Pentylenetetrazol and reflex activity of isolated frog spinal cord.

The superfused in vitro frog spinal cord preparation was used to investigate the effects of pentylenetetrazol (PTZ) on the spinal cord. PTZ depressed monosynaptic, but augmented polysynaptic reflexes, and decreased primary afferent deplorization. Concurrently, in Ringer's solution containing sufficient magnesium or cobalt ions to block synaptic transmission, PTZ antagonized the hyperpolarizing effects on motoneurons and the depolarizing effects on primary afferent fibers of the inhibitory amino acids GABA, beta-alanine, taurine, and glycine. PTZ did not affect responses to the excitatory amino acids glutamic acid and aspartic acid. Furthermore, PTZ did not alter high affinity uptake by cord slices, K+ -evoked release of [3H]GABA from them, or the spinal concentration of GABA. These data suggest that PTZ may produce its excitatory effects by postsynaptic blockade of inhibitory processes mediated by GABA (and possibly by other amino acids).     

Some actions of catechol on synaptic transmission in the isolated spinal cord of the frog.

The isolated frog spinal cord was used to investigate the synaptic effects of the convulsant agent catechol. Addition of the compound to the superfusate consistently enhanced orthodromic reflex activity recorded from ventral roots and augmented primary afferent depolarization. Concomitantly catechol altered the polarization changes produced in ventral and dorsal roots by putative neurotransmitter amino acids when these compounds were applied in Mg2+-containing Ringer. Catechol reduced the hyperpolarizations induced in motoneurons by the neutral amino acids, GABA, beta-alanine, taurine and glycine, but did not affect the depolarizations produced by the dicarboxylic amino acids, L-glutamate and L-aspartate. In contrast, catechol increased the dorsal root depolarizations elicited by both neutral and dicarboxylic amino acids and also the depolarizations produced by elevated potassium concentrations. Catechol did not bring about significant changes in the passive electrical properties of motoneurons or dorsal root fibers. In addition, it did not alter either the high affinity uptake or the depolarization-evoked release of tritiated GABA, glycine, L-glutamate and L-aspartate. It appears that the postsynaptic actions of catechol explain its ability to enhance spinal reflexes.     

Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system

Accumulation of [3H]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [3H]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [3H]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.     

The effect of glycine and gamma-aminobutyric acid on the excitatory postsynaptic potentials of the spinal motor neurons in the lamprey in the presence of antagonists

Effect of bath application of the inhibitory amino acids (glycine and GABA) on motoneurons of EPSPs was studied in the normal physiological solutions and after preliminary administration of antagonists: strychnine (10(-6) mol/l), bicuculline (10(-4) mol/l) or picrotoxin (10(-4) mol/l). All these antagonists diminished the depression of monosynaptic EPSPs which were elicited by both amino acids (glycine and GABA). Data obtained in this study and previously reported ones permit concluding that motoneuron membranes in the spinal cord of lamprey possess the unit receptor channel complex sensitive to both amino acids.     

Evidence that glycine mediates the postsynaptic potentials that inhibit lumbar motoneurons during the atonia of active sleep

Postsynaptic inhibition of somatic motoneurons underlies the atonia of active sleep. This inhibitory control depends, in large measure, on the bombardment of motoneurons during active sleep by a unique class of large-amplitude inhibitory postsynaptic potentials (IPSPs). These potentials are present only during this behavioral state and have therefore been designated as active sleep-specific IPSPs (AS-IPSPs). The present study was concerned with determining the neurotransmitter that mediates these AS-IPSPs. Lumbar motoneurons were recorded intracellularly during quiet and active sleep in intact, undrugged, normally respiring cats. The frequency and waveform parameters of the inhibitory postsynaptic potentials recorded from these motoneurons were examined following the microiontophoretic juxta-cellular administration of strychnine (a glycine receptor antagonist) and picrotoxin and bicuculline (GABA receptor antagonists). Microiontophoretically applied strychnine abolished the AS-IPSPs and a majority of smaller-amplitude IPSPs. Neither picrotoxin nor bicuculline modified the frequency, amplitude, or rising phase of the AS-IPSPs or the smaller-amplitude IPSPs. We conclude that the postsynaptic inhibitory drive that impinges on motoneurons during active sleep is principally mediated by glycine or a glycinergic substance.     

Effects of strychnine,bicuculline, and picrotoxin on inhibition of hypoglossal motoneurons

Postsynaptic potentials, evoked by lingual or hypoglossal nerve stimulation, were recorded from hypoglossal motoneurons of the cat with glass microelectrodes. Lingual nerve-evoked inhibitory postsynaptic potentials (LIPSPs) were recorded in 98% of hypoglossal motoneurons. Hypoglossal nerve stimulation caused a hyperpolarzing potential following the antidromic spike in all hypoglossal motoneurons tested. This potential was unaffected by depolarizing or hyperpolarizing currents, could not be evoked at a stimulus strength less than that which was threshold for the antidromic action potential, and did not change in shape or amplitude at stimulus strengths which were above threshold for antidromic invasion. This hyperpolarpolarizing potential was therefore considered to be an afterhyperpolarization. However, hypoglossal nerve-induced inhibitory postsynaptic potentials were recorded from hypoglossal units which had characteristics of interneurons, thus suggesting the presence of afferent fibers in the hypoglossal nerve. The hypoglossal nerve-induced afterhyperpolarization was not affected by strychnine, bicuculline, or picrotoxin. The LIPSP was antagonized by strychnine but unaffected by bicuculline or picrotoxin. The results suggest that inhibition of hypoglossal motoneurons via the lingual nerve is more likely to be mediated by glycine than gamma-aminobutyric acid (GABA) and is therefore similar to the strychnine-sensitive postsynaptic inhibition of spinal motoneurons.     

Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA

The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.     

Early development of glycine- and GABA-mediated synapses in rat spinal cord.

Motoneuron responses to the inhibitory amino acids glycine and GABA, and the contribution of inhibitory synapses to developing sensorimotor synapses were studied in rat spinal cords during the last week in utero. In differentiating motoneurons, glycine and GABA induced Cl(-)-dependent membrane depolarizations and large decreases in membrane resistance. These responses gradually decreased during embryonic development, and at birth they were significantly smaller than in embryos. In motoneurons of embryos and neonates, dorsal root stimulation produced only depolarizing potentials, some of which reversed at -50 mV membrane potential. Reduction of extracellular Cl- concentrations increased the amplitude of these potentials, suggesting that they are generated by Cl- current. Contribution of Cl(-)-dependent potentials to compound dorsal root-evoked potentials was studied by determining the effects of glycine and GABA antagonists on them. In motoneurons of embryos at days 16-17 of gestation (D16-D17), strychnine or bicuculline blocked dorsal root-evoked potentials. This suppression was neither the result of a decrease in neuronal excitability nor the inhibition of glutamate receptors. Strychnine-evoked depression was not blocked by atropine, indicating that it was not due to disinhibition of muscarinic synapses. By D19, strychnine and bicuculline significantly increased dorsal root-evoked potentials rather than blocking them. This reversed function did not result from an increase in neuronal excitability or changes in the specificity of strychnine and bicuculline antagonism. The number of glycine- and GABA-immunoreactive cells increased 20% between D17 and D19. The number of immunoreactive cells and fibers significantly increased in the motor nuclei and dorsal horn laminae. These morphological changes may contribute to establishment of new synaptic contacts on motoneurons, thus changing the actions of strychnine and bicuculline on dorsal root-evoked potentials.     

Specific antagonism of GABA-mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: a common mode of convulsant action

Mammalian spinal neurons grown in tissue culture were used to study the effects of the four convulsants-penicillin, pentylenetetrazol, picrotoxin, and bicuculline-on these neurons' responses to amino acids. Bath application of all four convulsants produced paroxysmal depolarizing events in the neurons; iontophoresis of the four convulsants selectively depressed responses produced by iontophoresis of the putative inhibitory transmitter GABA, and effected this depression without altering either inhibitory responses to beta-alanine or glycine, or excitation mediated by glutamate. These results support the hypothesis that the convulsant activity of these agents comes in part from selective antagonism of GABA-mediated postsynaptic inhibition.     

Bimodal action of glycine on frog spinal motoneurones.

In the presence of procaine the changes in electrical potential caused by glycine in the ventral root of the isolated hemisected spinal cord of the frog have been shown to be compounded of both hyperpolarizing and depolarizing responses to the amino acid. From a comparison of the effects of changes in the perfusion medium on the potentials produced by glycine, beta-alanine and L-glutamate in the presence and absence of strychnine, it was concluded that glycine acts on a similar receptor to beta-alanine and causes a hyperpolarizing response which is blocked by strychnine. However, glycine has an additional, depolarizing, action which is usually the major effect and masks the hyperpolarizing response. The depolarizations produced by L-glutamate and glycine could be differentiated by their different ionic dependencies. The glycine depolarization was selectively decreased by a lowered sodium ion concentration in the medium while L-glutamate depolarizations were selectively enhanced by lowered potassium ion concentration.     

In vitro sacral cord preparation and motoneuron recording from adult mice

We report the development of an intracellular recording technique for adult mouse motoneurons in sacral spinal cord. Based on a similar preparation for adult rat, we modified the cord preparation solution and filled the sharp electrode with a solution that has physiological osmolarity and pH. The viability of the preparation was examined by recording root reflexes. Short-latency reflexes mediated through monosynaptic transmission between S1 and S3 ventral root were reliably produced by dorsal root electrical stimuli and were stably recorded for more than eight hours. Long-lasting potentiation of the root reflex was observed by bath application of methoxamine, a noradrenergic alpha1 receptor agonist. Bath application of strychnine and picrotoxin, antagonists for glycine and GABA(A) receptors respectively, unmasked long-lasting reflexes that may contain polysynaptic components. In addition, on the background of strychnine and picrotoxin, adding methoxamine induced spontaneous ventral root activity. For intracellular recording, the motoneurons could be reliably penetrated and held for up to 30 min. In all 16 motoneurons recorded, resting membrane potential, input resistance, action potentials and repetitive firing were comparable to those of rat motoneurons. Thus, this preparation is viable and provides a new method for combined electrophysiological and genetic studies of the adult mouse spinal cord.     

Antagonists of the putative inhibitory transmitter effects of taurine and GABA in the retina

Intravitreal injection of taurine or GABA produces a marked decrease of the b-wave amplitude in the chicken ERG. Strychnine abolishes the depressant action of taurine, but not that of GABA. On the contrary, picrotoxin abolishes the depressant action of GABA, but not that of taurine. Taurine and GABA seem to act as inhibitory transmitters in the retina; taurine could be responsible for a postsynaptic inhibition, whereas GABA appears to be responsible for a presynaptic one. The complex nature of the effects of these amino acids on the tectal-evoked responses indicates that these substances act at more than one retinal site. The role of taurine considered as an inhibitory transmitter, or as a modulator, is also discussed.     

The effects of pentobarbital and related compounds on frog motoneurons

The effect of pentobarbital (PB) and related compounds on frog motoneurons was examined with sucrose gap recording from the ventral roots. PB was found to: (1) depress the action of glutamate, (2) selectively enhance the action of GABA, (3) reverse the non-competitive picrotoxin antagonism of GABA and the competitive strychnine antagonism of beta-alanine, but not the competitive bicuculline methiodide antagonism of GABA, and (4) elicit a GABAmimetic hyperpolarization. The first three actions had a threshold concentration of 10 microM, while the GABAmimetic action required a 10-fold higher concentration. The reversal of picrotoxin's action by PB suggests that PB might modify GABA mechanisms by combining to the picrotoxin recognition site. Phenobarbital shared all of the properties of PB but was approximately one-fifth as potent. The only property that phenytoin shared with PB was a weak depression of glutamate responses. Chlordiazepoxide selectively enhanced GABA responses but was devoid of the other actions of PB. These results suggest that the GABAmimetic effect of PB may be an important feature in the depressant and anesthetic properties of PB. The anesthetic chloralose, which is structurally unrelated to PB, nevertheless shared all of the actions of PB. This finding suggests that the properties described for PB may also be found in other general anesthetics.     

Opioid peptides decrease calcium-dependent action potential duration of mouse dorsal root ganglion neurons in cell culture

Opiate alkaloid and opioid peptide actions on spontaneous neuronal activity and postsynaptic amino acid responsiveness were assessed using intracellular recording techniques applied to murine spinal cord neurons in primary dissociated cell culture. Application of opiates was by superfusion and amino acids by iontophoresis. Glycine and GABA but not glutamate responses were antagonized by the opiate alkaloids. Since opiate effects on glycine and GABA responses were not naloxone-reversible, only weakly stereospecific, and not produced by the opioid peptide [d-Ala²]-Met-enkephalinamide, it is unlikely that these effects were mediated by opiate receptors. Opiate depression of glycine inhibition was correlated with the induction of paroxysmal depolarizations in cultured spinal cord neurons, suggesting that antagonism of inhibitory amino acid transmission may underlie the convulsant actions of high concentrations of the opiate alkaloids.     

The effect of glycine and gamma-aminobutyric acid on the evoked activity of the spinal cord motor neurons in the lamprey

The influence of bath application of glycine (10(-5)-10(-3) mol/l) and gamma-aminobutyric acid (10(-5)-10(-2) mol/l) on the monosynaptic EPSPs evoked in motoneurons by stimulation of a descending tract and individual Müller axons was studied in spinal cord-notochord preparation of lamprey (Lampetra fluviatilis). Both amino acids hyperpolarized the motoneuron membrane and depressed the evoked synaptic activity. But the inhibitory effect of glycine was stronger and it was revealed at lower concentrations as compared to that of GABA. It is concluded that glycine is more effective in blocking the postsynaptic motoneuron activity in comparison with GABA in the spinal cord of lamprey.     

Zinc co-localizes with GABA and glycine in synapses in the lamprey spinal cord

The presence of zinc in synaptic terminals in the lamprey spinal cord was examined utilizing a modification of the Timm's sulfide silver method and with the fluorescent marker 6-methoxy-8-quinolyl-p-toluenesulfonamide (TSQ). Axons labeled with a Timm's staining method were predominantly located in the lateral region of the dorsal column. This correlated with a maximum of TSQ fluorescence in this region of the spinal cord. Single labeled terminals accumulating Timm reaction product were also found throughout the gray matter and fiber tracts. At the ultrastructural level, zinc was located in a population of synaptic terminals that co-localized gamma-aminobutyric acid (GABA) and glycine. Possible effects of Zn2+ on neuronal activity were examined. In spinobulbar interneurons, which receive GABAergic input in the dorsal column, zinc potentiated responses to GABA application, but it did not affect responses to GABA in motoneurons. Responses in motoneurons to pressure application of glycine were also not affected by Zn2+. Zinc, however, potentiated monosynaptic glycinergic inhibitory postsynaptic potentials (IPSPs) evoked in motoneurons by inhibitory locomotor network interneurons and increased frequency, but not amplitude of spontaneous miniature IPSPs recorded in the presence of tetrodotoxin (TTX), suggesting presynaptic effects. Glutamate responses and the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons were reduced by zinc. These effects appeared to be mediated largely postsynaptically through an effect on the N-methyl-D-aspartate (NMDA) component of the glutamatergic input. Our results thus show that free zinc is present in inhibitory synaptic terminals in the lamprey spinal cord, and that it may function as a modulator of inhibitory synaptic transmission.     

Further evidence in support of taurine as a mediator of synaptic transmission in the frog spinal cord

It has been reported that 6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-1, 1-dioxide (AMBD, TAG) is a specific blocker of taurine and beta-alanine responses in the central nervous system. We have re-examined the effect of AMBD on amino acid and synaptically evoked responses recorded from isolated hemisected frog spinal cords by means of the sucrose gap technique. When indirect responses were blocked by adding tetrodotoxin (0.2 microM) or manganese chloride (2 mM) to the normal Ringer solution, AMBD (0.01-0.5 mM) selectively antagonized taurine, beta-alanine, hypotaurine and kojic amine evoked depolarizations of primary afferents at their intramedullary part (dorsal root terminals, DRT) and on dorsal root ganglia (DRG), without significantly affecting responses to glutamate (on DRT), glycine (on DRT) or GABA (on DRT and DRG). Depolarizing responses to taurine and beta-alanine (1 mM) were depressed by up to 50% with 0.1 mM AMBD and often completely antagonized with 0.25 mM AMBD. In normal Ringer solution, AMBD selectively antagonized the dorsal root potential evoked by ventral root stimulation (VR-DRP, threshold at 0.02 mM AMBD, 90% block with 0.25 mM); other synaptic potentials increased in duration and/or amplitude, demonstrating a strong convulsant effect of AMBD. Thus, the depolarizing responses of taurine, beta-alanine and hypotaurine on primary afferents are pharmacologically indistinguishable from the VR-DRP. These results are in agreement with the proposal that taurine or a taurine-like substance (possibly beta-alanine or hypotaurine) is the mediator of VR-DRP in amphibian spinal cord.     

Properties of stereotyped series of postsynaptic potentials in hypoglossal motoneurons

A stereotyped series of postsynaptic potentials produced in cat hypoglossal motoneurons by stimulation of the cerebral cortex, the inferior alveolar nerve or the lingual nerve was studied. These include an excitatory postsynaptic potentials (EPSP) and subsequently 3 different types of inhibitory postsynaptic potential (IPSPs). The first is a short-lasting IPSP which was blocked by strychnine administration. The second is a γ-aminobutyric acid (GABA) IPSP which was blocked by picrotoxin administration. This IPSP was sensitive to membrane polarization and dependent on a conductance increase. The third is a long-duration hyperpolarizing potential which was enhanced by the injection of picrotoxin and insensitive to membrane polarization. Moreover, we have demonstrated that the amplitude of cortically induced EPSPs decreased greatly with depolarization.     

Conductance changes during bath application of beta-alanine and taurine in giant interneurons of the isolated lamprey spinal cord.

(1) Input conductances of giant interneurons in the isolated spinal cord of lampreys were measured with two separate intracellular electrodes. Bath applications of 0.4--3 mM beta-alanine and taurine produced large, reversible conductance increases which were Cl-dependent. (2) Strychnine at 1--2.5 microM might be a competitive antagonist of both amino acids, but had a stronger effect on taurine. Bicuculline and picrotoxin were weak antagonists of beta-alanine and taurine in some cells. (3) A few giant interneurons desensitized after repeated application of the amino acids, but most became more sensitive. Responses to the amino acids also increase at low temperatures in in Na-free fluid, suggesting effect of uptake mechanisms.     

Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: A postembedding electron microscopic study

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of α-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal α-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies. © 1996 Wiley-Liss, Inc.