Application Of Glycine Research Articles

Membrane properties of an unusual intrinsically oscillating, wide-field teleost retinal amacrine cell.

In the retina, amacrine cells modulate the transfer of information from bipolar to ganglion cells. The nature of the modulation depends on the synaptic input and the membrane properties of the cells. In the retina of white bass, we identified a class of bistratified, wide-field amacrine cell characterized by immunopositive labelling for GABA and calmodulin. In isolation, the cells presented resting membrane potentials averaging -69 mV although some cells settled at more depolarized values (-30 mV). Injection of depolarizing current pulses induced oscillatory membrane responses. When elicited from depolarized cells, the oscillations were short-lived (< 40 ms). For the most part, the oscillatory potentials of hyperpolarized cells remained unattenuated throughout the depolarizing pulse. The frequency of the oscillations increased logarithmically with mean membrane potential, ranging from 74 to 140 Hz. Cells exhibiting depolarized membrane potentials oscillated at twice that rate. When the membrane potential of these cells was hyperpolarized to -70 mV, the oscillations became unattenuated and slowed. We found the cells expressed voltage-gated sodium, potassium and calcium currents and calcium-dependent potassium currents. We demonstrate that the oscillatory potentials arose as a result of the interplay between calcium and potassium currents. The cells responded to local application of GABA and glycine, both of which modulate the oscillatory potentials. Glutamate and its analogues depolarized the cell and induced oscillatory potentials. Our results indicate that oscillatory responses of a type of wide-field amacrine cell are an intrinsic feature of the cell and not due to circuit properties.

Effects of GABA A and glycine receptor agonists on the medullary inspiratory neuronal activity during spontaneous augmented breaths in anesthetized rats

To clarify whether GABAergic or glycinergic transmission alters the activity of inspiratory neurons during spontaneous augmented breaths, we recorded the single unit activity from inspiratory neurons in the dorsal and ventral respiratory groups in the medulla of pentobarbital anesthetized rats and applied GABA A and glycine receptor agonists by iontophoresis using multibarrel microelectrodes. The spontaneous augmented breath was divided into two different phases; the first phase (phase I) resembled a normal inspiration but the second phase (phase II) indicated a marked increase in diaphragm electromyogram activity. During application of either muscimol or glycine, the discharge of inspiratory neurons during the phase I of spontaneous augmented breaths was suppressed, but the augmenting discharge of the phase II did not change significantly in any cell type of the neurons (I-augmenting, I-decrementing and I-other). These results suggested that the excitatory inputs to inspiratory neurons during the phase II of augmented breaths may not be significantly influenced by the activation of either GABA A receptors or glycine receptors.

Auditory response properties in the superior paraolivary nucleus of the gerbil.

The ascending auditory pathway is characterized by parallel processing. At the brain stem level, several structures are involved that are known to serve different well-defined functions. However, the function of one prominent brain stem nucleus, the rodent superior paraolivary nucleus (SPN) and its putative homologue in other mammals, the dorsomedial periolivary nucleus, is unknown. Based on extracellular recordings from anesthetized gerbils, we tested the role of the SPN in sound localization and temporal processing. First, the existence of binaural inputs indicates that the SPN might be involved in sound localization. Although almost half of the neurons exhibited binaural interactions (most of them excited from both sides), effects of interaural time and intensity differences (ITD; IID) were weak and ambiguous. Thus a straightforward function of SPN in sound localization appears to be implausible. Second, inputs from octopus and multipolar/stellate cells of the cochlear nucleus and from principal cells of the medial nucleus of the trapezoid body could relate to precise temporal processing in the SPN. Based on discharge types, two subpopulations of SPN cells were observed: about 60% of the neurons responded to pure tones with sustained discharges, with irregular spike patterns and no phase-locking. Only four neurons showed a regular spike pattern ("chopping"). About 40% of the neurons responded with phasic ON or OFF discharges. Average first spike latency observed in neurons with sustained discharges was significantly shorter than that of ON responders, but had a considerably higher trial-to-trial variation ("jitter"). A subpopulation of ON responders showed a jitter of less than +/-0.1 ms. Most neurons (66%) responded to sinusoidally amplitude-modulated sounds (SAM) with an ongoing response, phase-locked to the stimulus envelope. Again, ON responders showed a significantly higher temporal precision in the phase-locked discharge compared with the sustained responders. High variability was observed among spike-rate-based modulation transfer functions. Histologically, a massive concentration of cytochemical markers for glycinergic input to SPN cells was demonstrated. Application of glycine or its blockade revealed profound effects of glycinergic inhibition on the auditory responses of SPN neurons. The existence of at least two subpopulations of neurons is in line with different subsets of SPN cells that can be distinguished morphologically. One temporally less precise population might modulate the processing of its target structures by providing a rather diffuse inhibition. In contrast, precise ON responders might provide a short, initial inhibitory pulse to its targets.

