Characterization of spinal amino acid release and touch-evoked allodynia produced by spinal glycine or GABA(A) receptor antagonist

Intrathecal strychnine (glycine antagonist) or bicuculline (GABA(A) antagonist) yields a touch-evoked agitation that is blocked by N-methyl-D-aspartate receptor antagonism. We examined the effects of intrathecal strychnine and bicuculline on touch-evoked agitation and the spinal release of amino acids. Fifty-two Sprague-Dawley rats were prepared under halothane anesthesia with a lumbar intrathecal catheter and a loop dialysis catheter. Four days after implantation, rats were randomized to receive an intrathecal injection of N-methyl-D-aspartate (3 microg), strychnine (3 microg) or bicuculline (10 microg), or a combination of N-methyl-D-aspartate with bicuculline or strychnine. The agitation produced by brief light tactile stroking of the flank (tactile allodynia), and the spontaneous spinal release of glutamate, taurine and serine was measured. Intrathecal N-methyl-D-aspartate, strychnine and bicuculline produced similar touch-evoked allodynia. Intrathecal bicuculline and N-methyl-D-aspartate alone evoked a transient spinal release of glutamate and taurine, but not serine, in the 0- 10 min sample, while strychnine did not affect spinal transmitter release at any time. As GABA(A) but not glycine receptor inhibition at equi-allodynic doses increases glutamate release, while the allodynia of both is blocked by N-methyl-D-aspartate receptor antagonism, we hypothesize that GABA(A) sites regulate presynaptic glutamate release, while glycine regulates the excitability of neurons postsynaptic to glutamatergic terminals.     

Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes

Xenopus oocytes were injected with mRNA from adult or neonatal rat spinal cord, or from adult cerebral cortex, and examined electrophysiologically to measure currents elicited by various receptor agonists. The mRNAs induced the oocytes to acquire similar types of amino acid receptors, albeit with different potencies. In oocytes injected with adult cord mRNA the responses to kainate and gamma-aminobutyrate were much smaller, whilst the currents elicited by glycine and beta-alanine were markedly larger, than those in oocytes injected with cortex mRNA. For these receptors, the expressional potency of neonatal spinal cord mRNA is similar to that of the adult cord mRNA, except for a lower sensitivity to beta-alanine.     

The hyperpolarization of spinal motoneurones by glycine and related amino acids

Summary Electrophoretically administered glycine, -alanine and GABA hyperpolarize spinal motoneurones in cats anaesthetized with pentobarbitone. The reversal potential for these hyperpolarizations is similar to that of inhibitory postsynaptic potentials. Alterations in intracellular K⁺ and Cl^– ion concentrations, and intracellular injection of a series of anions of different hydrated ion size, affect inhibitory and amino acid potentials in the same fashion. Hence it is probable that glycine, -alanine and GABA produce an alteration in membrane permeability similar to that produced by spinal inhibitory synaptic transmitters. Strychnine reversibly blocks the action of inhibitory transmitters, glycine and -alanine, but is without effect on the hyperpolarizing action of GABA.These results indicate that glycine may be a major spinal inhibitory transmitter, in which case strychnine affects spinal postsynaptic inhibition by limiting the action of glycine upon subsynaptic inhibitory receptors.Supported by a grant from the Swiss Academy of Medical Sciences.     

GABAA and strychnine-sensitive glycine receptors modulate N-methyl-D-aspartate-evoked acetylcholine release from rat spinal motoneurons: a possible role in neuroprotection

Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders. As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [(3)H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca(2+), tetrodotoxin, Cd(2+) ions and omega-conotoxin GVIA, but not on omega-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [(3)H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [(3)H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA. It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABA(A) and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABA(A) and glycine receptors.     

