Tetsuya Shirasaki

Serotonin suppressesN-methyl-d-aspartate responses in acutely isolated spinal dorsal horn neurons of the rat

In acutely isolated spinal dorsal horn neurons of the rat, effects of serotonin (5-hydroxytryptamine, 5-HT) on inward current induced by excitatory amino acids were studied under whole-cell voltage-clamp condition. 5-HT suppressed the response to N-methyl-D-aspartate (NMDA), but not the response to kainate or quisqualate. This inhibitory effect of 5-HT on NMDA response was present at 5-HT concentrations as low as 10(-15) M. Although the 5-HT effect exhibited similar pharmacology to the 5-HT1A-type receptors, it was not mimicked by increasing intracellular concentration of adenosine 3,5-cyclic monophosphate that is the common second messenger for 5-HT1A receptors in the mammalian central nervous system. Glycine strongly antagonized this inhibitory effect of 5-HT, and 5-HT reduced opening of NMDA-gated single channels recorded from the outside-out membrane patch. These lines of evidence are consistent with a possibility that 5-HT might directly modulate the NMDA receptor-ion channel complex, either by interacting with the regulatory site(s) or by acting on a distinct site.

Differential blockade of bicuculline and strychnine on GABA- and glycine-induced responses in dissociated rat hippocampal pyramidal cells.

The inhibitory effects of bicuculline (BIC) and strychnine (STR) on GABA- and glycine-induced responses were studied in the rat dissociated hippocampal CA1 pyramidal neurons in whole-cell mode by using the conventional patch-clamp technique. Both GABA and glycine elicited inward Cl- currents in a dose-dependent manner and had almost the same maximal responses. The half-maximum dose (Ka) and Hill coefficient were 6.4 microM and 1.1 for the GABA response, and 74 microM and 1.5 for the glycine response. BIC and STR antagonized both GABA and glycine responses in a competitive manner. The blocking potency of BIC and STR on the GABA response was comparable. The half inhibition dose (IC50) was 2.7 microM for BIC and 6.7 microM for STR. STR blocked the glycine response about 3,000 x more effectively than BIC. The IC50 was 28 nM for STR and 100 microM for BIC. The BIC and STR did not have voltage-dependent blocking effects on either GABA or glycine responses. Neither GABA nor glycine showed outward rectification in their current-voltage relationships. The functional role of glycine in the rat hippocampal CA1 region is discussed.

Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat.

1. Effect of intracellular ATP on Cl‐ current (ICl) mediated by the GABA (gamma‐aminobutyric acid) receptor subtype, GABAA, was studied in dissociated nucleus tractus solitarii (NTS) neurones using the whole‐cell mode of patch clamp. A concentration‐jump technique termed ‘Y tube’ was used to rapidly apply agents externally. Dissociated neurones were obtained from 1‐ to 3‐week‐old rats. 2. When the patch‐pipette solution contained 2 mM‐ATP, the amplitude of ICl elicited by 10(‐5) M‐GABA did not show any time‐dependent decrease (apparent run‐down), for more than 60 min after the initial recording. In the presence of ATP, the half‐maximum concentration (KD) and Hill coefficient calculated from the GABA concentration‐response curve were 9.12 microM and 1.47, respectively. 3. In the absence of intracellular ATP, the amplitude of GABA‐induced ICl decreased with time. The relative peak amplitudes after 20 and 60 min from the initial recording were 0.40 +/‐ 0.09 (n = 11) and 0.16 +/‐ 0.05 (n = 8) with respect to the initial response. 4. Removal of Mg2+ from the internal solution induced run‐down of the GABA response even in the presence of 2 mM‐intracellular ATP, suggesting that both intracellular ATP (2 mM or more) and Mg2+ are necessary to prevent run‐down of the GABA response. 5. Activation of dephosphorylation processes by alkaline phosphatase (100‐200 microM) did not affect the GABA response in neurones perfused with internal solution containing 2 mM‐ATP and 3 mM‐Mg2+. Blocking the dephosphorylation process by okadaic acid, a phosphatase inhibitor, did not prevent the run‐down of the GABA response. 6. Calcium influxes passing through both the voltage‐dependent L‐type Ca2+ channel and the glutamate receptor‐operated cation channel did not affect ICl induced by GABA. 7. GABA‐induced ICl was also maintained by adding 2 mM‐ADP or ATP gamma S (adenosine‐5‐O‐3‐thiotriphosphate) to the internal solution containing Mg2+. Addition of 2 mM‐adenosine, AMP, cyclic AMP, AMP‐PNP (adenylimido‐diphosphate) or ADP beta S (adenosine‐5‐O‐2‐thiodiphosphate) to the internal solution did not prevent the run‐down of the GABA response even in the presence of 3 mM‐intracellular Mg2+. Based on the chemical specificity of these ATP analogues, it is suggested that there is an ATP‐sensitive binding site (ATP receptor) in the cytoplasmic side of the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabotropic glutamate response in acutely dissociated hippocampal CA1 pyramidal neurones of the rat.