Precise inhibition is essential for microsecond interaural time difference coding.

Microsecond differences in the arrival time of a sound at the two ears (interaural time differences, ITDs) are the main cue for localizing low-frequency sounds in space. Traditionally, ITDs are thought to be encoded by an array of coincidence-detector neurons, receiving excitatory inputs from the two ears via axons of variable length ('delay lines'), to create a topographic map of azimuthal auditory space. Compelling evidence for the existence of such a map in the mammalian lTD detector, the medial superior olive (MSO), however, is lacking. Equally puzzling is the role of a--temporally very precise glycine--mediated inhibitory input to MSO neurons. Using in vivo recordings from the MSO of the Mongolian gerbil, we found the responses of ITD-sensitive neurons to be inconsistent with the idea of a topographic map of auditory space. Moreover, local application of glycine and its antagonist strychnine by iontophoresis (through glass pipette electrodes, by means of an electric current) revealed that precisely timed glycine-controlled inhibition is a critical part of the mechanism by which the physiologically relevant range of ITDs is encoded in the MSO. A computer model, simulating the response of a coincidence-detector neuron with bilateral excitatory inputs and a temporally precise contralateral inhibitory input, supports this conclusion.

Openings of the rat recombinant alpha 1 homomeric glycine receptor as a function of the number of agonist molecules bound.

The functional properties of rat homomeric α1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat α1 subunit cDNA. Whole-cell dose-response curves gave EC 50 estimates between 30 and 120 μM and a Hill slope of ∼3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 μM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ∼40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human alpha(1)beta glycine receptors.

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR beta subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the beta subunit was examined using site-directed mutagenesis and expression of recombinant (alpha(1)beta(Y413F)) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents (I(Gly)) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I(Gly) in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC(50) = 143 microM; cSrc EC(50) = 74 microM). Exogenous cSrc, applied intracellularly, upregulated heteromeric alpha(1)beta receptors but not homomeric alpha(1) receptors. Substitution mutation of the tyrosine to phenylalanine at position beta-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type alpha(1)beta but not alpha(1)beta(Y413F) receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the beta subunit.

Pharmacological characterization of glycine-gated chloride currents recorded in rat hippocampal slices.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus has been overlooked, largely due to the misconception that GlyR expression ceases early during development and to few functional studies demonstrating their presence. As a result, little is known regarding the physiological and pharmacological properties of native GlyRs expressed by hippocampal neurons. In this study, we used pharmacological tools and whole cell patch-clamp recordings of CA1 pyramidal cells and interneurons in acutely prepared hippocampal slices from 3- to 4-wk old rats to characterize these understudied receptors. We show that glycine application to recorded pyramidal cells and interneurons elicited strychnine-sensitive chloride-mediated currents (I(gly)) that did not completely desensitize in the continued presence of agonist but reached a steady state at 45-60% of the peak amplitude. Additionally, the inhibitory amino acid, taurine, which has been shown to activate GlyRs in other systems, activated GlyRs expressed by both pyramidal cells and interneurons, although with much less potency than glycine, having an EC(50) 10-fold higher. To examine the potential subunit composition of hippocampal GlyRs, we tested the effect of the GABA(A) receptor antagonist, picrotoxin, on I(gly) recorded from both cell types. At low micromolar concentrations of picrotoxin (< or =100 microM), which selectively block alpha homomeric GlyRs, I(gly) was partially attenuated in both cell types, indicating that alpha homomeric receptors are expressed by pyramidal cells and interneurons. At picrotoxin concentrations < or =1 mM, approximately 10-20% of the whole cell current remained, suggesting that alphabeta heteromeric GlyRs are also expressed because this subtype of GlyR is relatively resistant to picrotoxin antagonism. Finally, we examined whether hippocampal GlyRs are modulated by zinc. Consistent with previous reports in other preparations, zinc elicited a bidirectional modulation of GlyRs, with physiological zinc concentrations (1-100 microM) increasing whole cell currents and concentrations >100 microM depressing them. Furthermore, the same concentration of zinc that potentiates I(gly) suppressed currents mediated by the N-methyl-D-aspartate subtype of the glutamate receptor. Thus we provide a pharmacological characterization of native GlyRs expressed by both major neuron types in hippocampus and show that these receptors can be activated by taurine, an amino acid that is highly concentrated in hippocampus. Furthermore, our data suggest that at least two GlyR subtypes are present in hippocampus and that GlyR-mediated currents can be potentiated by zinc at concentrations that suppress glutamate-mediated excitability.

