Acid-evoked Responses Research Articles

PH-dependent modulation of TRPV1 by modality-selective antagonists.

Antagonists of TRPV1 that inhibit all activation modes cause hyperthermia, hampering their medical use as novel analgesics. TRPV1 antagonists that do not (fully) inhibit responses to low pH do not cause hyperthermia, but it remains incompletely understood how such antagonists affect channel gating. We tested the hypothesis that pH-sparing antagonists act in a modality-selective manner on TRPV1, differentially affecting channel activation by protons and capsaicin. Using whole-cell patch-clamp and calcium imaging to measure channel activity in cells expressing wild type human TRPV1 or the pH-insensitive mutant F660A. Responses to protons and capsaicin were measured at different pH values in the presence of antagonists that reportedly partially spare (A-1165442) or potentiate (AMG7905) acid-evoked channel activation. At pH5.5, A-1165442 was equipotent at blocking acid- and capsaicin-evoked responses of wild type TRPV1. Its potency to inhibit acid-evoked responses was attenuated at pH ≤ 5.0. AMG7905, at a concentration (1μM) that fully inhibits capsaicin-evoked responses, potentiated proton-evoked (pH5.5) responses of wild type TRPV1. In the F660A mutant, the inhibitory efficacy of A-1165442 and AMG7905 towards capsaicin-evoked responses was reduced at lower pH values and AMG7905 acted as a partial agonist. Our findings show that A-1165442 and AMG7905 interact in a pH-dependent manner with TRPV1, but this pH dependence is not strictly modality-selective. Reduced TRPV1 antagonism at acidic pH may limit analgesic efficacy in injured tissue and needs to be considered in models explaining the effects of antagonists on core body temperature.

Extracellular pH regulates excitability of vomeronasal sensory neurons.

The mouse vomeronasal organ (VNO) plays a critical role in semiochemical detection and social communication. Vomeronasal stimuli are typically secreted in various body fluids. Following direct contact with urine deposits or other secretions, a peristaltic vascular pump mediates fluid entry into the recipient's VNO. Therefore, while vomeronasal sensory neurons (VSNs) sample various stimulatory semiochemicals dissolved in the intraluminal mucus, they might also be affected by the general physicochemical properties of the "solvent." Here, we report cycle stage-correlated variations in urinary pH among female mice. Estrus-specific pH decline is observed exclusively in urine samples from sexually experienced females. Moreover, patch-clamp recordings in acute VNO slices reveal that mouse VSNs reliably detect extracellular acidosis. Acid-evoked responses share the biophysical and pharmacological hallmarks of the hyperpolarization-activated current Ih. Mechanistically, VSN acid sensitivity depends on a pH-induced shift in the voltage-dependence of Ih activation that causes the opening of HCN channels at rest, thereby increasing VSN excitability. Together, our results identify extracellular acidification as a potent activator of vomeronasal Ih and suggest HCN channel-dependent vomeronasal gain control of social chemosignaling. Our data thus reveal a potential mechanistic basis for stimulus pH detection in rodent chemosensory communication.

Phospholipase C and Diacylglycerol Mediate Olfactory Responses to Amino Acids in the Main Olfactory Epithelium of an Amphibian

The semi-aquatic lifestyle of amphibians represents a unique opportunity to study the molecular driving forces involved in the transition of aquatic to terrestrial olfaction in vertebrates. Most amphibians have anatomically segregated main and vomeronasal olfactory systems, but at the cellular and molecular level the segregation differs from that found in mammals. We have recently shown that amino acid responses in the main olfactory epithelium (MOE) of larval Xenopus laevis segregate into a lateral and a medial processing stream, and that the former is part of a vomeronasal type 2 receptor expression zone in the MOE. We hypothesized that the lateral amino acid responses might be mediated via a vomeronasal-like transduction machinery. Here we report that amino acid-responsive receptor neurons in the lateral MOE employ a phospholipase C (PLC) and diacylglycerol-mediated transduction cascade that is independent of Ca2+ store depletion. Furthermore, we found that putative transient receptor potential (TRP) channel blockers inhibit most amino acid-evoked responses in the lateral MOE, suggesting that ion channels belonging to the TRP family may be involved in the signaling pathway. Our data show, for the first time, a widespread PLC- and diacylglycerol-dependent transduction cascade in the MOE of a vertebrate already possessing a vomeronasal organ.

