Glycine And Γ-aminobutyric Acid Research Articles

Neurosteroids as Selective Inhibitors of Glycine Receptor Activity: Structure-Activity Relationship Study on Endogenous Androstanes and Androstenes.

The ability of androstane and androstene neurosteroids with modifications at C-17, C-5, and C-3 (compounds 1-9) to influence the functional activity of inhibitory glycine and γ-aminobutyric acid (GABA) receptors was estimated. The glycine- and GABA-induced chloride current (IGly and IGABA) were measured in isolated pyramidal neurons of the rat hippocampus and isolated rat cerebellar Purkinje cells, correspondingly, using the patch-clamp technique. Our results demonstrate that all the nine neurosteroids display similar biological activity, namely, they strongly inhibited IGly and weakly inhibited IGABA. The threshold concentration of neurosteroids inducing effects on IGly was 0.1 μM, and for effects on IGABA was 10–50 μM. Moreover, our compounds accelerated desensitization of the IGly with the IC50 values varying from 0.12 to 0.49 μM and decreased the peak amplitude with IC50 values varying from 16 to 22 μM. Interestingly, our study revealed that only compounds 4 (epiandrosterone) and 8 (dehydroepiandrosterone) were able to cause a significant change in IGABA in 10 μM concentration. Moreover, compounds 3 (testosterone), 5 (epitestosterone), 6 (dihydroandrostenedione), and 9 (etiocholanedione) did not modulate IGABA up to the concentration of 50 μM. Thus, we conclude that compounds 3, 5, 6, and 9 may be identified as selective modulators of IGly. Our results offer new avenues of investigation in the field of drug-like selective modulators of IGly.

Structure-Function Relationships of Glycine and GABAA Receptors and Their Interplay With the Scaffolding Protein Gephyrin.

Glycine and γ-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ∼93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission.

The endogenous agonist, β-alanine, activates glycine receptors in rat spinal dorsal neurons

β-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether β-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of β-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. β-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately −70 mV, which was close to the equilibrium potential of Cl−. Pharmacological analysis revealed that β-alanine activates glycine receptors, but not GABAA receptors. These results suggest that β-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl− channels through glycine receptors. Our findings raise the possibility that β-alanine may modulate pain sensation through glycine receptors.

Distinct development of the glycinergic terminals in the ventral and dorsal horns of the mouse cervical spinal cord

In the spinal cord, glycine and γ-amino butyric acid (GABA) are inhibitory neurotransmitters. However, the ontogeny of the glycinergic network remains unclear. To address this point, we examined the developmental formation of glycinergic terminals by immunohistochemistry for glycine transporter 2 (GlyT2), a marker of glycinergic terminals, in developing mouse cervical spinal cord. Furthermore, the developmental localization of GlyT2 was compared with that of glutamic acid decarboxylase (GAD), a marker of GABAergic terminals, and vesicular GABA transporter (VGAT), a marker of inhibitory terminals, by single and double immunolabeling. GlyT2-positive dots (glycinergic terminals) were first detected in the marginal zone on embryonic day 14 (E14). In the ventral horn, they were detected at E16 and increased in observed density during postnatal development. Until postnatal day 7 (P7), GAD-positive dots (GABAergic terminals) were dominant and GlyT2 immunolabeling was localized at GAD-positive dots. During the second postnatal week, GABAergic terminals markedly decreased and glycinergic terminals became dominant. In the dorsal horn, glycinergic terminals were detected at P0 in lamina IV and P7 in lamina III and developmentally increased. GlyT2 was also localized at GAD-positive dots, and colocalizing dots were dominant at P21. VGAT-positive dots (inhibitory terminals) continued to increase until P21. These results suggest that GABAergic terminals first appear during embryonic development and may often change to colocalizing terminals throughout the gray matter during development. The colocalizing terminals may remain in the dorsal horn, whereas in the ventral horn, colocalizing terminals may give rise to glycinergic terminals.

