threshold K⁺ current, which contribute to decay of the EPSP. These results suggest that I_h might affect the resolution of sound localization by regulating the EPSP time course in the NL, including the modulation of I_h by NA. [Jpn J Physiol 54 Suppl:S154 (2004)]

View full abstract

Responses of brain regions lining the third ventricle to extracellular Na+ changes observed in the mouseYoshida, S.1, Mohri, T.2, Sutani, K.31Dept. Life Sci., Kinki Univ., Higashi-Osaka; 2Natl. Inst. Physiol. Sci., Okazaki; 3Natl. Inst. Adv. Ind. Sci. & Tech., Ikeda, JapanIntroduction: Neurons in the brain are particularly susceptible to high Na+ level, and they may possess some mechanism for sensing the extracellular Na+ concentration ([Na+]o). The aim of the present work is to search for such a mechanism. Methods: Coronally-sectioned brain slices (200 μm thickness) were obtained from adult mice (C57BL/6J) at the level of the median eminence (ME). Slices were loaded with SBFI (Na+-sensitive fluorescent dye) and Na+ images were monitored using an image analyzer ARGUS-50. Results: 1) In all regions lining the third ventricle (3V) and ME, [Na+]i was greatly increased by ouabain and decreased by amiloride, 2) when [Na+]o was raised from a control of 140 mM to 160-200 mM, a sustained elevation in [Na+]i was observed in ME but only a transient or little response was observed in the other regions, and 3) the transitional area between ME and the lateral wall of 3V responded inversely. Discussion: The data indicate that the basal level of [Na+]i in the brain regions lining 3V and ME is maintained by Na+ pumps and epithelial channels (ENaCs). The ME, which is devoid of a blood-brain barrier, seems to work as a sensor of [Na+]o. [Jpn J Physiol 54 Suppl:S154 (2004)]

View full abstract

Excitatory synaptic transmission from the reticular formation around the trigeminal motor nucleus (MoV) to trigeminal motoneurons (TMNs) in rat brain stem slice preparations was studied by high-speed optical recording techniques and gramicidin perforated-patch recordings. Slices were stained with the voltage-sensitive dye Di-4-ANEPPS and optical signals were analyzed by a CCD camera system (MiCAM01, Brain Vision). Electrical stimulation of the reticular formation located dorsal to MoV (dRF), evoked excitatory responses in MoV. Stimulation of MoV antidromically evoked an excitatory response in dRF under suppression of synaptic transmission by substitution of external Ca²⁺ with Mn²⁺. Application of CNQX (20 μM) and APV (20 μM) reduced the excitatory response in MoV evoked by dRF stimulation in both neonatal and juvenile rats. Application of strychnine (5 μM) to MoV also suppressed the excitatory response in MoV of neonatal rats. Gramicidin perforated-patch recordings from TMNs revealed that glycinergic postsynaptic potentials in TMNs evoked by dRF stimulation were depolarizing. In contrast to neonatal animals, application of strychnine to MoV in juveniles enhanced the excitatory response in MoV. We conclude that interneurons in dRF excite TMNs through activation of glutamate or glycine receptors in neonatal rats, whereas glycine receptor activation in TMNs becomes inhibitory in juveniles. Such developmental change of synaptic transmission from dRF to MoV might be involved in the transition from suckling to mastication. [Jpn J Physiol 54 Suppl:S155 (2004)]