Selective antagonist activity of 5-aminohex-2-enedioic acid on amino acid excitation of cat spinal neurones

D- and L-5-aminohex-2-enedioic acid (AHED) and the DL-2-bromo derivative, which are conformationally restricted analogues of the excitatory amino acid antagonist D-alpha-aminoadipic acid (D alpha AA), were tested microelectrophoretically on cat spinal neurones. D-AHED reduced the excitatory action of N-methyl-D-aspartate (NMDA) to a greater extent than that of L-glutamate and the potency of this action was a half to a third that of D alpha AA. The DL-2-bromo derivative excited neurons, an effect greater than that of L-glutamate and not blocked by either D alpha AA or L-glutamic acid diethyl ester.     

A pharmacological study of the depression of spinal neurones by glycine and related amino acids

Summary An analysis has been made in anaesthetised cats of the depression by glycine and related amino acids of the firing of spinal dorsal horn interneurones, Renshaw cells and cortical neurones. In general, electrophoretically administered glycine was a more potent depressant of interneurones than GABA. The reverse was true for cortical neurones, whereas these two amino acids were approximately equally effective upon Renshaw cells. Strychnine blocked the depressant action of - and -amino acids, but not that of - and higher -amino acids. Only convulsants having a strychnine-like effect on spinal post-synaptic inhibition blocked the action of glycine. The depression of spinal neurones produced by glycine or GABA was not affected by structural analogues of glycine and GABA that were not depressants, or by substances influencing amino acid transport systems. Some evidence was obtained for the enzymic inactivation of electrophoretically administered glycine in spinal tissue. The results are discussed in terms of the involvement of a glycine-like amino acid as a major spinal inhibitory transmitter.Supported by a grant from the Swiss Academy of Medical Sciences.     

Role of spinal GABAA receptor reduction induced by stress in rat thermal hyperalgesia

The mechanisms underlying stress-induced hyperalgesia (SIH) remain poorly understood. Recent findings have provided strong evidence indicating that SIH could be related, at least in part, to alterations in spinal cord GABA activity. In the present study, we first investigated how acute restraint stress impacted pain responses as assessed using the tail flick immersion test. These results showed that rats developed hyperalgesia at 6 h after being subjected to 1-h acute restraint stress. Second, we measured the activation of spinal neurons and alterations in expression of GABA_A receptor β2 and β3 subunits as related to stress-induced hyperalgesia. Results from Western blot and immunofluorescence assays showed that c-fos protein increased in the dorsal horn of the lumbar spinal cord and GABA_A receptor β2 and β3 subunit proteins decreased significantly at 6 h after exposure to 1 h of acute restraint stress. Finally, the effects of spinal GABA_A receptor alteration on SIH were evaluated. These results showed that intrathecal administration of muscimol inhibited hyperalgesia induced by stress while bicuculline enhanced hyperalgesia in the control groups. Taken together, the present data reveal that GABA_A receptor β2 and β3 decrease following 1 h of acute restraint stress and may play a critical role in SIH.     

AMPA-evoked acetylcholine release from cultured spinal cord motoneurons and its inhibition by GABA and glycine

The release of [(3)H]acetylcholine evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and its inhibition mediated by GABA(A) and glycine receptors were studied in superfused cultured rat embryo spinal cord motoneurons prelabeled with [(3)H]choline. AMPA elicited tritium release, possibly representing [(3)H]acetylcholine release in a concentration-dependent manner. The release was external Ca(2+)-dependent and was sensitive to Cd(2+) ions, omega-conotoxin GVIA and omega-conotoxin MVIIC, but not to nifedipine, suggesting the involvement of N-, P/Q-, but not L-type Ca(2+) channels. The AMPA effect was insensitive to tetrodotoxin. The glutamate receptors involved are AMPA type since the AMPA-evoked [(3)H]acetylcholine release was blocked by LY303070 and was potentiated by the antidesensitizing agent cyclothiazide. Muscimol inhibited completely the AMPA effect on [(3)H]acetylcholine release; muscimol was potentiated by diazepam and antagonized by SR95531, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Glycine, acting at strychnine-sensitive receptors, also inhibited the effect of AMPA, but only in part. The inhibitory effects of muscimol and glycine are additive.We conclude that glutamate can act at AMPA receptors sited on spinal motoneurons to evoke release of acetylcholine. GABA and glycine, possibly released as cotransmitters from spinal interneurons, inhibit glutamate-evoked acetylcholine release by activating GABA(A) and glycine receptors on motoneurons.     

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA_A, GABA_C and glycine receptor Cl⁻ channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA_A, GABA_C and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I⁻ ions, driven by an external I⁻ concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration–responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels.     

Comparison of receptive field expansion produced by GABAB and GABAA receptor antagonists in raccoon primary somatosensory cortex

Recordings were made from 62 neurons in the forepaw representation of primary somatosensory cortex in anesthetized raccoons. Microiontophoretic administration of a specific GABA(B) receptor antagonist, CGP 55845, produced receptive field expansion in 74% of 46 neurons, in which it was tested first. The mean receptive field area was approximately doubled, with increases ranging from 12 to 500%. The GABA(B) receptor agonist baclofen reduced the receptive field in most (11 of 16) neurons, but increased the size in 4 neurons. Comparison of the effects of GABA(B) and GABA(A) antagonists in the same cells showed that GABA(A) receptor blockade produced greater expansion than GABA(B) blockade (144% vs 114%, respectively). Simultaneous administration of the two antagonists produced additional expansion in 16 of 25 neurons. There was no evidence of separate skin regions being masked by the two GABA receptor subtypes, as the larger expansion usually included the skin that was unmasked by the less effective drug. These results indicate that both GABA(B) and GABA(A) receptors play a role in shaping the normal receptive fields in somatosensory cortex.     

The specificity of strychnine as a glycine antagonist in the mammalian spinal cord

Summary An investigation was made of the influence of strychnine on the depression of the firing of spinal interneurones and Renshaw cells by glycine, GABA, nor-adrenaline and 3-hydroxytyramine. Administered electrophoretically or intravenously, strychnine blocks the effect of glycine more readily than that of the other depressants. Such specific antagonism of glycine action by relatively low concentrations of strychnine may be competitive in nature, but technical difficulties precluded a full assessment of the type of antagonism. The effects of relatively high concentrations of strychnine on the action of the other depressants probably result from interference with membrane permeability changes. The findings are considered to support previous proposals that glycine is the transmitter at spinal strychnine-sensitive inhibitory synapses.     

GABAA receptor antagonist bicuculline alters response properties of posteroventral cochlear nucleus neurons.

1. The role of GABAergic inhibitory inputs onto posteroventral cochlear nucleus (PVCN) neurons in the anesthetized chinchilla was investigated through iontophoretic application of the GABAA receptor agonist muscimol and the GABAA receptor antagonist bicuculline. The majority of the neurons studied displayed phasic temporal response patterns. 2. All the neurons were sensitive to bicuculline and displayed an increase in discharge rate, which was greatest during the post-onset portion of the response. Most of the tested neurons were also sensitive to muscimol, which appeared to mimic the putative effect of endogenous GABA. 3. Bicuculline reduced the average first-spike latency and the average variability of the first-spike latency. Muscimol had the opposite effect. 4. Bicuculline did not significantly alter the threshold but rather increased discharge rate at suprathreshold intensities. 5. The width of the excitatory response area was not significantly increased by application of bicuculline. The increase in discharge rate occurred within the units' excitatory response areas. 6. The shape of the rate-intensity functions was not altered by bicuculline application. 7. We conclude that GABAergic inhibitory inputs control the post-onset discharge rate of some PVCN neurons. They may suppress tonic activity, resulting in more phasic discharge patterns.     