1. The metabotropic glutamate (mGlu) response was investigated in dissociated rat hippocampal CA1 pyramidal neurones using conventional and nystatin‐perforated whole‐cell modes of the patch recording configuration. 2. In the perforated patch recording configuration, the application of glutamate (Glu), quisqualate (QA), aspartate (Asp) and N‐methyl‐D‐aspartate (NMDA) induced a slow outward current superimposed on a fast ionotropic inward current, whereas alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) and kainate (KA) induced only an ionotropic inward current at a holding potential (VH) of ‐20 mV. A specific agonist of the mGlu receptor (mGluR), trans‐1‐aminocyclopentane‐1,3‐dicarboxylate (tACPD), induced an outward current in approximately 80% of the neurones tested. Asp‐ and NMDA‐induced outward currents were antagonized by D‐2‐amino‐5‐phosphonopentanoate (D‐AP5) whereas Glu‐, QA‐ and tACPD‐induced outward currents were not antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) and D‐AP5, indicating that the mGlu response is an outward current component. 3. L‐2‐Amino‐3‐phosphonopropionate (L‐AP3) and DL‐2‐amino‐4‐phosphonobutyrate (AP4) did not block the mGlu response. 4. The relative potencies of mGlu agonists were QA > Glu > tACPD. The threshold and EC50 values of metabotropic outward currents were 10‐100 times lower than those of the ionotropic inward current (iGlu response). 5. The reversal potential of the mGlu response (EmGlu) was close to EK (K+ equilibrium potential), and it shifted 59.5 mV for a tenfold change in extracellular K+ concentration. 6. In Ca(2+)‐free external solution, the mGlu response was elicited by an initial application of Glu, but subsequent applications failed to induce the response. There was also an increase in the intracellular free Ca2+ concentration ([Ca2+]i) during the application of Glu and QA but not of AMPA, indicating Ca2+ release from an intracellular Ca2+ store. 7. During the activation of a Ca(2+)‐dependent K+ current (IK(Ca)) by inositol trisphosphate (IP3) in the internal solution, the mGlu response was suppressed. Addition of GDP‐beta‐S, neomycin or heparin to the internal solution also suppressed the mGlu response, but staurosporine had no effect. The mGlu response was abolished by pretreatment with either caffeine or ryanodine, but treatment with pertussis toxin (IAP) for 6‐8 h had no effect. 8. The mGlu response was suppressed by tetraethylammonium, but not by either apamin or iberiotoxin, suggesting that intermediate‐conductance Ca(2+)‐dependent K+ (KCa+) channels are involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat

The excitatory amino-acid-induced currents in nucleus tractus solitarii neurons freshly isolated from rats were investigated in a whole-cell recording mode using a conventional patch-clamp technique. At a holding potential of -70 mV, L-glutamate (Glu), N-methyl-D-aspartate (NMDA) with 10(-9) M glycine, kainate (KA), quisqualate (QA) and L-aspartate (Asp) evoked inward currents. The currents increased in a sigmoidal fashion with increasing agonists concentration. The half-maximum concentration (EC50) values were 5 x 10(-5) M for Glu, 10(-6) M for QA, 10(-4) M for KA, 6 x 10(-5) M for NMDA and 5 x 10(-5) M for Asp. The Hill coefficients of the Glu-, QA-, KA-, NMDA- and Asp-induced responses were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. The Glu-, QA-, NMDA- and Asp-induced currents consisted of a transient initial peak and a successive steady-state component showing no desensitization. These currents had the same reversal potential near +5 mV. In the current-voltage (I-V) relationships for the Glu-, NMDA- and Asp-induced currents, slight outward rectifications were observed in Mg2(+)-free external solution at membrane potentials negative to 0 mV. In the presence of extracellular Mg2+, the currents induced by Glu, NMDA and Asp were suppressed at negative membrane potentials, but the suppression was less for the Glu response. The I-V relationships for QA- and KA-induced responses were almost linear at a membrane potential between -90 and +50 mV with or without the presence of Mg2+.

Differential properties of type I and type II benzodiazepine receptors in mammalian CNS neurones

1 The effects of benzodiazepine receptor (BZR) partial agonists, Y‐23684 and CL218,872, were compared with its full agonist, diazepam, on γ‐aminobutyric acid (GABA)‐induced Cl− current (ICl) in acutely dissociated rat cerebral cortex (CTX), cerebellar Purkinje (CPJ) and spinal ventral horn (SVH) neurones, by the whole‐cell mode patch‐clamp technique. 2 The GABA‐induced responses were essentially the same in both SVH and CPJ neurones, but the KD value of the GABA response in CTX neurone was lower than those in the other two brain regions. 3 Enhancement of the GABA response by the two partial agonists was about one‐third of that by diazepam in the SVH neurones (where type II subtype of BZR, BZ2, is predominant), whereas these partial agonists potentiated the GABA response as much as diazepam in CPJ neurones (where the type I subtype of BZR, BZ1, is predominant). In CTX neurones where both type I and II variants are expressed, the augmentation ratio of the GABA response by diazepam was between the values in CPJ and SVH neurones. 4 In concentration‐response relationships of BZR partial agonists, the threshold concentrations, KD values and maximal augmentation ratio of the GABA response were similar in all CTX, CPJ and SVH neurones. Also, in all preparations, the threshold concentration and KD values of diazepam action were 10 fold less than those induced by partial agonists. 5 All BZR agonists shifted the concentration‐response relationship for GABA to the left without changing the maximum current amplitude, indicating that activation of both BZ1 and BZ2 increase the affinity of the GABAA receptor for GABA. 6 The results are important in clarifying the mechanism of anxiety and might explain the anxioselectivity of BZR partial agonists.

Glycine-insensitive desensitization of N-methyl-d-aspartate receptors in acutely isolated mammalian central neurons

Acutely isolated rat central neurons were recorded by whole-cell voltage-clamp and responses to a class of excitatory amino acid N-methyl-D-aspartate (NMDA) were examined. Rapid application of NMDA evoked inward current consisted of a fast initial peak followed by a sustained component. Glycine potentiated both initial and desensitized states of the NMDA response with identical concentration-dependence. The initial response, but not the sustained component, was abolished when low concentration of NMDA was pre-applied, and glycine could not reverse the desensitization. This evidence suggests that the NMDA receptor desensitization is sensitive to NMDA but not to glycine, and support the hypothesis that glycine initiates the activation of NMDA receptors rather than that glycine prevents desensitization at NMDA receptors in these cells.

