Frog Sensory Neurons Research Articles

Amiloride inhibition of gamma-aminobutyric acid(A) receptors depends upon the alpha subunit subtype.

gamma-Aminobutyric acid(A) (GABA(A)) receptors (GABARs) are responsible for most fast inhibitory neurotransmission in the mammalian brain. The GABARs contain several allosteric modulatory sites, many of which are useful clinically. The activity of most of these modulators depends upon the subunit composition of the receptor. The diuretic amiloride was previously reported to inhibit GABARs in frog sensory neurons. We measured its effects on recombinant GABARs to determine its mechanism of action at mammalian receptors and to examine the effect of subunit composition. Amiloride acted primarily as a competitive antagonist, reducing the sensitivity of the receptor to GABA without affecting the maximal current amplitude. Receptors containing an alpha6 subunit were about 10-fold more sensitive to amiloride than those containing other alpha subunits. In contrast, the identity of the beta or gamma subtype had little effect on amiloride sensitivity. Although several other modulators have specific effects at alpha6-containing receptors, amiloride is the first inhibitor to be reported with no additional dependence on the identity of the beta or gamma subunit. Therefore, it probably represents a unique modulatory site on the GABAR, which could be useful for developing drugs targeting these receptors. The selective activity of amiloride could also be helpful for isolating the contribution of receptors composed of alpha6 subtypes in heterogeneous native GABAR populations.

Serotonin‐ and proton‐induced and modified ionic currents in frog sensory neurons

Using the whole-cell patch-clamp technique, the effects of serotonin (5-HT) and increased acidity to produce membrane currents and to modify high threshold voltage-dependent calcium currents were studied in isolated dorsal root ganglion (DRG) cells of the frog maintained in short-term culture. DRG cells were classified by morphology into two types: (1) cells with a large number of dark rusty brown granules, and (2) cells devoid of these granules or with few scattered pale granules. Fast application of 5-HT (10-30 microM) induced a rapidly desensitizing inward current with a reversal potential at about 0 mV in 38 of 50 granule-containing neurons (76%) which was never observed (0/35) in "clear" neurons. This current was blocked by 10 nM (+)-tubocurarine. In addition, a small noninactivating outward current was also observed in most DRG neurons during 5-HT superfusion. A sudden decrease of pH from 7.4 to 6 or 5.8 induced a fast inactivating inward current of 100-300 pA in 74% of the "clear" neurons and only 24% of the granule-containing neurons. Small noninactivating membrane currents induced by lowering pH were observed in all neurons. Both 5-HT and increased extracellular H+ reduced the magnitude of high threshold calcium currents in all DRG neurons. It is suggested that the 5-HT receptors are expressed on a morphologically distinct population of neurons while the cells with channels responsible for the fast inactivating proton-induced current cannot be related to any distinct morphological cell type.

Synthesis and structure-activity analysis of 2-(4-chlorophenyl)-5,6-dihydrothieno[2′,3′:2,3]thiepino[4,5- c]pyridazin-3(2 H)-ones as ligands for benzodiazepine receptors

A series of 2-(4-chlorophenyl)-5,6-dihydrothieno[2′,3′:2,3]thiepino[4,5-c]pyridazin-3(2H)-ones were synthesized and evaluated in vitro for their affinity toward benzodiazepine receptors (BZRs) in rats and for their intrinsic efficacy in the augmentation of the γ-aminobutyric acid (GABA)-induced chloride currents in the dissociated frog sensory neurons. Compounds in which the 9-position of the condensed-ring system was substituted with alkyl group or bromine had a high affinity toward BZRs. The substituents at the same position also influenced significantly the GABA-induced chloride currents. As the result, 9-alkyl and 9-bromo substituents would interact with the lipophilic area of BZRs. A series of 2-(4-chlorophenyl)-5,6-dihydrothieno[2′,3′:2,3]thiepino[4,5-c]pyridazin-3(2H)-ones -ones exhibited partial and full agonistic activities toward BZRs.