View full abstract

At the brainstem auditory synapse in rodents, high fidelity (HI-FI) transmission is acquired through postnatal development, and plays crucial roles in sound source localization. At the calyx of Held synapse in auditory brainstem, a variety of molecules undergo developmental changes during the first postnatal month. Among them the endogenous Ca2+ buffer calretinin was immunocytochemically detectable in the calyceal presynaptic terminal only after postnatal day 10 (P10) in mice. To address the role of calretinin in synaptic development, we compared the properties of synaptic transmission between calretinin-knockout homogygous (CRKO) mice and wild type (WT) mice. During the postnatal period from P7 to P21, paired-pulse ratio (PPR) of EPSCs increased in WT mice, whereas this increase was much smaller in CRKO mice. We estimated release probability (p) and pool size of readily releasable synaptic vesicles (N) using the high frequency stimulation protocol. In WT mice p decreases and N increases with development as reported in rats. In CRKO mice, however, the developmental increase in p was largely impaired, whereas the developmental increase in N was similar to WT mice. Furthermore, in CRKO mice, the developmental acquisition of HI-FI transmission in response to high frequency synaptic inputs was markedly impaired. We conclude that a developmental increase in the expression of calretinin in the nerve terminal increases synaptic efficacy to high frequency inputs via decreasing p, thereby contributing to the developmental acquisition of HI-FI synaptic transmission. [Jpn J Physiol 54 Suppl:S155 (2004)]

View full abstract

We have reported that the pattern of the Trigemino-Facial reflex (T-F R) changes during axonal regeneration after facial nerve (MB; trunk of the temporal-zygomatico-orbital and both superior and inferior buccolabial branch) suture in the cat. The T-F R is induced only in the ipsilateral MB but never in PA (posterior auricular branch) in normal cats, but the reflex can be detected in PA as well as in MB during axonal regeneration after MB suture. The pattern of T-F R returned to normal at 3 to 4 months after suture. In the present study, we examined the relation between the pattern of T-F R and functional recovery by comparing the number of retrogradely labeled MB motoneurons and the antidromic latencies. At various postoperative days, 5% fast blue (FB) was injected into the distal part of the sutured MB and the labeled MB neurons were examined and compared with in normal animals. The labeled MB neurons were first detected at 6 days after suture, and the number increased until 85 days after suture. The number of labeled neurons returned to the normal values at 129 days (2909±140 vs. 3027 at 129 days). The antidromic latency was 1.37ms at 10 days and shortened progressively until around 4-5 months after suture. At 165 days, the latency (1.05 msec, N=4) also returned to the normal (1.0±0.2msec, N=95). The present results indicate that the pattern of T-F R returned to the normal, when the axonal regeneration and functional recovery have completed. The neural mechanisms of the appearance of aberrant T-F R in PA should be studied further. [Jpn J Physiol 54 Suppl:S155 (2004)]

View full abstract

Morphological and electrophysiological analyses were carried out to investigate the fate of neuroepithelial stem cells grafted in the striatum. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of transgenic GFP-carrying rat embryos, and stereotaxically implanted into the striatum of normal adult rats. 4-6 weeks after the implantation, grafted neuroepithelial stem cells differentiated mostly into neurons together with some astrocytes. Some donor-derived neurons stained positively for tyrosine hydroxylase (TH), but no donor-derived neurons stained positively for dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32). However, 6 months after the grafting, DARPP-32-positive neurons could be observed. Action potentials and both glutamatergic and GABAergic spontaneous postsynaptic currents were recorded with whole-cell recording from donor-derived neurons in the striatal slices 4-6 weeks after the implantation. Furthermore, both glutamatergic and GABAergic postsynaptic currents were evoked by the electrical stimulation applied to the host area. These results suggest that grafted neuroepithelial stem cells differentiate to functionally active neurons in the host brain tissues and make synaptic contacts to the environment, indicating their feasibility in the functional repair of damaged neural circuitry. [Jpn J Physiol 54 Suppl:S155 (2004)]