Non-competitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones

1 The interaction of a new class of quinolone antimicrobials (new quinolones) and non‐steroidal anti‐inflammatory agents (NSAIDs) with the GABAA receptor‐Cl− channel complex was investigated in frog sensory neurones by use of the internal perfusion and ‘concentration clamp’ techniques. 2 The new quinolones and the NSAIDs (both 10−6−10−5 m) had little effect on the GABA‐induced chloride current (ICl) when applied separately. At a concentration of 10−4 m the new quinolones, and to a lesser degree the NSAIDs, produced some suppression of the GABA response. 3 The co‐administration of new quinolones and some NSAIDs (10−6−10−14 m) resulted in a marked suppression of the GABA response. The size of this inhibition was dependent on the concentration of either the new quinolone or the NSAID tested. The inhibitory potency of new quinolones in combination with 4‐biphenylacetic acid (BPAA) was in rank order norfloxacin (NFLX) ≫ enoxacin (ENX) > ciprofloxancin (CPFX) ≫ ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA ≫ indomethacin = ketoprofen > naproxen > ibuprofen > pranoprofen. Diclofenac, piroxicam and acetaminophen did not affect GABA responses in the presence of ENX. 4 In the presence of ENX or BPAA, there was a small shift to the right of the concentration‐response curve for GABA without any effect on the maximum response. However, the co‐administration of these drugs suppressed the maximum of the GABA concentration‐response curve, indicating a non‐competitive inhibition, for which no voltage‐dependency was observed. 5 Simultaneous administration of ENX and BPAA also suppressed pentobarbitone (PB)‐gated ICl. On the other hand, both PB and phenobarbitone reversed the inhibition of GABA‐induced ICl by co‐administration of ENX and BPAA. 6 The effect on GABAA responses of co‐administration of new quinolones and NSAIDs was not via an interaction with benzodiazepine receptors coupled to the GABAA receptor, since this effect was not reversed by Ro15–1788 or diazepam. 7 It is concluded that the co‐administration of new quinolones and some of the NSAIDs inhibit GABAergic transmission, and could result in convulsions.

Effects of antagonists on N-methyl-d-aspartate response in acutely isolated nucleus tractus solitarii neurons of the rat

The effect of antagonists on N-methyl-D-aspartate (NMDA)-induced response was investigated in isolated nucleus tractus solitarii (NTS) neurons freshly isolated from the rat using a conventional pathclamp technique. The NMDA-induced inward current consisted of an initial peak followed by a steady-state component. The competitive antagonists of NMDA receptor, D-2-amino-5-phosphonovalerate (APV), D-2-amino-4-phosphonoheptanoate (APH) and 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphate (CPP), selectively suppressed the initial peak of NMDA-induced current more than the steady-state component at low concentrations. The non-competitive antagonists, MK-801, ketamine, Zn2+ and Mg2+, equally blocked both peak and steady-state components.

Interaction of various non-steroidal anti-inflammatories and quinolone antimicrobials on GABA response in rat dissociated hippocampal pyramidal neurons

Interaction of various non-steroidal anti-inflammatory drugs (NSAIDs) and quinolone antimicrobials with gamma-aminobutyric acid (GABA)A receptor-Cl- channel complex was investigated in rat dissociated hippocampal CA1 neurons by using whole-cell mode patch-clamp technique. Neither NSAIDs nor quinolones alone affected the GABA-induced chloride current (ICl). In the presence of norfloxacin (NFLX), one of quinolones, some NSAIDs suppressed the GABA response in a concentration-dependent manner. The inhibitory potency of NSAIDs was 4-biphenylacetic acid (BPA), a metabolite of fenbufen much greater than indomethacin = naproxen greater than mefenamic acid greater than diclofenac greater than piroxicam. The results suggest that fenbufen, indomethacin and naproxen in the presence of quinolones may induce the epileptogenic neurotoxicities via pharmacological interaction with GABAA receptor.