Effects of lindane (γ-BHC) and related convulsants on GABA A receptor-operated chloride channels in frog dorsal root ganglion neurons

Effects of lindane (γ-benzenehexachloride; γ-BHC) on GABA-evoked Cl − current ( I GABA) in freshly dissociated frog sensory (dorsal root ganglion) neurons were studied and compared with those of tert-butylbicycloortho benzoate (TBOB) and picrotoxin by the use of the suction-pipette method [13]. Drugs were applied with a rapid drug-application method, “Concentration-clamp” technique. At concentration of GABA of > 3 × 10 −6M, at least two components of the I GABA were recognized distinct degree of desensitization. Those were defined as the peak and plateau components in the text. At low concentration (> 3 × 10 −7M of γ-BHC, only the plateau component of I GABAat 10 −5M were depressed without changing the peak amplitude. While γ-BHC at high concentration (> 3 × 10 −5M) depressed both the peak and plateau current components. The γ-BHC-induced depression of I GABA seemed to be I GABA-component-dependent. A detailed analysis of the γ-BHC action in the concentration-response relationship for GABA revealed that the I GABA with strong desensitization was preferentially blocked by γ-BHC (> 3 × 10 −5M). The rate of recovery of the I GABA from γ-BHC-induced block depended on the concentration of GABA. The lower the concentration of GABA, the slower the recovery. The GABA A receptor Cl − channels were proposed to be classified into two types of the γ-BHC-sensitive and -resistant ones. The interposed application of GABA + γ-BHC during generation of I GABA has shown that γ-BHC as well as TBOB or picrotoxin exerted depression of the I GABA under this condition, suggesting that both compounds can act on the GABA A receptor in the activated state. γ-BHC and TBOB were found to compete with picrotoxin in depression of the I GABA. Presence of a specific site within the GABA A receptor-Cl − channel for binding of γ-BHC, TBOB and picrotoxinin, where the convulsants presumably inhibit the Cl −-binding.

Penicilin-induced triphasic modulation of GABA A receptor-operated chloride current in frog sensory neuron

Effects of penicillin-G(PPCN) on GABA-evoked Cl − current ( I GABA) were investigated in freshly dissociated frog sensory neurons by the use of the concentration-clamp technique combined with the suction-pipette method. Under conditions where the internal and external solutions allowed only Cl − permeability, PCN elicited triphasic modulation on I GABA, consisting of two modes of blockade on I GABA and a following rebound (rebound-like transient I GABA). Simultaneously applied PCN and GABA depressed I GABA immediately (phasic blockade), with the depressed I GABA slightly recovering in amplitude to achieve a stable level of blockade (tonic blockade). When a solution containing a mixture or PCN and GABA was quickly replaced by one containing GABA alone, a rebound-like transient Cl − current ( I GABA) was evoked. Each component of the PCN actions on I GABA was PCN- and GABA-concentration-dependent. The reversal potential for each component of the PCN actions on GABA was close to the chloride equilibrium potential ( E Cl calculated using the Nernst equation. The current-voltage ( I− V) relations for both the phasic and tonic blockade revealed inward rectification, while I− V curves for the control I GABA and the I R were outwardly rectified. The degree of I GABA-desensitization and the amplitude of the I R correlated well. The data suggest that partial removal of the GABA A receptor-desensitization may result in generation of the I R.

Blockade of 5-HT3 receptor-mediated currents in dissociated frog sensory neurones by benzoxazine derivative, Y-25130.