View full abstract

Two tetrapeptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), have been identified as endogenous ligands for the μ-opioid receptors. In order to clarify details of the actions of EM-1 and EM-2 on nociceptive transmission, we examined their effects on synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole-cell patch-clamp technique. Each of EM-1 and EM-2 produced outward currents at -70 mV in a dose-dependent manner (EC₅₀ = 0.13 μM and 0.15 μM, respectively). Currents induced by them reversed their polarity at potentials which were close to the equilibrium potential for K⁺, and were reduced in amplitude by a μ-opioid receptor antagonist, CTAP (1 μM). Spontaneous glutamatergic excitatory postsynaptic currents decreased in frequency by EM-1 and EM-2 (1 μM, each) to about 50％ of control with no notable change in the amplitude in almost all neurons tested. Glycinergic and GABAergic focally-evoked inhibitory postsynaptic currents were reduced in amplitude by EM-1 and EM-2 (1 μM, each) to about 50% of control. It is concluded in SG neurons that endomorphins hyperpolize membranes by opening K⁺ channels, and depress excitatory and inhibitory transmission through the activation of μ-opioid receptors; these actions would contribute to at least a part of the modulation of nociceptive transmission in the spinal dorsal horn. [Jpn J Physiol 54 Suppl:S156 (2004)]

View full abstract

It is well known that referred pain is provoked by noxious stimulation of skeletal muscle in human. In this study, we examined the plastic changes of rat dorsal horn neurons after myositis induced by carrageenan. The experiments were performed on 25 SD rats with urethane anesthesia. The spinal cord was exposed by laminectomy and the activity of single neuron at the L3-L5 level was extracellularly recorded. The neurons were searched by applying mechanical stimulation. The type of neurons and the area of receptive fields (RFs) were determined, 2% carageenan was injected into the skeletal muscle which was separated from the original RF by a major joint. Five low-threshold mechanosensitive (LTM), 15 wide dynamic range (WDR) and five noxious specific neuron (NS) were examined. All neurons had RFs both skin and deep tissues. After injection of carageenan into the muscle, none of the neurons changed the responsiveness and character in original RF. The new RFs appeared or expanded in five out of 15 WDR and two out of 5 NS neurons. In one WDR neuron, the RF area reduced slightly, and no change of RF in the LTM neuron. The expansion of RFs was continued during experiment and reversed after lidocaine injection into the myositic muscle in 2 (one WDR and 1 NS) out of three neurons tested. These results suggest that the myositis produced by intramuscular injection of carageenan might be activated the silent synaptic connection at the spinal dorsal horn. [Jpn J Physiol 54 Suppl:S156 (2004)]

View full abstract

We have previously reported an inhibition by cannabinoids of excitatory transmission in substantia gelatinosa (SG) neurons. In order to know further a role of cannabinoids in regulating nociceptive transmission, their actions on inhibitory transmission in SG neurons were examined using whole-cell voltage-clamp recordings. In 18 of 23 neurons examined, superfusing an endocannabinoid, anandamide (10μM), for 2 min resulted in reducing the amplitude of glycinergic focally-evoked IPSCs (eIPSCs); this reduction persisted over at least 6 min with an extent of 27± 3% (2 min after its washout). A similar reduction was also seen for GABAergic eIPSC amplitudes in 17 of 24 neurons tested; this extent was 42± 4%. These inhibitons were reversed by superfusing a cannabinoid-receptor antagonist, SR141716A (5μM), following anandamide washout. A ratio of the second to first glycinergic or GABAergic eIPSC amplitude in paired-pulse experiments was increased by anandamide. Glycinergic and GABAergic spontaneous IPSCs were also reduced in frequency by anandamide (10μM) by 46± 5 and 35± 7% (each n = 5), respectively, without a change in the amplitudes. Another endocannabinoid, 2-arachdonoyl glycerol (20μM), and a synthetic cannabinoid-receptor agonist, WIN55212-2 (5μM), also reduced glycinergic and GABAergic IPSC amplitudes. It is concluded that cannabinoids presynaptically inhibit both glycinergic and GABAergic transmission to SG neurons; this would contribute to a modulation of nociceptive transmission. [Jpn J Physiol 54 Suppl:S156 (2004)]