Changes in extracellular amino acid neurotransmitters produced by focal cerebral ischemia

Excitatory amino acids (EAAs) have been implicated in the pathophysiology of cellular injury after brain ischemia. Changes in extracellular levels of amino acids in rat cerebral cortex after permanent proximal middle cerebral artery (MCA) occlusion were examined using microdialysis. Significant increases were found in dialysate concentrations of glutamate, aspartate and gamma-aminobutyric acid (GABA) from the ischemic cortex during the first 90 min after MCA occlusion compared to pre-ischemic concentrations and contralateral hemispheric controls. Total tissue levels of these amino acids in the infarcted hemisphere 90 min after onset of ischemia were not different from the contralateral hemisphere. These results are consistent with the hypothesis that the release of EAAs may contribute to tissue damage in focal cerebral ischemia.     

Modulation of the GABAA receptor by steroids

The functioning of the GABA_A receptor-chloride ion channel is subject to several modulatory influences. Facilitation by benzodiazepines and barbiturates and antagonism by picrotoxin involve separate sites on the GABA_A protein that are each distinct from the γ-aminobutyric acid (GABA) recognition site. To these can now be added another site for the facilitatory action of hypnotic steroids. A clear steroid structure-activity profile has been described and among the most active steroids are some naturally occurring reduced metabolites of progesterone and deoxycorticosterone. In many respects, these steroids have a barbiturate-like action but they do not share a common recognition site with the barbiturates.     

Characterization of type C virus induction by amino acid analogs

Several amino acid analogs were tested for their ability to induce type C virus from a cloned Balb/c mouse cell line transformed by Kirsten sarcoma virus. O-Methylthreonine and hydroxynorvaline, analogs of isoleucine and threonine, respectively, were found to induce type C virus with the host range of Balb:virus-2. Type C virus was also activated by lysine deprivation. When either O-methylthreonine or hydroxynorvaline was used in conjunction with the arginine analog canavanine, the induction equalled or surpassed the maximum induction by cycloheximide. Using synchronized cells, induction of virus by canavanine was found to be cell-cycle dependent. Depriving cells of certain single amino acids alone or in combination with specific analogs markedly reduced protein synthesis without inducing virus. The results suggest that a general reduction in protein synthesis cannot account for the induction and imply that the inducing analogs may affect the functionality or degradation of proteins involved in the regulation of virus expression.     

Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor.

Recently we demonstrated that [3H]dehydroepiandrosterone sulfate binds specifically to two populations of sites in rat brain membranes [Majewska et al. (1990) Eur. J. Pharmac. 189, 307-315]. As an extension of this work, we studied the biochemical and pharmacological properties of [3H]dehydroepiandrosterone sulfate binding to brain membranes and the effects of dehydroepiandrosterone sulfate on GABA-induced currents in cultured neurons. [3H]Dehydroepiandrosterone sulfate binding depended upon incubation time, pH, protein concentration, and incubation temperature. Thermal denaturation or pretreatment of the membranes with protease or phospholipase A2 reduced the binding by 54-85%. The higher affinity [3H]dehydroepiandrosterone sulfate binding sites appeared to be associated with protein and with the GABAA receptor complex. Among substances known to interact with the GABAA receptor complex, pregnenolone sulfate, pentobarbital, and phenobarbital inhibited the binding of [3H]dehydroepiandrosterone sulfate. High micromolar concentrations of dehydroepiandrosterone sulfate inhibited [3H]muscimol and [3H]flunitrazepam binding to rat brain membranes, primarily by reducing the binding affinities. Dehydroepiandrosterone sulfate also produced a concentration-dependent block of GABA-induced currents in cultured neurons from ventral mesencephalon (IC50 = 13 +/- 3 microM). The results of this study are consistent with an action of dehydroepiandrosterone sulfate as a negative noncompetitive modulator of the GABAA receptor. Because concentrations of dehydroepiandrosterone sulfate in the brain undergo physiological variations, this neurosteroid may play a vital role in regulation of neuronal excitability in the central nervous system.