1. The effect of Y-25130, ((+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dih ydr o- 2H-1,4-benzoxazine-8-carboxamide hydrochloride), a high affinity 5-hydroxytryptamine3 (5-HT3) receptor ligand, was examined on the 5-HT-induced response in dissociated frog dorsal root ganglion (DRG) neurones by use of the extremely rapid concentration-jump ('concentration-clamp') and the conventional whole-cell patch-clamp techniques. 2. 5-HT induced a rapid transient inward current associated with an increase in membrane conductance at a holding potential of -70 mV. The current amplitude increased sigmoidally as 5-HT concentration increased. The half-maximum value (Ka) and the Hill coefficient estimated from the concentration-response curve were 1.7 x 10(-5) M and 1.7, respectively. 3. The current-voltage (I-V) relationship of 5-HT-induced current (I5-HT) showed inward rectification at potentials more positive than -40 mV. The reversal potential (E5-HT) was -11 mV. The E5-HT value was unaffected by total replacement of intracellular K+ by Cs+, indicating that the 5-HT-gated channels might be large cation channels. 4. Both the activation and inactivation phases of I5-HT were single exponentials. The time constants of activation and inactivation (tau a and tau i) decreased with increasing 5-HT concentration. 5. The 5-HT response was mimicked by a selective 5-HT3 receptor agonist, 2-methyl-5-HT, but the maximum response induced was approximately 25% that of 5-HT. The 5-HT response was reversibly antagonized by the 5-HT3 receptor antagonists, ICS 205-930, metoclopramide and Y-25130, but not by a 5-HTIA receptor antagonist, spiperone, and a 5-HT2 receptor antagonist, ketanserin. The half-inhibition concentrations (IC50) were 4.9 x 10-10 M for Y-25130, 4.8 x 10-10 M for ICS 205-930 and 8.6 x 10-9 M for metoclopramide.6. Y-25130 (5 x 10-10 M) caused a rightward shift of the concentration-response curve for 5-HT while decreasing the maximum response.7. The results suggest that Y-25130 is a potent antagonist of the 5-HT3 receptor-channel complex.

P-066 - Penicillin-induced modulation of GABAA receptor operated chloride current in frog sensory neurons

P-066 - Penicillin-induced modulation of GABAA receptor operated chloride current in frog sensory neurons

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 (ICI) 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) much greater than enoxacin (ENX) greater than ciprofloxancin (CPFX) much greater than ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA much greater than indomethacin = ketoprofen greater than naproxen greater than ibuprofen greater than 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 Ic, by coadministration 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 Rol5-1788 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.

Studies on the Synthesis of Condensed Pyridazine Derivatives. IV. Synthesis and Anxiolytic Activity of 2-Aryl-5,6-dihydro-(1)benzothiepino(5,4-c)pyridazin-3(2H)-ones and Related Compound.

A series of 2-aryl-5,6-dihydro-(1)benzothiepino[5,4-c]pyridazin-3(2H)- ones and related compounds were synthesized and evaluated for their ability to displace 3H-diazepam from rat brain membranes in vitro, and to prevent bicuculline induced convulsions in mice in vivo. Compounds with a 4'-methoxyphenyl (36) or 4'-chlorophenyl group (37, 39--42) as 2-aryl substituents showed prominent activities in both the in vitro and in vivo tests. Among them, 2-(4'-chlorophenyl)-5,6-dihydro- (37) and 2-(4'-chlorophenyl)-5,6-dihydro-10-fluoro-(1)benzothiepino[5,4-c]+ ++pyridazin- 3(2H)-one 7-oxides (41) showed activity twice as potent as diazepam in an anticonflict test (Vogel type, rats) while exhibiting less muscle relaxation (rotarod test, mice) and augmentation of gamma-aminobutyric acid-induced chloride current (Icl) in isolated frog sensory neurones than diazepam. Compound 37 (Y-23684) was selected from this series as a candidate for further development. The structure-activity relationships are discussed.

Augmentation of GABA-induced current in frog sensory neurons by pentobarbital

The effect of pentobarbital sodium (PB) on the gamma-aminobutyric acid (GABA)-induced macroscopic and microscopic Cl- currents (ICl and iCl, respectively) was studied in the soma membrane of isolated frog sensory neurons using a rapid concentration-jump and patch-clamp technique. The GABA-induced ICl was composed of a transient peak and a steady plateau. PB shifted the concentration-response curve of the GABA-induced peak ICl to the left without affecting the maximum value. The apparent dissociation constant (KD) decreased from 13 microM at control to 8.0, 4.8, and 2.9 microM by adding 10, 30, and 100 microM PB, respectively. PB also shifted the concentration-response curve for the plateau ICl to the left and augmented the maximum value of the plateau, indicating an increase in the available receptor-channel complex. The Hill coefficient (n = 2) in concentration-response curves of both peak and plateau responses was not changed by adding PB. Both the activation and desensitization phases of GABA-induced ICl consisted of two exponential components. PB significantly increased the time constant of slow desensitization component at all concentrations of GABA used. In the "inside-out" configuration, PB markedly increased the open probability (Po) of a GABA-gated single Cl- channel having a conductance of 14.57 +/- 2.3 pS (n = 123) without affecting the single-channel conductance. The increase of Po was due to the prolongation of mean open time (tau of the tau os) and shortening of mean closed time (tau cf and tau cs), resulting in the increase of channel-opening events.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in protein and tubulin mRNA synthesis in frog sensory neurons treated with the adenylate cyclase activator, forskolin

We are interested in the role cyclic AMP may play as a mediator of growth factor-induced gene expression in regenerating peripheral nerves. As a first step, we investigated mRNA levels and protein synthesis in intact frog dorsal root ganglia (DRG) treated with the adenylate cyclase activator, forskolin in vitro. Forskolin (10-7 M) increased intraganglionic cyclic AMP concentration 3-fold within 40 min of application and maintained this concentration through 60 min, when the drug was withdrawn. Addition of forskolin to isolated DRG neurons for 1 h increased the incorporation of [3H]leucine into TCA-insoluble material beginning 12 h after the withdrawal of forskolin. Axonally transported labeled material was increased almost 2-fold by 12 h. The effect of forskolin could be blocked by the simultaneous addition of actinomycin D, but not if actinomycin D was added 1 h later. Northern and dot-blot analysis of RNA extracted from the treated ganglia indicated that an mRNA coding for an α-subunit of tubulin was increased by treatment with forskolin. 2D PAGE also demonstrated an increase in an α-subunit of tubulin. An increase in neuronal cyclic AMP appears to selectively increase the production of specific proteins and may contribute to the production of macromolecules involved in the initiation and stimulation of axonal regeneration.

Differential effects of extra- and intracellular anions on GABA-activated currents in bullfrog sensory neurons

1. Kinetic properties of gamma-aminobutyric acid (GABA)-gated inward and outward anion currents were investigated in the frog sensory neurons perfused internally and externally with various anions with the use of a rapid concentration-jump (termed as 'concentration-clamp') technique. 2. Extracellular Br- [( Br-]o) shifted the dose-response curves of GABA-induced inward anion currents to the left without affecting the maximum values, whereas [Cl-]o, [I-]o, [No3-]o, [HCOO-]o, and [CH3COO-]o altered the rate of desensitization differently without shifting the GABA dose-response curves, indicating that the kinetics of desensitization phase are affected differently by various extracellular anions. 3. [CH3COO-]o suppressed the maximum current of the dose-response curve of the GABA-induced inward ICl without affecting Kd. 4. Both activation and desensitization phases of GABA-induced ICl consisted of fast and slow components, respectively. [Br-]o, [I-]o, and [NO3-]o significantly prolonged the slow desensitization component, whereas both [HCOO-]o and [CH3COO-]o shortened it. The fast desensitization and the fast and slow activation components were also affected by these foreign anions. 5. GABA dose-response curves of inward currents carried by various intracellular anions (Cl-, Br-, NO3-, I-, SCN-, HCOO-, F-, CH3COO-, CH3CH2COO-, BrO3-, and ClO3-) while keeping a constant [Cl-]o had a constant Kd value but different saturating maximum currents. There were no marked differences among their current kinetics except in the case of SCN-, indicating that the current kinetics is not affected by replacing intracellular Cl- [( Cl-]i) with various foreign anions. 6. The configuration and amplitude of GABA-gated outward anion currents at a constant [Cl-]i reflected the extracellular action of individual anions on the anion-binding site of GABA receptor associated with the anion-selective channel. 7. The relative conductances of the various anions, calculated from the maximum peak currents in dose-response curves of the GABA-induced inward anion currents at a constant [Cl-]o, was in the sequence: I- greater than Br- greater than or equal to NO3- greater than ClO3- greater than SCN- greater than or equal to Cl- greater than HCOO- greater than BrO3- greater than CH3COO- greater than F- greater than CH3CH2COO-.(ABSTRACT TRUNCATED AT 400 WORDS)

Influences of external Ca 2+ on the GABA-induced chloride current and the efficacy of diazepam in internally perfused frog sensory neurons

The effects of extracellular Ca 2+ on the γ-aminobutyric acid (GABA)-induced Cl - current and the efficacy of diazepam in the facilitation of GABA response were studied in frog isolated sensory neurons, using a ‘concentration clamp’ technique which combines a suction pipette (internal perfusion and voltage clamp) and a rapid drug application system. When nominal Ca 2+-free external solution was changed to the solution containing 2 mM Ca 2+, the response elicited by 1 × 10 −5 M GABA was reduced by about 40% of the control obtained in nominal Ca 2+-free solution. The dose-response curve for GABA was shifted to the right without affecting the maximum response. It can be suggested that the application of external Ca 2+ modulates the affinity of the GABA receptor to its agonist, GABA. Diazepam at the concentration of 3 × 10 −6 M shifted the dose-response curve for GABA to the left without changing the maximum reponse with or without external Ca 2+. However, the augmentatory action of diazepam on the GABA response was reduced in the presence of external Ca 2+. Possible mechanisms for inhibitory action of external Ca 2+ on the GABA-gated response and the reduced efficacy of diazepam are discussed.

Augmentation of GABA-induced chloride current in frog sensory neurons by diazepam.

The effect of diazepam (DZP) on the GABA-induced macroscopic and microscopic Cl- current was investigated in isolated frog sensory neurons using both 'concentration-clamp' and patch-clamp techniques. At concentration range between 10(-9) and 10(-4) M, DZP itself evoked no response but potentiated time- and dose-dependently the subthreshold GABA responses, though at high DZP concentrations beyond 10(-5) M the potentiation ratio decreased. The potentiation effect was long-lasting and desensitized slowly over the course of several 10 minutes after washing-out of DZP. DZP potentiated GABA response without shifting the GABA reversal potential. The entire GABA dose-response curve was shifted in a parallel manner to the left by adding DZP without changing cooperatively: the Hill slope was 2.0. The potentiation of GABA response by DZP did not depend on either inward or outward direction of the Cl- current but slightly on the membrane potential. The time constants of activation of desensitization of GABA-gated Cl- current consisted of fast and slow components, respectively. The slow components were concentration-dependent, and significantly changed in the presence of DZP, while DZP had little effects on fast components. In the 'inside-out' configuration, the addition of DZP activated GABA-receptor ionophore complexes under subthreshold without changing the single Cl- channel conductance. It is concluded that DZP may act at a site to modulate GABA binding, in which DZP increases GABA binding affinity and also affects the kinetics of GABA-gated Cl- channels, indicating that DZP has dual action on the GABA-induced responses.

Serotonin modulates calcium-dependent plateau of action potentials recorded from bull frog A-type sensory neurons which is ω-conotoxin GVIA-sensitive, but dihydropyridine-insensitive

Tetraethylammonium-treated bull frog sensory neurons exhibit a calcium-dependent plateau on the falling limb of the action potential, which is reduced in duration by 5-HT. The portion of the calcium-dependent plateau which is reduced by 5-HT is blocked by omega-conotoxin GVIA but is not reduced by nifedipine or enhanced by Bay K 8644. The lack of effect of 5-HT, when retested after exposure of a previously 5-HT sensitive neuron to conotoxin favors the hypothesis that 5-HT reduces the duration of the calcium-dependent plateau by inactivating voltage-dependent calcium channels, rather than by increasing a voltage-dependent potassium current.

O-115 - Interaction of quinolone antimicrobial agents and 4-biphenylacetic acid on GABA-induced chloride current in frog sensory neurons.

O-115 - Interaction of quinolone antimicrobial agents and 4-biphenylacetic acid on GABA-induced chloride current in frog sensory neurons.

Inhibition of the voltage-dependent calcium currents in isolated frog sensory neurons by GABA-related agonistic compounds

Effects of GABA A-, barbiturate- and benzodiazepine receptor agonists and GABA B agonist, baclofen, on voltage-dependent Ca 2+ current ( I Ca) were studied in isolated frog sensory neurons after suppression of Na + and K + currents using single-electrode voltage-clamp. GABA, muscimol, taurine and pentobarbital (PB) dose-dependently induced a transient Cl − current ( I Cl), while baclofen and diazepam (DZP) did not elicit any currents. With GABA A agonists such as GABA, muscimol and taurine, I Ca was suppressed transiently, and the maximum inhibition of I Ca occurred within 1 min. The suppression of I Ca by all GABA A agonists was neither voltage dependent nor attenuated in the presence of either bicuculline or picrotoxin. In addition, there was no correlation between GABA- and baclofen-induced suppressions of I Ca. The results suggest that the inhibition of I Ca by GABA A receptor agonists is not due to either GABA A or GABA B receptor activation at least. The inhibition of I Ca by baclofen, PB and DZP was persistent. PB suppressed the amplitude of I Ca and also facilitated the inactivation process, suggesting that PB behaves as a Ca channel blocker. However, the mechanisms of I Ca suppression by baclofen and DZP are the subject for a future study. The potency order of the drugs in reducing I Ca was muscimol > GABA = DZP > baclofen > PB > taurine.

Intracellular picrotoxin blocks pentobarbital-gated Cl − conductance

Using the ‘inside-out’ configuration of frog sensory neurons, we studied the effect of intracellular picrotoxin on the pentobarbital-gated single channel response of Cl −-current ( i Cl). The pentobarbital-induced i Cl showed no voltage-dependency and the single channel conductance ( γ Cl) was 16±3.1 pS ( n = 6). Picrotoxin caused the pentobarbital-gated Cl − channels to react in a flickering pattern and then finally caused them to cease their opening altogether. This inhibitory action of picrotoxin was reversible.

Three putative 5-HT 1A agonists act as 5-HT antagonists in frog sensory neurons

The putative 5-HT 1A agonists 8-hydroxy-dipropylaminotetralin (DPAT), buspirone and p-aminophenylethyl-m-trifluoromethylphenyl piperazine (PAPP) were tested for their ability to narrow the action potentials of bullfrog sensory neurons, a serotonergic response observed to be blocked by the 5-HT 1A antagonists spiperone and spiroxitrine. Given alone, DPAT, buspirone, and PAPP were ineffective 5-HT agonists. When applied concomitantly with 5-HT, however, they significantly antagonized the original response. Thus, in some systems these putative 5-HT 1A agonists function as antagonists.

Inhibition of drug-gated chloride currents by calcium influx in frog sensory neurons

In isolated and internally perfused frog sensory neurons, muscimol-, pentobarbital- and α-chloralose-induced Cl − currents were suppressed by voltage-dependent Ca 2+ currents. The amount of chloride currents inhibited by Ca 2+ influx developed in a hyperbolic manner as a function of Ca 2+ influx. The increase in intracellular Ca 2+ concentration also suppressed the currents carried by monovalent anions, such as Br −, I −, NO 3 − and HCOO −.