The effects of avermectin on amino acid neurotransmitters and their receptors in the pigeon brain

The objective of this study was to examine the effects of avermectin (AVM) on amino acid neurotransmitters and their receptors in the pigeon brain. Four groups two-month-old American king pigeons (n=20/group) were fed either a commercial diet or an AVM-supplemented diet (20mg/kg·diet, 40mg/kg·diet, or 60mg/kg·diet) for 30, 60, or 90days. The contents of aspartic acid (ASP), glutamate (GLU), glycine (GLY), and γ-aminobutyric acid (GABA) in the brain tissues were determined using ultraviolet high-performance liquid chromatography (HPLC). The expression levels of the GLU and GABA receptor genes were analyzed using real-time quantitative polymerase chain reaction (qPCR). The results indicate that AVM exposure significantly enhances the contents of GABA, GLY, GLU, and ASP in the cerebrum, cerebellum, and optic lobe. In addition, AVM exposure increases the mRNA expression levels of γ-aminobutyric acid type A receptor (GABAAR), γ-aminobutyric acid type B receptor (GABABR), N-methyl-d-aspartate 1 receptor (NR1), N-methyl-d-aspartate 2A receptor (NR2A), and N-methyl-d-aspartate 2B receptor (NR2B) in a dose- and time-dependent manner. Moreover, we found that the most damaged organ was the cerebrum, followed by the cerebellum, and then the optic lobe. These results show that the AVM-induced neurotoxicity may be associated with its effects on amino acid neurotransmitters and their receptors. The information presented in this study will help supplement the available data for future AVM toxicity studies.

Cumulus Cells Gene Expression Profiling in Terms of Oocyte Maturity in Controlled Ovarian Hyperstimulation Using GnRH Agonist or GnRH Antagonist

In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

Regulation of spinal interneuron differentiation by the paracrine action of glycine.

Glycine and γ-aminobutyric acid (GABA) are depolarizing during early development but the purpose is unclear. We tested the effect of altering glycine signaling in zebrafish embryos by overexpressing the potassium-chloride co-transporter type 2 (KCC2) to reverse the chloride gradient or by blocking glycine receptors with strychnine or by selectively knocking down the embryonic glycine receptor (GlyR KD). Using a variety of markers we observed in all three cases a reduction of all types of spinal interneuron populations examined, indicating that glycine modulates their overall differentiation rather than choice of cell fate. Other cell populations (motor, sensory, and glial cells) were unaffected. As glycine appeared to act preceding neural and synaptic development, we examined the bandoneon (beo) mutant in which glycine receptors are functional but not clustered at synapses. Neural populations in beo embryos appeared normal, suggesting a paracrine action of circulating glycine in promoting interneuron differentiation.

Density functional studies of endosulphan and its interaction with glycine and GABA#

Density functional theoretic (DFT) methods are employed to study the interactions between endosulphan and two amino acids, namely glycine and γ-aminobutyric acid (GABA). Two conformers of each isomer α- and β-endosulphan are considered in the study. The DFT methods B3LYP, M05, M05–2X, M06 and M06–2X in conjunction with the basis set 6–31+ +G** are used. The complexes of α- and β-endosulphan with amino acids are stabilized by a strong hydrogen bond. In addition, there are several weak C–H⋯O interactions present between the two moieties. Among the DFT methods used, M06–2X method shows the highest stabilization energy for all the complexes. The M06–2X/6–31+ +G** method predicts that among the four conformers of endosulphan, the α conformer in which the S=O points up, forms the most stable complex with both glycine and GABA, with stabilization energies −15.24 kcal/mol and −14.39 kcal/mol, respectively. The β conformer in which the S=O points down, forms the least stable complex with both amino acids with stabilization energies −7.14 and −7.85 kcal/mol, respectively. Interactions between various conformers of the organo-chloro pesticide endosulphan and the amino acids (glycine and GABA) have been studied using the MP2 and density functional methods. The results indicated that endosulphan binds strongly with both glycine and GABA. In addition to the one strong hydrogen bond, several weak interactions (C–H…O and C–H...N) participate in stabilizing the complex.

Distribution of glycine immunoreactivity in the brain of the Siberian sturgeon (Acipenser baeri): Comparison with γ-aminobutyric acid

Glycine and γ-aminobutyric acid (GABA) are the main inhibitory neurotransmitters in the central nervous system (CNS) of vertebrates. Studies on the distribution of glycinergic neurons and fibers have been carried out mainly in rodents and lampreys. With the aim of discovering more about the early evolution of this system in vertebrates, we analyzed the distribution of glycine-immunoreactive (Gly-ir) neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), by use of immunohistochemical techniques. We also compared the distribution of glycine and GABA by the use of double-immunofluorescence techniques and confocal microscopy. Our results revealed the presence of Gly-ir cells in different regions of the CNS, such as olfactory bulbs, preoptic area, hypothalamus, thalamus, pretectum, optic tectum, tegmentum and rostral spinal cord, although most of the Gly-ir cells and the most intensely immunoreactive cells were located in the rhombencephalon, mainly in the octavolateral area and reticular formation. In addition, coronet cells of the basal hypothalamus and saccus vasculosus were Gly-ir. Glycinergic fibers coursed along most brain regions and were more abundant in the thalamus, hypothalamus, optic tectum, tegmentum, isthmic region, and basal rhombencephalon. The Mauthner cell perikaryon was richly innervated by Gly-ir boutons, as reported for teleosts. With regard to the colocalization of glycine and GABA, double-immunoreactive cells were located mainly in the rhombencephalon. The results enable us to conclude that the distribution of glycine in the sturgeon brain is more similar to that observed in lampreys than that observed in mammals.