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To study the inhibitory mechanisms of the ventral longitudinal muscle fibers of the mealworm larva Tenebrio molitor, the effects of several inhibitory compounds on the excitatory post-synaptic potentials (EPSPs), membrane potentials and L-glutamate-induced potentials (GLU-Ps) were investigated by means of a microelectrode. Application of GABA, glycine, L-α-alanine, β-alanine, taurine and picrotoxin were used in the muscle preparations. None of these compounds had an effect on the EPSP and membrane potentials. In contrast, GLU-Ps were potentiated by picrotoxin, GABAA antagonist and chloride channel blocker, whereas GABA, glycine, L-α-alanine, β-alanine and taurine had no effect. This potentiation of GLU-Ps may result from a blockade of glutamate-gated chloride channels by picrotoxin. These results suggested the presence of extrajunctional chloride-channel-type glutamate receptors in the ventral longitudinal muscle fibers of the larval mealworm. A similar result was obtained with a glutamate agonist, quisqualate, suggesting the chloride channel may be under the control of a quisqualate like receptor.

Early expression of glycine and GABAA receptors in developing spinal cord neurons. Effects on neurite outgrowth

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABAA-activated currents were demonstrated during applications of glycine and GABA (50–100 μM) in 5 days in vitro (DIV) neurons. Long application (≥10 min) of 100 μM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg2+, at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca2+ indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca2+ events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABAA receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca2+ at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABAA and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 μM), nimodipine (4 μM) and bicuculline (20 μM) completely blocked it. On the other hand, APV (50 μM) and CNQX (20 μM) were unable to affect neurite outgrowth.These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Potentiation of L-Type Calcium Channels Reveals Nonsynaptic Mechanisms that Correlate Spontaneous Activity in the Developing Mammalian Retina

Although correlated neural activity is a hallmark of many regions of the developing nervous system, the neural events underlying its propagation remain largely unknown. In the developing vertebrate retina, waves of spontaneous, correlated neural activity sweep across the ganglion cell layer. Here, we demonstrate that L-type Ca(2+) channel agonists induce large, frequent, rapidly propagating waves of neural activity in the developing retina. In contrast to retinal waves that have been described previously, these L-type Ca(2+) channel agonist-potentiated waves propagate independent of fast synaptic transmission. Bath application of nicotinic acetylcholine, AMPA, NMDA, glycine, and GABA(A) receptor antagonists does not alter the velocity, frequency, or size of the potentiated waves. Additionally, these antagonists do not alter the frequency or magnitude of spontaneous depolarizations that are recorded in individual retinal ganglion cells. Like normal retinal waves, however, the area over which the potentiated waves propagate is reduced dramatically by 18alpha-glycyrrhetinic acid, a blocker of gap junctions. Additionally, like normal retinal waves, L-type Ca(2+) channel agonist-potentiated waves are abolished by adenosine deaminase, which degrades extracellular adenosine, and by aminophylline, a general adenosine receptor antagonist, indicating that they are dependent on adenosine-mediated signaling. Our study indicates that although the precise spatiotemporal properties of retinal waves are shaped by local synaptic inputs, activity may be propagated through the developing mammalian retina by nonsynaptic pathways.

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.

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.

Interaction of the neuroprotective drug riluzole with GABA A and glycine receptor channels

Riluzole is used as therapeutic agent in amyotrophic lateral slerosis. We investigated the interaction of riluzole with recombinant GABA (γ-aminobutyric acid) A receptor channels (α 1β 2γ 2-subunits) and glycine receptor channels (α 1β-subunits) transiently expressed in HEK293 cells. For electrophysiological experiments, the patch–clamp technique in combination with tools for ultrafast solution exchange was used. Saturating concentrations of GABA or glycine were applied with different concentrations of riluzole to outside-out patches containing α 1β 2γ 2 GABA A receptor channels or α 1β-glycine receptor channels on their surface, respectively. The current declined after application of GABA or glycine with three time constants of desensitization to a steady-state current amplitude. Application of riluzole resulted in a shift to fast desensitized states at both receptors. The proportion of the time constants of fast desensitization increased and the time constants of slow desensitization and the steady-state current decreased whereas the maximal current amplitudes were not affected by riluzole. The data of the study demonstrate for the first time interaction of GABAergic and glycinergic currents with riluzole under physiological conditions.