View full abstract

A current response induced by adenosine was examined in substantia gelatinosa (SG) neurons by using the whole-cell patch-clamp technique. In 78% of neurons examined, superfusing adenosine induced an outward current at -70 mV in a dose-dependent manner (EC₅₀ = 177 μM). The adenosine current reversed its polarity at a potential which was close to the equilibrium potential for K⁺ and exhibited a linear current-voltage relationship in a range of -150 to -40 mV. The adenosine current was suppressed by Ba²⁺ (100 μM) and 4-AP (5 mM) but not by TEA (5 mM). The adenosine current was mimicked by an A₁ agonist, CPA (1 μM), and was completely blocked by an A₁ antagonist, DPCPX (1 μM). The adenosine current was enhanced in duration by an equilibrative nucleoside-transport (rENT1) inhibitor, S-(4-nitrobenzyl)-6-thioinosine, in a dose-dependent manner in a range of 0.2-5 μM (by 29% at 1 μM) and also by an adenosine-deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (1 μM), by 23%, and slowed in falling phase by an adenosine-kinase inhibitor, iodotubercidine (1 μM). It is concluded in SG neurons that a Ba²⁺- and 4-AP-sensitive K⁺ channel exhibiting no rectification is opened through the activation of A₁ receptors by adenosine whose level near the receptors is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. This hyperpolarizing action of adenosine might contribute to its antinociceptive effect. [Jpn J Physiol 54 Suppl:S156 (2004)]

View full abstract

We examined the possible role of lactate as an energy substrate by using ³¹P-NMR in rat brain slices from pre-stressed rats. Preconditioned brain slices were obtained from rats, the brain of which had been made ischemic by occluding contralateral middle cerebral artery, from heat stroked rats (42-43°C,15 min.) or from rats pretreated with a low dose of 3-nitropropionic acid (3-NP) 48 hours before the preparation, respectively. Stimulation of the metabolism in brain slices was induced by changing the medium to a high-K solution (60 mM K⁺). Both glucose and lactate were able to support the recovery of high-energy phosphates (PCr etc.) levels in brain slices from control rats (7-10 weeks) following the high-K⁺ stimulation at 25°C. After brain slices were treated with fluorocitrate (FC), a glial toxin, glucose was able, while lactate was unable, to support the recovery of PCr following the high-K⁺ stimulation. In FC-treated brain slices obtained from pre-treated rats (ischemic, heat-stressed or pretreated with 3-NP), however, lactate supported the recovery of PCr substantially following the high-K⁺ stimulation. These results suggest that neurons themselves are unable to take up and metabolize lactate, but after pre-conditioning neurons acquire the ability to take up and utilize lactate as an energy substrate. Even if the way of preconditioning is different, the same process may be involved in inducing similar changes in energy metabolism of the neuron. [Jpn J Physiol 54 Suppl:S157 (2004)]

View full abstract

Neural stem (progenitor) cells (NSCs) differentiate to neurons, astrocytes and oligodendrocytes. From the viewpoint of differentiation to neurons, the regulation of cell cycle in NSCs seems to be important. However, how cell cycle relates to neuronal differentiation is poorly understood. We investigated whether NSCs derived from E12.5 rat ventral mesencephalon can efficiently differentiate into neurons or not under the control of cell cycle. We first checked the toxicity (optimal concentration and duration of treatment) of cell cycle blockers (agent that blocked before G1/S phase, S and G2/M). FACScan analysis showed that G1 phase blockers such as mimosine (100 μM, 8 h), deferoxamine (500 μM, 8 h) and aphidicolin (1.5 μM, 8 h) blocked NSCs proliferation without cell toxicity. BrdU analysis showed that BrdU-positive cells (proliferationg NSCs) were significantly decreased compared with non treated ones. When NSCs were blocked their cell cycle in the G1 phase and were differentiated in DMEM/F12+1% FCS for 3days, the numbers of β-tubulin III-positive cells were increased to approximately twice compared with non-treated ones. By the treatment the mRNA of MAP-2 was increased while that of S-100 was not changed. Western immunoblot analysis showed the increase of MAP-2 protein and decrease of GFAP protein. No programmed cell death was detected by TUNEL reaction. Data suggest that, by the treatment with the G1 phase blockers, NSCs were promoted to differentiate to neural phenotype without apoptosis. [Jpn J Physiol 54 Suppl:S157 (2004)]

View full abstract