Pharmacology of Intracisternal or Intrathecal Glycine, Muscimol, and Baclofen in Strychnine-induced Thermal Hyperalgesia of Mice

Glycine and γ-aminobutyric acid (GABA) are localized and released by the same interneurons in the spinal cord. Although the effects of glycine and GABA on analgesia are well known, little is known about the effect of GABA in strychnine-induced hyperalgesia. To investigate the effect of GABA and the role of the glycine receptor in thermal hyperalgesia, we designed an experiment involving the injection of muscimol (a GABAA receptor agonist), baclofen (a GABAB receptor agonist) or glycine with strychnine (strychnine sensitive glycine receptor antagonist). Glycine, muscimol, or baclofen with strychnine was injected into the cisterna magna or lumbar subarachnoidal spaces of mice. The effects of treatment on strychnine-induced heat hyperalgesia were observed using the pain threshold index via the hot plate test. The dosages of experimental drugs and strychnine we chose had no effects on motor behavior in conscious mice. Intracisternal or intrathecal administration of strychnine produced thermal hyperalgesia in mice. Glycine antagonize the effects of strychnine, whereas, muscimol or baclofen does not. Our results indicate that glycine has anti-thermal hyperalgesic properties in vivo; and GABA receptor agonists may lack the binding abilities of glycine receptor antagonists with their sites in the central nervous system.

Expression of the glycinergic system during the course of embryonic development in the mouse spinal cord and its co-localization with GABA immunoreactivity

To understand better the role of glycine and gamma-aminobutyric acid (GABA) in the mouse spinal cord during development, we previously described the ontogeny of GABA. Now, we present the ontogeny of glycine-immunoreactive (Gly-ir) somata and fibers, at brachial and lumbar levels, from embryonic day 11.5 (E11.5) to postnatal day 0 (P0). Spinal Gly-ir somata appeared at E12.5 in the ventral horn, with a higher density at the brachial level. They were intermingled with numerous Gly-ir fibers reaching the border of the marginal zone. By E13.5, at the brachial level, the number of Gly-ir perikarya sharply increased throughout the whole ventral horn, whereas the density of fibers declined in the marginal zone. In the dorsal horn, the first Gly-ir somata were then detected. From E13.5 to E16.5, at the brachial level, the density of Gly-ir cells remained stable in the ventral horn, and after E16.5 it decreased to reach a plateau. In the dorsal horn, the density of Gly-ir cells increased, and after E16.5 it remained stable. At the lumbar level, maximum expression was reached at E16.5 in both the ventral and dorsal horn. Finally, the co-localization of glycine and GABA was analyzed, in the ventral motor area, at E13.5, E15.5, and E17.5. The results showed that, regardless of developmental stage studied, one-third of the stained somata co-expressed GABA and glycine. Our data show that the glycinergic system matures 1 day later than the GABAergic system and follows a parallel spatiotemporal evolution, leading to a larger population of glycine cells in the ventral horn.