Development of gill and lung breathing in amphibia

In the 25 morphological stages of larval bullfrog development there exists a gradual transition from gill to lung ventilation associated with a developmental decrease in the contribution of the skin in gas exchange. Bath application of GABA and/or glycine inhibited gill but not lung burst activities of cranial nerve (CN) VII in the premetamorphic (stages 16–19) in vitro tadpole brainstem preparation [1]. It was proposed that the neural basis of gill rhythmogenesis involved network inhibition, whereas lung rhythmicity was pacemaker driven [1]. Bath application of a bicuculline/strychnine solution abolished gill and enhanced lung bursting in stages 16–19 in vitro [1]. Bath application of the GABAB receptor antagonist 2-hydroxy-saclofen disinhibited the lung central pattern generator (CPG) resulting in precocious lung bursting patterns as early as developmental stage 6 [2]. We recorded efferent activity from CNVII and spinal nerve (SN)II in the in vitro tadpole brainstem preparation in three successive developmental groups (3–9; 10–15; 16–19) before and after bath application of a 10 μM bicuculline and 5 μM strychnine solution. We also exposed the brainstem to bath pH7.4, 7.8, and 8.2 before and after bath application of bicuculline/strychnine. Bicuculline/strychnine produced lung ventilatory bursts in all developmental stages tested, indicating the presence of the lung CPG as well as excitatory synapses to respiratory motor neurons as early as stage 3. We also designed an experiment to examine the importance of lung ventilation on the developmental shift from gill to lung bursting. Two groups of tadpoles were hatched from eggs. Control tadpoles had free access to the air-water interface throughout development, whereas tadpoles were denied access to the air-water interface via the placement of Plexiglas 2.5 cm below the surface of the water. CNVII and SNII efferent activities were recorded in vitro at bath pH7.4, 7.8, and 8.2 before and after bath application of 10 μM bicuculline and 5 μM strychnine. Postmetamorphic barrier tadpoles exhibited different motor patterns than stage-matched controls. Tadpoles reared in the barrier condition to stages 20–25 possessed fictive gill and lung ventilatory activities of premetamorphic tadpoles. All barrier tadpole preparations exhibited robust, spontaneous lung burst activity following bath application of bicuculline/strychnine. We propose that developmentally dependent GABA- and glycinergic mechanisms lead to disinhibition of the lung CPG such that early in development gill motor patterns are the dominant respiratory rhythm, whereas in late development the lung CPG is the dominant respiratory rhythm. Denying the tadpole the ability to practice lung breathing during metamorphosis produces a morphologically correct postmetamorphic tadpole with an immature, or premetamorphic respiratory rhythm. We propose that prevention of lung inflation in barrier tadpoles leads to premetamorphic levels of GABA- and glycinergic inhibition, and that this inhibition may be altered by pulmonary stretch receptor feedback in control tadpoles.

Group II and group III metabotropic glutamate receptor agonists depress synaptic transmission in the rat spinal cord dorsal horn

The effects of group II and group III metabotropic glutamate receptor agonists on synaptic responses evoked by primary afferent stimulation in the dorsal horn, but mostly substantia gelatinosa, neurons were studied in the spinal cord slice preparation using conventional intracellular recording technique. Bath application of a potent metabotropic glutamate receptor 2- and 3-selective agonist (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine reversibly suppressed monosynaptic and polysynaptic excitatory postsynaptic potentials evoked by A primary afferent fibers stimulation, the effect likely mediated by mGlu3 receptor subtype. This suppressing effect of (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine on primary afferent neurotransmission was dose dependent and reduced by (S)-α-ethylglutamate, a group II metabotropic glutamate receptor antagonist. (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine suppressed excitatory postsynaptic potentials without inducing detectable changes of postsynaptic membrane potential and neuronal input resistance in dorsal horn neurons. The paired-pulse depression at excitatory synapses between primary afferent fibers and dorsal horn neurons was reduced by (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine application, suggesting a presynaptic site of action. The selective group III metabotropic glutamate receptor agonist (S)-2-amino-4-phosphonobutanoate also depressed A afferent fibers-evoked monosynaptic and polysynaptic excitatory postsynaptic potentials in a dose-dependent and reversible manner. The concentration-dependence of (S)-2-amino-4-phosphonobutanoate-mediated depression was most consistent with activation of mGlu receptor subtypes 4 and 7. However, on the basis of anatomical distribution of mGlu 4 and 7 subtypes, it is also possible that the (S)-2-amino-4-phosphonobatanoate effect is due to interaction with mGlu 7 receptor alone. (RS)-α-cyclopropyl-4-phosphonophenylglycine a preferential antagonist at group III metabotropic glutamate receptors, completely reversed the depressant effects of (S)-2-amino-4-phosphonobutanoate on both monosynaptic and polysynaptic responses. (S)-2-amino-4-phosphonobutanoate reduced the paired-pulse depression at excitatory synapses between primary afferent fibers and dorsal horn neurons, but did not alter their postsynaptic membrane potential and input resistance. A clear facilitation of the (S)-2-amino-4-phosphonobutanoate-induced depression of monosynaptic and polysynaptic excitatory postsynaptic potentials in the absence of gamma-aminobutyric acid-subtype A receptor- and glycine-mediated synaptic inhibition was shown. Besides the depressant effect on excitatory synaptic transmission, inhibitory actions of group II and III metabotropic glutamate receptor agonists on the inhibitory postsynaptic potentials evoked by primary afferent stimulation in dorsal horn neurons were observed.These results suggest that group II and group III metabotropic glutamate receptors are expressed at primary afferent synapses in the dorsal horn region, and activation of the receptors suppresses synaptic transmission by an action on the presynaptic site.

Glycine induced calcium concentration changes in vestibular type I sensory cells

Glutamate is the neurotransmitter of the synapse between vestibular type I hair cells and the afferent nerve calyx. This calyx may also be involved in local feedback, which may modify sensory cell activity via N-methyl- D-aspartate (NMDA) receptors. Glycine is the co-agonist of glutamate in NMDA receptor activation. Both agents have been detected by immunocytochemistry in the nerve calyx. Glutamate and NMDA stimulations cause changes in the intracellular calcium concentration ([Ca 2+] i) of isolated type I sensory cells. We investigated the effect of glycine stimulation on [Ca 2+] i in guinea pig type I sensory cells by spectrofluorimetry with fura-2. Glycine application to isolated type I sensory cells induced a rapid and transient increase in [Ca 2+] i. The fluorescence ratio increased by 55% above the resting level. The peak was reached in 9 s and the return to basal level took about 20 s. A specific antagonist of the glycine site on NMDA receptors, 7-chlorokynurenate (10 μM), decreased the calcium response to glycine by 60%. Glycine may activate NMDA receptors. Glycine may also activate the strychnine-sensitive glycine receptor-gated channel. Strychnine (50 μM) decreased the calcium response to glycine by 60%. Thus, glycine probably induces calcium concentration changes in type I vestibular sensory cells via NMDA receptors and/or glycine receptors.

GABACReceptors Control Adaptive Changes in a Glycinergic Inhibitory Pathway in Salamander Retina

We studied the role of GABA in adaptive changes in a lateral inhibitory system in the tiger salamander retina. In dark-adapted retinal slice preparations picrotoxin caused a slow enhancement of glycine-mediated IPSCs in ganglion cells. The enhancement of glycinergic IPSCs developed slowly over the course of 5-20 min, even though picrotoxin blocked both GABA(A) and GABA(C) receptors within a few seconds. The slow enhancement of glycinergic IPSCs by picrotoxin was much weaker in light-adapted preparations. The slow enhancement of glycinergic inhibitory inputs was not produced by bicuculline, indicating that it involved GABA(C) receptors. The responses of ganglion cells to direct application of glycine were not enhanced by picrotoxin, indicating that the enhancement was not caused by an action on glycine receptors. In dark-adapted eyecup preparations picrotoxin caused a slow enhancement of glycinergic IPSPs and transient lateral inhibition produced by a rotating windmill pattern, similar to the effect of light adaptation. The results suggest that the glycinergic inhibitory inputs are modulated by an unknown substance whose synthesis and/or release is inhibited in dark-adapted retinas by GABA acting at GABA(C) receptors.

Effects of inhibitory neurotransmitters on the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

Effects of inhibitory neurotransmitters on the locomotor rhythm and pattern generation were investigated using an in vitro preparation isolated from the mudpuppy (Necturus maculatus). The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial nerves. During N-methyl-d-aspartate (NMDA)-induced locomotion, the rhythmic motor output (EMG) was recorded unilaterally from elbow flexor and extensor muscles. While neither glycine nor gamma-aminobutyric acid (GABA)-related substances induced locomotion in the absence of NMDA, they modulated NMDA-induced locomotion. Bath application of glycine and GABA suppressed the rhythmic motor pattern induced by NMDA. Addition of glycine receptor antagonist strychnine or GABA(A) receptor antagonist bicuculline disrupted the phase relationship between antagonistic motor pools during ongoing locomotion, thereby changing the normal alternating pattern into synchronous EMG bursts. Both the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen mimicked the effects of GABA as they either slowed down or stopped locomotion. Nipecotic acid, a GABA uptake blocker, had a similar effect. This suggested that an endogenous release of GABA modulated the locomotor rhythm. The endogenous release was antagonized by the GABA(A) and GABA(B) receptor antagonists bicuculline and CGP-35348, respectively. Immunocytochemistry revealed that glycine and GABA-positive neurons and fibers were present in mudpuppy spinal cord. Although the GABAergic neurons were more numerous than glycinergic neurons, both cell types contributed processes directed towards the white matter and occasionally towards the ependymal lining of the central canal. Our results suggest that inhibitory neurotransmitters exert powerful actions upon the neuronal network governing forelimb locomotion in the mudpuppy. The effects we observed may be mediated by a network of segmentally distributed glycinergic and GABAergic spinal neurons.

Voltage dependence of the glycine receptor-channel kinetics in the zebrafish hindbrain.

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from -50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of approximately 5 and approximately 30 ms (holding potential of -50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 +/- 2.8% of the total current at -50 mV and 54.1 +/- 10% at +20 mV. The 20-80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20-80% rise time was 0.2 ms at -70 mV and 0.22 ms at +20 mV. Responses evoked by 100-200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation.

1. The inhibitory neurotransmitter glycine can elicit depolarizing responses in immature neurones. We investigated the changes in glycine responses and their ionic mechanism in developing neurones of the rat lateral superior olive (LSO), an auditory brainstem nucleus involved in sound localization. 2. Whole-cell and gramicidin perforated-patch recordings were performed from visually identified LSO neurones in brain slices and glycine was pressure applied for 3-100 ms to the soma. Glycine-evoked currents were reversibly blocked by strychnine. They were mostly monophasic, but biphasic responses occurred in approximately 30 % of P8-11 neurones in perforated-patch recordings. 3. In whole-cell recordings from P2-11 neurones, the reversal potential of glycine-evoked currents (EGly) was determined by the transmembranous Cl- gradient and corresponded closely to the Nernst potential for Cl-, regardless of age. This indicates that Cl- is the principle ion permeating glycine receptors, but is also consistent with a low relative (10-20 %) permeability for HCO3-. The Cl- gradient also determined the polarity and amplitude of glycine-evoked membrane potential changes. 4. Leaving the native intracellular [Cl-] undisturbed with gramicidin perforated-patch recordings, we found a highly significant, age-dependent change of EGly from -46.8 +/- 1.8 mV (P1-4, n = 28) to -67.6 +/- 3.3 mV (P5-8, n = 10) to -82.2 +/- 4.1 mV (P9-11, n = 18). The majority of P1-4 neurones were depolarized by glycine ( approximately 80 %) and spikes were evoked in approximately 30 %. In contrast, P9-11 neurones were hyperpolarized. 5. In perforated-patch recordings, EGly was influenced by the voltage protocol and the glycine application interval; it could be shifted in the positive and negative direction. For a given application interval, these shifts were always larger in P1-4 than in P8-11 neurones, pointing to less effective Cl- regulation mechanisms in younger neurones. 6. Furosemide (frusemide), a blocker of cation-Cl- cotransporters, reversibly shifted EGly in the negative direction in P2-4 neurones, yet in the positive direction in P8-10 neurones, suggesting the blockade of net inward and net outward Cl- transporters, respectively. 7. Taken together, age-dependent changes in active Cl- regulation are likely to cause the developmental shift from depolarizing to hyperpolarizing glycine responses. A high intracellular [Cl-] is generated in neonatal LSO neurones which decreases during maturation.