Gustatory modulation of the responses of trigeminal subnucleus caudalis neurons to noxious stimulation of the tongue in rats

Certain tastants inhibit oral irritation by capsaicin, whereas anesthesia of the chorda tympani (CT) enhances oral capsaicin burn. We tested the hypothesis that tastants activate the CT to suppress responses of trigeminal subnucleus caudalis (Vc) neurons to noxious oral stimuli. In anesthetized rats, we recorded Vc unit responses to noxious electrical, chemical (pentanoic acid, 200μm) and thermal (55°C) stimulation of the tongue. Electrically evoked responses were significantly reduced by a tastant mix and individually applied NaCl, monosodium glutamate (MSG), and monopotassium glutamate. Sucrose, citric acid, quinine and water (control) had no effect. Pentanoic acid-evoked responses were similarly attenuated by NaCl and MSG, but not by other tastants. Responses to noxious heat were not affected by any tastant. Transection and/or anesthesia of the CT bilaterally affected neither Vc neuronal responses to electrical or pentanoic acid stimulation, nor the depressant effect of NaCl and MSG on electrically evoked responses. Calcium imaging showed that neither NaCl nor MSG directly excited any trigeminal ganglion cells or affected their responses to pentanoic acid. GABA also had no effect, arguing against peripheral effects of GABA, NaCl or MSG on lingual nocicepive nerve endings. The data also rule out a central mechanism, as the effects of NaCl and MSG were intact following CT transection. We speculate that the effect is mediated peripherally by the release from taste receptor cells (type III) of some mediator(s) other than GABA to indirectly inhibit trigeminal nociceptors. The results also indicate that the CT does not exert a tonic inhibitory effect on nociceptive Vc neurons.

Airway irritation and cough evoked by acid: from human to ion channel.

Inhalation or aspiration of acid solution evokes airway defense responses such as cough and reflex bronchoconstriction, resulting from activation of vagal bronchopulmonary C-fibers and Aδ afferents. The stimulatory effect of hydrogen ion on these sensory nerves is generated by activation of two major types of ion channels expressed in these neurons: a rapidly activating and inactivating current mediated through ASICs, and a slow sustaining current via activation of TRPV1. Recent studies have shown that these acid-evoked responses are elevated during airway inflammatory reaction, revealing the potential convergence of a wide array of inflammatory signaling on these ion channels. Since pH in the airway fluid drops substantially in patients with inflammatory airway diseases, these heightened stimulatory effects of acid on airway sensory nerves may play a part in the manifestation of airway irritation and excessive cough under those pathophysiological conditions.

Activation of Polycystic Kidney Disease-2-like 1 (PKD2L1)-PKD1L3 Complex by Acid in Mouse Taste Cells

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca(2+) imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca(2+) increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.

Regulation of acid signaling in rat pulmonary sensory neurons by protease-activated receptor-2

Airway acidification has been consistently observed in airway inflammatory conditions and is known to cause cardiorespiratory symptoms that are, at least in part, mediated through the activation of bronchopulmonary C fibers and the subsequent reflexes. Protease-activated receptor-2 (PAR(2)) is expressed in a variety of cells in the lung and airways and is believed to play a role in airway inflammation and hyperresponsiveness. This study was carried out to investigate the effect of PAR(2) activation on the acid signaling in rat bronchopulmonary C-fiber sensory neurons. Our RT-PCR results revealed the expression of mRNAs for transient receptor potential vanilloid receptor 1 (TRPV1) and four functional acid-sensing ion channel (ASIC) subunits 1a, 1b, 2a, and 3 in these sensory neurons. Preincubation of SLIGRL-NH(2), a specific PAR(2)-activating peptide, markedly enhanced the Ca(2+) transient evoked by extracellular acidification. Pretreatment with PAR(2) agonists significantly potentiated both acid-evoked ASIC- and TRPV1-like whole cell inward currents. Activation of PAR(2) also potentiated the excitability of these neurons to acid, but not electrical stimulation. In addition, the potentiation of acid-evoked responses was not prevented by inhibiting either PLC or PKC nor was mimicked by activation of PKC. In conclusion, activation of PAR(2) modulates the acid signaling in pulmonary sensory neurons, and the interaction may play a role in the pathogenesis of airway inflammatory conditions, where airway acidification and PAR(2) activation can occur simultaneously.