Characterization of the glycinergic input to bipolar cells of the mouse retina

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory transmitters of the mammalian retina, and bipolar cells receive GABAergic and glycinergic inhibition from multiple amacrine cell types. Here we evaluated the functional properties and subunit composition of glycine receptors (GlyRs) in bipolar cells. Patch-clamp recordings were performed from retinal slices of wild-type, GlyRalpha1-deficient (Glra1(spd-ot)) and GlyRalpha3-deficient (Glra3(-/-)) mice. Whole-cell currents following glycine application and spontaneous inhibitory postsynaptic currents (IPSCs) were analysed. During the recordings the cells were filled with Alexa 488 and, thus, unequivocally identified. Glycine-induced currents of bipolar cells were picrotoxinin-insensitive and thus represent heteromeric channels composed of alpha and beta subunits. Glycine-induced currents and IPSCs were absent from all bipolar cells of Glra1(spd-ot) mice, indicating that GlyRalpha1 is an essential subunit of bipolar cell GlyRs. By comparing IPSCs of bipolar cells in wild-type and Glra3(-/-) mice, no statistically significant differences were found. OFF-cone bipolar (CB) cells receive a strong glycinergic input from AII amacrine cells, that is preferentially based on the fast alpha1beta-containing channels (mean decay time constant tau = 5.9 +/- 1.4 ms). We did not observe glycinergic IPSCs in ON-CB cells and could elicit only small, if any, glycinergic currents. Rod bipolar cells receive a prominent glycinergic input that is mainly mediated by alpha1beta-containing channels (tau = 5.5 +/- 1.6 ms). Slow IPSCs, the characteristic of GlyRs containing the alpha2 subunit, were not observed in bipolar cells. Thus, different bipolar cell types receive kinetically fast glycinergic inputs, preferentially mediated by GlyRs composed of alpha1 and beta subunits.

Cadmium toxicity in synaptic neurotransmission in the brain

Chronic exposure to cadmium causes central nervous system disorders, e.g. olfactory dysfunction. To clarify cadmium toxicity in synaptic neurotransmission in the brain, the movement of cadmium in the synapses was examined using in vivo microdialysis. One and 24 h after injection of 109CdCl 2 into the amygdala of rats, 109Cd release into the extracellular space was facilitated by stimulation with high K +, suggesting that cadmium taken up by amygdalar neurons is released into the synaptic clefts in a calcium- and impulse-dependent manner. Moreover, to examine the action of cadmium in the synapses, the amygdala was perfused with artificial cerebrospinal fluid containing 10–30 μM CdCl 2. The release of excitatory neurotransmitters, i.e. glutamate and aspartate, into the extracellular space was decreased during perfusion with cadmium, while the release of inhibitory neurotransmitters, i.e. glycine and γ-amino butyric acid (GABA), into the extracellular space was increased during the period. These results suggest that cadmium released from the amygdalar neuron terminals affect the degree and balance of excitation–inhibition in synaptic neurotransmission.

Enhancement of Glycine Receptor Function by Ethanol Is Inversely Correlated with Molecular Volume at Position α267

Glycine and gamma-aminobutyric acid (GABA)A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the alpha1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-Ralpha1 and gamma-aminobutyric acid rho1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-Ralpha1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-Ralpha1 (Ser-267 --> Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position alpha267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%)of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.

Roles of monoaminergic, glycinergic and GABAergic inhibitory systems in the spinal cord in rats with peripheral mononeuropathy

The current study was designed to determine if the monoaminergic descending inhibitory system and the glycinergic and GABAergic inhibitory systems were activated in the spinal cord in the presence of peripheral mononeuropathy produced by loose ligatures around the common sciatic nerve. The time course of withdrawal latencies to thermal stimuli were assayed in lesioned and sham-operated rats. The levels of monoamines (serotonin; 5-HT, noradrenaline, and dopamine), glycine and γ-aminobutyric acid (GABA) in the dorsal half of the spinal cord were measured using HPLC with electrochemical detection. Furthermore, on day 7 after nerve ligation, intrathecal methysergide, yohimbine, strychnine or bicuculline was administered in order to investigate the roles of these inhibitory neuromodulators in this pathological pain state. The levels of 5-HT and noradrenaline significantly increased in both ipsi- and contralateral sides of the dorsal half of the lumbar spinal cord in the lesioned, but not sham-operated animals. The levels of glycine and GABA in the ipsilateral dorsal half of the spinal cord increased significantly and were significantly higher than in the contralateral side. Intrathecal antagonists of 5-HT, noradrenaline, glycine and GABA produced enhancement of the magnitude of hyperalgesia on the lesioned hindpaw. We also examined the effects of four daily single treatments with intrathecal MK-801 beginning 15 min prior to nerve ligation on the development of thermal hyperalgesia and on the contents of the neuromodulators in the ligation model. MK-801 treatment effectively abolished the increases in 5-HT, noradrenahne, glycine and GABA levels as well as preventing the development of hyperalgesia. The results of the present study suggest that the pathological pain state activates or increases the activity of these inhibitory systems.

Effects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization.

Hypothermia slows the increase in extracellular excitatory amino acid (EAA) concentrations during temporary cerebral ischemia. However, it is unclear whether hypothermia slows the rate of EAA release or just delays the time until the first sharp increase (which occurs coincident with terminal depolarization). