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ABSTRACT: Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA(A) receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na(+) or Ca(2+) channels. Exogenously applied muscimol (0.1-3 microM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10 microM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr, a specific GABA(A) receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA(A) receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.
Neuroscience 02/2006; 138(1):25-35. · 3.38 Impact Factor
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ABSTRACT: Single CNS neurons could be dissociated with adherent functional synaptic boutons without using any enzyme, namely when preparing
a “synaptic bouton.” This allows experimenters to investigate the effects of presynaptic modulators of synaptic transmission
with unprecedented case and accuracy. Moreover, a single bouton can be visualized using fluorescent markers and can also be
focally stimulated with electrical pulses. In this communication, high voltage-dependent Ca2+ channels of nerve endings, as one of experimental examples using the “synaptic bouton” preparation, are described. Ca2+ channels belonging to different subtypes, which trigger GABA release from nerve terminals (boutons) projecting to rat hippocampal
CA1 pyramidal neurons, were studied. GABA-ergic evoked inhibitory postsynaptic currents (eIPSCs) were recorded; these currents
were evoked by focal stimulation of single boutons in mechanically dissociated neurons and by stimulation of a nerve bundle
in slice preparations. Nilvadipine, an L-type Ca2+ channel blocker, completely inhibited eIPSCs evoked by stimulation of single boutons but exerted no effect on eIPSCs evoked
by low-frequency stimulation of the nerve bundle. Nilvadipine did, however, prevent potentiation of the eIPSC amplitude following
high-frequency stimulation of the nerve bundles in slice preparations. ω-Conotoxin-GVIA, an N-type Ca2+ channel blocker, and ω-Agatoxin-IVA, a P/Q-type Ca2+ channel blocker, completely inhibited the eIPSCs in 33.3 and 83.3% of the recordings from single boutons, respectively. In
response to low-frequency nerve bundle stimulation in the slice preparation, both ω-Conotoxin-GVIA and ω-Agatoxin-IVA partially
reduced the amplitude of eIPSC, and the residual component could be abolished by Cd2+. From these results, the following hypotheses could be drawn. (i) The distribution of P/Q- and N-type Ca2+ channels at a single bouton is nonuniform; (ii) when a focal stimulation is applied to a single bouton, L-type Ca2+ channels play a significant role in generation of action potentials, which subsequently activate P/Q- and N-type Ca2+ channels at GABA release sites; and (iii) action potentials conducted through axons in the slice preparation are sufficient
to depolarize the bouton membrane, even when L-type Ca2+ channels are suppressed.
Neurophysiology 02/2005; 37(2):163-165. · 0.47 Impact Factor
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ABSTRACT: Disynaptic GABAergic inputs from Schaffer collateral (SC) afferents on to the soma of glutamatergic CA1 pyramidal neurons are involved in feed-forward inhibition in the hippocampal neural circuits. Here we report the functional roles of presynaptic GABA(A) receptors on SC afferents projecting to CA1 pyramidal neurons. Muscimol (0.5 microM), a selective GABA(A) receptor agonist, increased SC-evoked EPSC amplitude and decreased paired-pulse ratio in the slice preparation, in addition, it facilitated spontaneous glutamate release on to mechanically dissociated CA1 pyramidal neurons in an external Ca2+-dependent manner. In field recordings, muscimol at low concentrations (< or = 0.5 microM) increased not only the excitability of SC afferents but glutamate release, however, it at high concentrations (> or = 1 microM) changed bidirectionally. These results suggest that the moderate activation of presynaptic GABA(A) receptors depolarizes SC afferents and enhances SC-mediated glutamatergic transmission. When endogenous GABA was disynaptically released by brief trains of stimulation of SC afferents, the axonal excitability in addition to glutamate release was increased. The effects of endogenous GABA on the excitability of SC afferents were blocked by either SR95531 or AMPA receptor blockers, which would be expected to block disynaptic feed-forward neural circuits. The present results provide a novel form of presynaptic modulation (feed-forward facilitation) of glutamatergic transmission by presynaptic GABA(A) receptors within the intrinsic hippocampal neural circuits.
Neuroscience 02/2005; 135(3):737-48. · 3.38 Impact Factor
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ABSTRACT: We investigated P2X purinoceptors in rat dorsomedial hypothalamic (DMH) neurons using nystatin-perforated patch-clamp recordings and fura-2 microfluorometry. Adenosine triphosphate (ATP) concentration-dependently evoked an inward current and increased cytosolic Ca(2+) ([Ca](i)). The rise in [Ca](i) was dependent on external Ca(2+) and Na(+), was blocked by Ca(2+) channel antagonists and had pharmacological properties consistent with P2X2 receptors. These results suggest that P2X receptor-mediated depolarization activates voltage-gated Ca(2+) channels, resulting in an increase in [Ca](i).
Brain Research 06/2004; 1009(1-2):234-7. · 2.73 Impact Factor
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ABSTRACT: 5-HT is known to modify the excitability of GABAergic interneurons projecting to hippocampal CA1 neurons. In this study we investigate the presence and functionally characterize the 5-HT receptor subtypes found on the presynaptic nerve terminals of these GABAergic neurons. Using conventional whole-cell patch recording, we confirmed that the 5-HT(1A) agonist, 8-hydroxy-2-dipropylaminotetralin, presynaptically decreased electrically evoked GABA release while the 5-HT(3) agonist, m-chlorophenylbiguanide (mCPBG), presynaptically facilitated release. Using the 'synaptic bouton preparation', where CA1 neurons are acutely isolated with functional nerve terminals/boutons remaining adherent, we next showed that these receptor subtypes are found presynaptically. We next used the technique of focal stimulation of a single bouton in this preparation to further investigate the distribution of these 5-HT receptor subtypes. We found that all boutons contained inhibitory 5-HT(1A) receptors while a subset of boutons showed both 5-HT(1A) and excitatory 5-HT(3) receptors. No boutons were detected which contained only 5-HT(3) receptors. Our studies show that presynaptic 5-HT receptor subtypes are found presynaptically and are not uniformly distributed. This provides another potential mechanism whereby 5-HT can modulate GABA release and hence the excitability of hippocampal neurons.
Neuropharmacology 07/2003; 44(8):1022-30. · 4.81 Impact Factor
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ABSTRACT: The modulation of spontaneous miniature GABAergic inhibitory postsynaptic currents (mIPSC) by the metabotropic glutamate receptors was investigated in the mechanically dissociated rat nucleus basalis of Meynert neurons using the conventional whole-cell patch recording configuration. An application of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) reversibly reduced the frequency of mIPSC without affecting the current amplitude distribution. The application of K+ channel blockers such as 4-aminopyridine, Cs+, Ba2+ or tetraethylammonium increased the mIPSC frequency, but failed to inhibit the tACPD action on mIPSC. Although the removal of Ca2+ from the extracellular solution reduced the mIPSC frequency, the inhibitory effect of tACPD on mIPSC was unaltered. These results suggested that neither voltage-dependent K+ or Ca2+ channels are involved in the inhibitory effect of tACPD on mIPSC frequency. Forskolin, an activator of adenylate cyclase, facilitated the mIPSC frequency in a concentration-dependent manner and inhibited the tACPD-induced suppression of mIPSC frequency. 8-Br-cAMP, a membrane permeable analog of cAMP, also prevented the inhibitory action of tACPD. However, Sp-cAMP, an activator of protein kinase A, could not prevent the inhibitory action of tACPD. L-CCG-I and (2R,4R)-APDC, group II mGluR agonists, mimicked the tACPD action on mIPSC frequency, but L-AP4, a group III mGluR agonist, had no such effect. MCCG, a group II mGluR antagonist, fully blocked the tACPD action. It was concluded that the activation of group II mGluR on the GABAergic presynaptic nerve terminals projecting to the rat nucleus basalis of Meynert neurons therefore inhibits the GABA release by reducing the activity of the cAMP-dependent pathway.
Neuroscience 02/2002; 109(2):299-311. · 3.38 Impact Factor
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ABSTRACT: The effects of a volatile anesthetic, isoflurane, on glycinergic miniature inhibitory postsynaptic currents (IPSCs) were investigated in mechanically dissociated rat trigeminal nucleus neurons with intact glycinergic interneuronal presynaptic nerve terminals. The nystatin-perforated patch recording configuration was used to record the miniature IPSCs under voltage-clamp conditions. Isoflurane shifted in a parallel fashion the glycine (Gly) concentration-response curve of enzymatically dissociated neurons to the left without changing the maximum response. Isoflurane reversibly increased the frequency of the miniature IPSCs and prolonged the decay time constant without affecting the mean amplitude. The increase in the frequency of miniature IPSCs in the presence of isoflurane was also observed in Ca(2+)-free external solution. Thapsigargin prohibited the facilitatory effect of isoflurane on the miniature IPSC frequency. It is concluded that isoflurane increases the Ca(2+) concentration in the glycinergic presynaptic nerve terminal by enhancing the release and/or suppressing the uptake of Ca(2+) into stores.
European Journal of Pharmacology 12/2001; 431(3):269-76. · 2.52 Impact Factor
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ABSTRACT: 1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.
The Journal of Physiology 11/2001; 536(Pt 2):505-19. · 4.72 Impact Factor
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ABSTRACT: Opioids have been thought to induce analgesia by activating the descending pain control system, especially at the level of periaqueductal gray, and regulate the neurotransmitter release through the inhibition of calcium channel. In the present study, the modulatory effects of protein kinase C and protein kinase A on the mu-opioid agonist-induced inhibition of the high-voltage activated calcium current were examined in the acutely dissociated rat periaqueductal gray neurons with the nystatin-perforated patch-clamp technique. Among 505 neurons tested, the barium current passing through the high-voltage activated calcium channels of 172 neurons (34%) were inhibited by 32+/-3% with the application of an mu-opioid agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM). The barium currents itself and the DAMGO-induced inhibitory effects were not affected by the application of either an adenylate cyclase activator (forskolin, 1 microM) or a protein kinase inhibitor (staurosporin, 10 nM) for 2 min. The DAMGO inhibition was completely and irreversibly antagonized by the application of a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 1 microM) for 2 min without any alteration of the barium current itself. However, the antagonizing effect of PMA was completely abolished by the application of 10 nM staurosporin for 2 min. After then, PMA did not show the antagonizing effect any more. Inversely, when staurosporin was applied before PMA, the antagonizing effect of PMA was also not shown. These results demonstrate that the mu-opioid agonist-induced inhibition of the periaqueductal gray neuronal high-voltage activated calcium current can be antagonized by protein kinase C activation. This finding may provide us a significant clue to understand the action mechanism of opioid-induced analgesia in the periaqueductal gray.
Brain Research 11/2001; 916(1-2):61-9. · 2.73 Impact Factor
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ABSTRACT: The periaqueductal gray (PAG) plays a critical role in descending antinociception. In mechanically dissociated rat PAG neurons, pharmacologically separated spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the nystatin-perforated patch technique. Both DAMGO, a specific mu-opioid receptor agonist, and serotonin inhibited mIPSC frequency in a dose-dependent manner without affecting mIPSC amplitude, respectively, in the same PAG neurons. The presynaptic opioid effect was blocked by a specific mu-opioid receptor antagonist, CTOP. The presynaptic serotonergic effect was mimicked by a specific 5-HT(1A) receptor agonist, 8-OH-DPAT, and blocked by the specific antagonist, NAN-190. These opioidergic and serotonergic inhibitions of GABA release employed the similar intracellular mechanism of opening 4-AP-sensitive K(+) channels via GTP-binding proteins (G-proteins). Subthreshold concentrations of DAMGO (3 nM) significantly decreased mIPSC frequency with subthreshold concentrations of serotonin (3 nM) and this effect was completely blocked by pretreatment with N-ethylmaleimide (NEM), a PTX-sensitive G-protein inhibitor. In contrast, maximum doses of DAMGO (10 microM) did not further inhibit mIPSC frequency with maximum doses of serotonin (10 microM). In conclusion, activation of presynaptic mu-opioid and 5-HT(1A) receptors synergistically inhibited GABA release. These results suggest a cellular mechanism within PAG for the analgesic effectiveness of combined therapies using opioids in conjunction with classes of anti-depressants which increase synaptic serotonin levels.
Neuropharmacology 11/2001; 41(5):529-38. · 4.81 Impact Factor
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ABSTRACT: Tri-n-butyltin (TBT), an environmental pollutant, is accumulated in edible mollusks and fishes. It has also become a health concern in today's society. In the present study, to elucidate the possible neurotoxic action of TBT, the effect on spontaneous gamma-aminobutyric acid (GABA) release from GABAergic nerve terminals projecting to rat ventromedial hypothalamic neurons was examined using "synaptic bouton" preparation with a nystatin perforated patch recording mode under voltage-clamp conditions. The threshold concentration of TBT to affect the synaptic transmission was 10 to 30 nM. TBT at 30 nM or higher concentrations increased the frequency of GABAergic miniature inhibitory postsynaptic currents in a dose-dependent manner, whereas the current amplitude and current kinetics were not affected. The removal of either external Ca2+ or application of Cd2+ attenuated the TBT-induced facilitation of neurotransmission. TBT at 1 microM induced an inward current in more than one-half of the cells. This current persisted even after TBT was washed out. The present results indicate that TBT at environmentally relevant concentrations (30-100 nM) facilitates the GABAergic neurotransmission in the mammalian brain and the external Ca2+ is needed in this facilitation. Because the concentration of TBT accumulated in some mollusks and fishes has been reported to reach levels of 100 nM or more, such accumulation of TBT in some mollusks and fishes is thus suggested to be hazardous to the health of humans.
Journal of Pharmacology and Experimental Therapeutics 11/2001; 299(1):171-7. · 3.83 Impact Factor
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ABSTRACT: 1. Nicotinic effects on glycine release were investigated in slices of lumbar spinal cord using conventional whole-cell recordings. In most of the substantia gelatinosa (SG) neurons tested, nicotine increased the frequency of the glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs). In a smaller proportion, nicotine evoked not only this same presynaptic response but also a postsynaptic response. 2. Nicotinic facilitation of glycinergic mIPSCs was investigated in mechanically dissociated SG neurons using nystatin-perforated patch recordings. Nicotine (3 x 10(-6) to 10(-5) M) reversibly enhanced the frequency of glycinergic mIPSCs without altering their amplitudes, thus indicating that nicotine facilitates glycine release through a presynaptic mechanism. 3. Choline, a selective alpha7 subunit of nicotinic acetylcholine receptor (nAChR) agonist, had no effect on the mIPSC frequency while anatoxin A, a broad-spectrum agonist of nAChR, facilitated the mIPSC frequency. 4. alpha-Bungarotoxin, a selective alpha7 subunit antagonist, failed to block the nicotinic facilitatory action. Mecamylamine, a broad-spectrum nicotinic antagonist, reversibly inhibited nicotinic action. Dihydro-beta-erythroidine, a selective antagonist of nAChRs containing alpha4-beta2 subunits, completely blocked nicotinic action. 5. Ca(2+)-free but not Cd(2+)-containing bath solutions blocked nicotinic actions. 6. We therefore conclude that nicotine facilitates glycine release in the substantia gelatinosa of the spinal dorsal horn via specific nAChRs containing alpha4-beta2 subunits. This action on a subset of presynaptic nAChRs may underlie nicotine's modulation of noxious signal transmission and provide a cellular mechanism for the analgesic function of nicotine.
The Journal of Physiology 11/2001; 536(Pt 1):101-10. · 4.72 Impact Factor
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ABSTRACT: GABA(A) receptor-mediated responses manifest as either hyperpolarization or depolarization according to the intracellular Cl(-) concentration ([Cl(-)](i)). Here, we report a novel functional interaction between the Na-K-Cl cotransporter (NKCC) and GABA(A) receptor actions on glutamatergic presynaptic nerve terminals projecting to ventromedial hypothalamic (VMH) neurons. The activation of presynaptic GABA(A) receptors depolarizes the presynaptic nerve terminals and facilitates spontaneous glutamate release by activating TTX-sensitive Na(+) channels and high-threshold Ca(2+) channels. This depolarizing action of GABA was caused by an outwardly directed Cl(-) driving force for GABA(A) receptors; that is, the [Cl(-)](i) of glutamatergic nerve terminals was higher than that predicted for a passive distribution. The higher [Cl(-)](i) was generated by bumetanide-sensitive NKCCs and was responsible for the GABA-induced presynaptic depolarization. Thus, GABA(A) receptor-mediated modulation of spontaneous glutamatergic transmission may contribute to the development and regulation of VMH function as well as to the excitability of VMH neurons themselves.
Journal of Neuroscience 09/2001; 21(16):5962-72. · 7.11 Impact Factor
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ABSTRACT: The modulatory effects of Zn(2+) and other divalent cations on the ATP-induced responses of preganglionic neurons acutely dissociated from the rat dorsal motor nucleus of the vagus (DMV) were examined using a nystatin-perforated patch technique under voltage-clamp. DMV neurons were identified by back-filling of DiI placed on the vagal bundle at the neck. Zn(2+) exerts a concentration-dependent effect on P2X receptor-mediated current (I(ATP)): a potentiation by low concentrations of Zn(2+) (< or = 50 microM) and an inhibition by high concentrations (> 50 microM). Inhibition of the ATP response was associated with a prolongation of the rising phase of I(ATP). Cu(2+) mimicked Zn(2+) regarding the biphasic modulation of I(ATP). On the other hand, Ni(2+) potentiated, but failed to inhibit, the ATP response even at a concentration of 3 mM. Quantitative RT-PCR revealed the similarity of P2X(2) mRNA expression between the DMV and superior cervical ganglion (SCG) but not in the dorsal root ganglion (DRG) and hypoglossal nucleus (XII). The results from the electrophysiological and molecular approaches suggest that functional P2X receptors expressed in DMV neurons are characterized mainly by the P2X(2) and P2X(2/6) subtype. DMV neurons possess similar P2X receptor characteristics to SCG neurons.
Journal of Neurochemistry 09/2001; 78(5):1009-18. · 4.06 Impact Factor
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ABSTRACT: 1. The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus in the homeostatic regulation of neuroendocrine and behavioural functions. In mechanically dissociated rat VMH neurones with attached native presynaptic nerve endings, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded using the nystatin perforated patch recording mode under voltage-clamp conditions. 2. Histamine reversibly inhibited the sIPSC frequency in a concentration-dependent manner without affecting the mean current amplitude. The selective histamine receptor type 3 (H(3)) agonist imetit (100 nM) mimicked this effect and it was completely abolished by the selective H(3) receptor antagonists clobenpropit (3 microM) and thioperamide (10 microM). 3. The GTP-binding protein inhibitor N-ethylmaleimide (10 microM) removed the histaminergic inhibition of GABAergic sIPSCs. 4. Elimination of external Ca(2+) reduced the GABAergic sIPSC frequency without affecting the distribution of current amplitudes. In this condition, the inhibitory effect of imetit on the sIPSC frequency completely disappeared, suggesting that the histaminergic inhibition requires extracellular Ca(2+). 5. The P/Q-type Ca(2+) channel blocker omega-agatoxin IVA (300 nM) attenuated the histaminergic inhibition of the GABAergic sIPSC frequency, but neither the N-type Ca(2+) channel blocker omega-conotoxin GVIA (3 microM) nor the L-type Ca(2+) channel blocker nicardipine (3 microM) was effective. 6. Activation of adenylyl cyclase with forskolin (10 microM) had no effect on histaminergic inhibition of the sIPSCs. 7. In conclusion, histamine inhibits spontaneous GABA release from presynaptic nerve terminals projecting to VMH neurones by inhibiting presynaptic P/Q-type Ca(2+) channels via a G-protein coupled to H(3) receptors and this may modulate the excitability of VMH neurones.
The Journal of Physiology 09/2001; 534(Pt 3):791-803. · 4.72 Impact Factor
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ABSTRACT: 1. Spontaneous miniature outward currents (SMOCs) were observed in mechanically dissociated rat Meynert neurons using nystatin perforated patch recordings under voltage-clamp conditions. 2. SMOCs were blocked by apamin, a selective blocker of small conductance Ca(2+)-activated K(+) (SK) channels, but not by blockers for other types of Ca(2+)-activated K(+) channel. 3. Ryanodine (10-100 microM) reduced both the amplitude and frequency of SMOCs. Caffeine (1 mM) increased the SMOC frequency. Blockers of the sarco/endoplasmic reticulum Ca(2+)-ATPase completely abolished SMOCs, indicating a requirement for functioning sarco/endoplasmic reticulum (SR/ER) Ca(2+) stores. 4. Both Cd(2+)-containing and Ca(2+)-free solutions partially inhibited SMOC frequency, a result which suggests that Ca(2+) influx contributes to, but is not essential for, SMOC generation. 5. Thus, SMOCs are SK currents linked to ryanodine- and caffeine-sensitive SR/ER Ca(2+) stores, and are only indirectly influenced by extracellular Ca(2+) influx. The development of this new, minimally invasive mechanical dissociation method has revealed that SMOCs are common in native CNS neurons.
The Journal of Physiology 08/2001; 534(Pt 1):99-107. · 4.72 Impact Factor
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ABSTRACT: Glycine release was facilitated by the activation of presynaptic ATP receptors (P(2X)-type) in a preparation of dissociated trigeminal nucleus pars caudalis neurons in which the native synaptic boutons were preserved. The action of ATP was completely blocked by substance P (SP) without alteration of the miniature IPSC (mIPSC) amplitude distribution. SP itself had no effect on mIPSC frequency or amplitude. The inhibitory effect of SP on ATP action was blocked by CP99994, indicating that the SP receptors are of the neurokinin-1 type. The ATP-induced facilitation of the mIPSC frequency was unaffected by Cd(2+). Moreover, SP did not inhibit the increase in mIPSC frequency induced high K(+) application, suggesting that SP did not modulate voltage-dependent calcium channels or subsequent steps in the release process. KT5720 and phorbol 12-myristate 13-acetate did not block SP action, indicating that neither the cAMP-protein kinase A nor the protein kinase C pathway mediates the SP effects. However, in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), SP was no longer able to inhibit the ATP-induced stimulation of mIPSC frequency. 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine also suppressed the SP action, suggesting that SP modulates P(2X) receptors via a Ca(2+)/calmodulin-dependent protein kinase II-mediated pathway. In conventional whole-cell mode, the presence of W-7 in the patch pipette did not affect the SP inhibitory action. Thus, SP is not likely to be generating its modulation through the production of a retrograde signal (involving calmodulin) from the postsynaptic cell to the presynaptic boutons. These results are the first demonstration of the modulation of one presynaptic receptor by another. Because SP inhibits the ATP stimulation of glycine release, SP may play a significant role in hyperalgesia or chronic pain.
Journal of Neuroscience 06/2001; 21(9):2983-91. · 7.11 Impact Factor
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ABSTRACT: The effects of nitrooxy alkyl apovincaminate VA-045 ((+)-eburunamenine-14-carboxylic acid(2-nitroxy-ethyl ester), VA) were investigated in acutely dissociated rat neocortical neurons by using a nystatin-perforated patch recording configuration. VA activated a steady-state outward current in a concentration-dependent manner, with an EC50 of 0.65 microM. The reversal potential for the current shifted 56.5 mV with tenfold changes in the extracellular K+ concentration, suggesting that the current was carried by K+. The VA-induced current was not suppressed by apamin (1 microM), charybdotoxin (1 microM), Cs+ (3 mM), Ba2+ (3 mM), 4-aminopyridine (10 mM) or glibenclamide (10 microM), whereas tetraethylammonium suppressed the current with an IC50 of 1.4 mM. These pharmacological properties of the VA-induced current were compatible with a slowly inactivating delayed rectifier current (I(K)). It was suggested that the current activated by VA was I(K). The VA-induced current was not affected by Ca2+ depletion or by staurosporine (0.1 microM), quinacrine (10 microM), wortmanin (1 microM) or genistein (1 microM). The intracellular perfusion of GDPbetaS (0.4 mM) also had no significant effect. Thus, VA may directly activate the K+ channels.
The Japanese Journal of Pharmacology 03/2001; 85(2):124-32.
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ABSTRACT: The ATP-induced increase in the cytosolic Ca(2+) concentration ([Ca]i) and current in acutely dissociated ventromedial hypothalamic rats neurons were investigated using fura-2 microfluorometry and the nystatin-perforated patch recording method, respectively. The ATP-induced [Ca]i increase was mimicked by dimethyl-thio-ATP and ATPgammaS, and was inhibited by P2 purinoreceptor antagonists. The ATP-induced [Ca]i increase was markedly reduced by removal of external Na(+) or Ca(2+), and by addition of various Ca(2+) channel antagonists. ATP induced a transient inward current exhibiting a strong inward rectification at membrane potentials more positive than -20 mV. The ATP-induced current at a holding potential of -70 mV was concentration-dependent with a half-maximum effective concentration of 26 microM. Increasing the external Ca(2+) concentration to 10 mM shifted the dose-response relationship to the right. ATP induced only a small current and a small increase in [Ca]i, even at 10 mM Ca(2+), when external Na(+) was removed, suggesting the relatively low permeability to Ca(2+) of purinoceptor channels. These results suggest that ATP activates non-selective cation channels by acting on P2X purinoceptors on dissociated ventromedial hypothalamic neurons, which in turn increases [Ca]i by increasing Ca(2+) influx through voltage-dependent Ca(2+) channels.
Neuroscience 02/2001; 105(2):393-401. · 3.38 Impact Factor
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ABSTRACT: Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, alpha-methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10(-6) M) and L-AP4 (10(-6) M). In K(+)-free or K(+)-free external solution with Ba(2+) and Cs(+), Ca(2+)-free or Cd(2+) external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway.
Journal of Neurophysiology 02/2001; 85(1):332-40. · 3.32 Impact Factor