Na+-K+-2Cl−cotransporters and Cl−channels regulate citric acid cough in guinea pigs

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.

Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system.

To better understand the full extent of the odorant detection capabilities of fish, we investigated the olfactory sensitivity of zebrafish to a monoamine and several polyamines using electrophysiological and activity-dependent labeling techniques. Electro-olfactogram (EOG) recording methods established the relative stimulatory effectiveness of these odorants as: spermine >> spermidine approximately agmatine > glutamine > putrescine >or= cadaverine >or= histamine > artificial freshwater. The detection threshold for the potent polyamines was approximately 1 micromol l(-1). Cross-adaptation experiments suggested that multiple receptors are involved in polyamine detection. Three observations indicated that polyamine signaling may involve a transduction cascade distinct from those used by either amino acids or bile salts. Like bile salts and the adenylate cyclase activator forskolin, but unlike amino acid odorants, polyamines failed to stimulate activity-dependent labeling of olfactory sensory neurons with the cation channel permeant probe agmatine, suggesting a signaling pathway different from that used by amino acid stimuli. Also supporting distinct amino acid and polyamine signaling pathways is the finding that altering phospholipase C activity with the inhibitor U-73122 significantly reduced amino acid-evoked responses, but had little effect on polyamine- (or bile salt-) evoked responses. Altering cyclic nucleotide-mediated signaling by adenylate cyclase activation with forskolin, which significantly reduced responses to bile salts, failed to attenuate polyamine responses, suggesting that polyamines and bile salts do not share a common transduction cascade. Collectively, these findings suggest that polyamines are a new class of olfactory stimuli transduced by a receptor-mediated, second messenger signaling pathway that is distinct from those used by amino acids or bile salts.

Orphanin FQ (nociceptin) modulates responses of trigeminal neurons evoked by excitatory amino acids and somatosensory stimuli, and blocks the substance P-induced facilitation of N-methyl- d-aspartate-evoked responses

The present investigation details the modulation of medullary dorsal horn neuron responses to excitatory amino acids and peripheral cutaneous stimuli by orphanin FQ (nociceptin), an endogenous ligand for the opioid receptor-like 1 receptor. Effects of orphanin FQ, administered microiontophoretically or given intracerebroventricularly, were tested on the responses of nociceptive-specific, wide dynamic range and low threshold neurons recorded in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in anesthetized (urethane or pentobarbital) male rats. Microiontophoretic application of orphanin FQ reduced the N-methyl- d-aspartate-evoked responses in 86% (71/82) of neurons, and the (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses in 86% (30/35) of neurons. However, orphanin FQ produced a longer lasting inhibitory effect on the N-methyl- d-aspartate-evoked responses relative to the (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses. The inhibitory effect of orphanin FQ was not modality-specific, responses evoked by noxious as well as non-noxious stimuli were reduced in 22/23 neurons. However, the inhibitory effect was more pronounced on noxious stimulus-evoked responses. Naloxone applied at currents that antagonized the inhibitory effects of selective agonists at μ and κ opioid receptors failed to inhibit the effects of orphanin FQ. Microiontophoretic co-application of substance P with N-methyl- d-aspartate facilitated the N-methyl- d-aspartate-evoked responses in 52% (26/50) of nociceptive neurons. Orphanin FQ blocked or reduced the substance P-induced facilitation by 86±24.4% ( n=14). In order to compare electrophysiological data with previous behavioral observations, effects of orphanin FQ administered intracerebroventricularly were tested on the excitatory amino acid-evoked responses. Orphanin FQ reduced the N-methyl- d-aspartate-evoked responses in 85% (11/13) of neurons whereas the (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-evoked responses were facilitated in 69% (9/13) of neurons. We suggest that orphanin FQ produces a predominantly inhibitory effect on, (i) noxious stimuli evoked responses, (ii) excitatory amino acid receptor-mediated transmission and, (iii) the substance P-induced facilitation of the N-methyl- d-aspartate-evoked responses. We conclude that orphanin FQ primarily produced an antinociceptive action at the level of the dorsal horn of the medulla.

Effects of depolarizing stimuli on calcium homeostasis in cultured rat motoneurones.

Intracellular calcium concentrations in individual rat motoneurones in enriched primary cultures were measured by Indo-1 fluorimetry. Motoneurones in the cultures were characterized morphometrically and by cholineacetyltransferase immunocytochemistry. Depolarization of the cells with glutamic acid or veratridine increased intracellular calcium levels, which returned to baseline only slowly after removal of the depolarizing agent. The use of selective agonists (N-methyl-D-aspartic acid, AMPA, kainic acid, quisqualic acid and 1R-3S-ACPD) and antagonists (MK 801 and CNQX) showed that the excitatory amino acid-evoked responses were mediated by AMPA/kainate receptors rather than by NMDA receptors. Depolarization-evoked calcium transients in motoneurones are blocked by the neuroprotective drug riluzole Calcium transients reflected entry of calcium from without the cell, and their blockade by nitrendipine and lanthanum chloride suggested that this entry took place primarily through voltage-dependent calcium channels. These findings may be relevant for understanding the selective vulnerability of motoneurones to excitotoxicity in amyotrophic lateral sclerosis, and the therapeutic activity of riluzole in the treatment of this disease.

Activation of hamster suprachiasmatic neurons in vitro via metabotropic glutamate receptors.

The neurophysiological effects of ionophoretic application of the metabotropic glutamate receptor agonist (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid on the firing rate of single suprachiasmatic nucleus cells was studied in a hypothalamic slice preparation. In addition, the effects of the phenylglycine derivative (RS)-alpha-methyl-4-carboxyphenylglycine, a selective metabotropic glutamate receptor antagonist, on responses evoked by (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid and N-methyl-D-aspartate were assessed. (1S,3R)-1-Amino-1,3-cyclopentanedicarboxylic acid elicited current-dependent increases in neuronal activity in 65% of all suprachiasmatic nucleus cells studied, while N-methyl-D-aspartate activated 93% of the same cells. Cells in the ventrolateral suprachiasmatic nucleus were more sensitive to (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application (82% of cells activated) than those in the dorsomedial suprachiasmatic nucleus (28% of cells activated). In addition, responses evoked by (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid in the ventrolateral suprachiasmatic nucleus had a rapid onset and a prolonged recovery from agonist application, whereas responses elicited in the dorsomedial suprachiasmatic nucleus were slower in onset and recovered more quickly from agonist application. Co-application of (RS)-alpha-methyl-4-carboxyphenylglycine selectivity attenuated (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid-evoked responses but had no effect on N-methyl-D-aspartate-evoked activity in the same cells. These results indicate that (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application activates single cells in the hamster suprachiasmatic nucleus and these responses are selectively attenuated by (RS)-alpha-methyl-4-carboxyphenylglycine. Cells in the ventrolateral suprachiasmatic nucleus are more sensitive to (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application than those in the dorsomedial suprachiasmatic nucleus. These results are the first demonstration that metabotropic glutamate receptors can modulate spontaneous neuronal activity within the suprachiasmatic nucleus, the predominant mammalian circadian pacemaker.

Inhibitors of nitric oxide synthase enhance rat ileum contractions induced by ricinoleic acid in vitro

The effects of N G- nitro- L-arginine methyl ester (L-NAME) and N G- monomethyl- L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, were studied on ricinoleic acid-evoked contractions in rat isolated ileum. Ricinoleic acid (10 −5 to 10 −4 M) caused a concentration-dependent contraction. Addition of L-NAME (30–300 μM) or L-NMMA (30–300 μM) to the Tyrode's solution increased in a concentration-dependent fashion the amplitude of the ricinoleic acid-evoked responses. L-Arginine (900 μM), a natural substrate of NO synthase, but not D-arginine (900 μM), counteracted the effect of L-NAME (300 μM). The potentiating effect of L-NAME was also prevented by sodium nitroprusside (0.1–1 μM), a generator of NO. These results provide evidence that endogeno NO may modulate the contraction of rat ileum induced by ricinoleic acid. As the contraction induced by ricinoleic acid is not blocked by tetrodotoxin (0.6 and 6.0 μM) the contractile effect of ricinoleic acid results mainly from a direct action on the smooth muscle.

Physiological influence of lateral proisocortex on the midbrain periaqueductal gray: Evidence for a role of an excitatory amino acid in synaptic activation

Recent anatomical studies in this laboratory have demonstrated that the proisocortex cortex adjacent and dorsal to the rhinal sulcus is one of the major forebrain afferent inputs to the midbrain periaqueductal gray matter in the rat. The physiological influence(s) of this projection has not been examined. The present studies investigated the responses of periaqueductal gray neurons to chemical and electrical stimulation of proisocortex in chloral hydrate-anesthetized rats. In addition, the role of glutamate as a possible transmitter in excitatory proisocortex-periaqueductal gray synaptic responses was tested. Microinjection of d,l-homocysteate into proisocortex excited 44% (19/43), inhibited 37% (16/43) and had no effect on 19% of periaqueductal gray cells. The onset of d,l-homocystic acid-evoked responses ranged from 2 to 60 s; the duration of responses ranged from 1 to 18 min. Low-frequency, single-pulse electrical stimulation of proisocortex robustly altered neuronal discharge in 25% of periaqueductal gray neurons sampled; 10% (74/724) of neurons were excited and 15% (107/724) were inhibited. Insular cortex-evoked excitatory responses had a mean onset latency of19.5 ± 4.2 ms and a mean duration of38.5 ± 26.9 ms. Inhibitory responses had a mean onset latency of26.2 ± 15.6 ms and mean duration of108.0 ± 84.9 ms. Trains of high-frequency electrical stimulation of proisocortex excited 22% (13/59) and inhibited 25% (15/59) of periaqueductal gray cells tested. In separate experiments, stimulation electrodes were placed in periaqueductal gray to antidromically activate proisocortex neurons that project to periaqueductal gray. Stimulation of periaqueductal gray antidromically activated 10/55 cells at latencies ranging from 7 to 18 ms, and a mean latency of1 2.0 ± 0.9ms. Assuming a straight line trajectory for proisocortex fibers that project to periaqueductal gray, these values indicate a axonal impulse conduction velocity along proisocortex-periaqueductal gray fibers of 0.6 to 1.4 m/s, with an overall mean of 1.1 m/s. This relatively slow conduction velocity indicates that periaqueductal gray-IC fibers may be thin, unmyelinated axons. Microinjection of the broad-spectrum excitatory amino acid receptor antagonist kynurenic acid directly onto periaqueductal gray neurons blocked proisocortex-evoked excitation in 52% (24/46) of periaqueductal gray cells tested. In addition, kynurenic acid blocked inhibitory responses to proisocortex stimulation three of 11 periaqueductal gray cells tested. It is concluded that: (i) proisocortex neurons have a direct synaptic influence on periaqueductal gray neurons. (ii) high-frequency train stimulation or chemical stimulation of proisocortex is significantly more effective in altering the firing pattern of periaqueductal gray neuron than single pulse stimulation, indicating that modulation of periaqueductal gray cells by proisocortex requires some temporal and/or spatial summation. (iii) excitatory amino acid neurotransmission accounted for at least half of the excitatory responses mediated by this afferent system.

Modulation of extracellular pH by glutamate and GABA in rat hippocampal slices.

1. Alkaline extracellular pH transients evoked by afferent stimulation, and local pressure ejection of glutamate and gamma-aminobutyric acid (GABA), were studied in the CA1 region of rat hippocampal slices. Amino acid-evoked responses were obtained by use of a dual micromanipulator, with the tip of a double-barreled pH-sensitive microelectrode positioned 50 microns from a pressure ejection pipette. 2. At 31 degrees C, in Ringer solutions buffered with 26 mM HCO3- and 5% CO2, mean extracellular pH in submerged 300-microns slices was 7.15 +/- 0.12 (n = 27 slices), at a tissue depth of approximately 150 microns. In Ringer buffered with 35 mM HCO3- and 5% CO2, extracellular pH was 7.29 +/- 0.10 (n = 19 slices). 3. Repetitive stimulation of the Schaffer collaterals caused an extracellular alkaline shift in stratum oriens, pyramidale, and radiatum, averaging 0.05 +/- 0.03 pH units among all regions (n = 138), with a maximum response of 0.16 pH units. Alkaline transients of similar appearance were obtained by local ejection of glutamate (0.01-0.12 pH units, n = 110) and GABA (0.01-0.18 pH units, n = 137). Control ejection of these amino acids into dilute agar caused only small acid shifts. 4. Superfusion of 100 microM picrotoxin abolished the GABA-evoked alkaline shift but failed to inhibit the Schaffer collateral- and glutamate-evoked alkalinizations. 5. Superfusion of 10(-5)-10(-3) M acetazolamide acidified the baseline by 0.05-0.10 pH units and amplified the Schaffer collateral- and glutamate-evoked alkaline shifts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord.

Intracellular recording techniques have been used to provide information on the identity of excitatory transmitters released at synapses formed between dorsal root ganglion (DRG) and spinal cord neurones in two in vitro preparations. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurones and synaptic potentials recorded intracellularly from dorsal horn neurones after DRG explant stimulation. More than 80% of dorsal horn neurones received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations (5 mmol l-1 Ca2+, 3 mmol l-1 Mg2+) the acidic amino acid receptor agonists, L-glutamate, kainate (KA) and quisqualate (QUIS) excited all dorsal horn neurones which received a monosynaptic DRG neurone input, whereas L-aspartate and N-methyl-D-aspartate (NMDA) had little or no action. 2-Amino-5-phosphonovalerate (APV), a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and L-glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurones. Intracellular recordings from motoneurones in new-born rat spinal cord were used to study the sensitivity of the Ia excitatory postsynaptic potential (EPSP) to antagonists of excitatory amino acids. Superfusion of the spinal cord with APV did not inhibit the Ia EPSP but did suppress later, polysynaptic components of the afferent-evoked response. Kynurenate was a potent and selective inhibitor of the Ia EPSP, acting via a postsynaptic mechanism. These findings indicate that L-glutamate, or a glutamate-like compound, but not L-aspartate, is likely to be the predominant excitatory transmitter that mediates fast excitatory postsynaptic potentials at primary afferent synapses with both dorsal horn neurones and motoneurones.

Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate

Intracellular recording techniques have been used to provide information on the identity of excitatory sensory transmitters released at synapses formed between dorsal root ganglion (DRG) and dorsal horn neurons maintained in cell culture. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurons and synaptic potentials were recorded intracellularly from dorsal horn neurons after DRG explant stimulation. More than 80% of dorsal horn neurons within 1 mm of DRG explants received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations, the acidic amino acid receptor agonists, L-glutamate, kainate, and quisqualate excited all dorsal horn neurons which received a monosynaptic DRG neuron input, whereas aspartate and N-methyl-D-aspartate (NMDA) had little or no action. Several compounds reported to antagonize the actions of acidic amino acids were tested for their ability to block DRG-evoked synaptic potentials and glutamate-evoked responses in dorsal horn neurons. 2-Amino-5-phosphonovalerate, a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurons. These findings indicate that glutamate, or a glutamate-like compound, but not aspartate, is the excitatory transmitter that mediates fast excitatory postsynaptic potentials at the DRG-dorsal horn synapses examined in this study.

Influence of caffeine and midazolam on γ-aminobutyric acid-evoked responses in the frog spinal cord

The recently reported potentiation of γ-aminobutyric acid (GABA) evoked depolarizations by caffeine in the frog spinal cord might involve an interaction with GABA-linked benzodiazepine receptors. This possibility was investigated using a new potent benzodiazepine, midazolam, and a benzodiazepine antagonist, Ro 147437. Caffeine or midazolam enhanced the amplitude of submaximal GABA responses by about 50%; when equieffective enhancing doses of these compounds were simultaneously applied. GABA depolarizations were usually depressed below control levels. It was however possible to detect a narrow range of concentrations of midazolam which had an additive effect to the enhancement by caffeine. Ro 147437 did not block caffeine-induced potentiations of GABA responses. It is suggested that caffeine and benzodiazepines have distinct modes of action in modulating GABA-induced depolarizations in the in vitro spinal cord of the frog.

Effects of flurazepam on amino acid-evoked responses recorded from the lobster muscle and the frog spinal cord

The effects of flurazepam hydrochloride on GABA- or glutamate-evoked responses recorded from the lobster muscle fibre and the frog isolated spinal cord were studied using electrophysiological techniques. On lobster muscle flurazepam (up to 100 μM) reversibly antagonized responses to bath-applied or iontophoretically-applied glutamate without any effect on GABA responses. Higher concentrations of flurazepam (>200 μM) sometimes increased the resting membrane conductance, an effect different from that of GABA in being insensitive to picrotoxin (0.5 μM). In the tetrodotoxin (TTX)-treated frog spinal cord flurazepam (5 μM) reversibly antagonized both glutamate- and GABA-evoked dorsal root depolarizations although a smaller dose (2.5 μM) clearly potentiated the action of GABA. Spontaneous fluctuations of the dorsal root d.c. level in the presence of flurazepam were also observed. It is suggested that flurazepam antagonized amino acid responses by blocking receptor-activated Na + channels on the postsynaptic membranes. However, the potentiation of GABA action on the frog spinal cord may have involved either release of endogenous GABA or “sensitization” of spinal receptors to GABA, an action easily obscured by the predominant antagonistic effect of flurazepam at higher concentrations.

Antagonism by some antihistamines of the amino acid-evoked responses recorded from the lobster muscle fibre and the frog spinal cord.

1 The effects of some antihistamines on the lobster muscle fibre and the frog spinal cord were investigated using intracellular and extracellular recordings, respectively. 2. On lobster muscle, histamine H1-blockers reversibly antagonized responses to bath-applied glutamate, aspartate and quisqualate but not responses to gamma-aminobutyric acid (GABA). Iontophoretic glutamate potentials were also reduced. Histamine (up to 1 mM) had no effect on this preparation. 3 The H1-antagonists produced a small increase in muscle membrane conductance and a slight hyperpolarization. These effects were largely unchanged in a low C1- bathing solution. Procaine (1 mM) decreased membrane conductance and did not affect responses to GABA or glutamate. 4 The H2-antagonist burimamide blocked both glutamate and GABA-evoked responses on the lobster muscle without affecting resting potential or conductance. 5 In the frog cord, bath-applied histamine produced ventral root depolarizations and dorsal root hyperpolarizations (sometimes biphasic responses). These effects were reduced by tetrodotoxin (TTX) but not by antazoline (H1-blocker) or burimamide; the latter reversibly antagonized responses to both glutamate and GABA on TTX-treated cords while antazoline was ineffective. 6 It is suggested that antihistamines can act as non-specific amino acid antagonists by interacting at the level of the receptor-coupled ionophores.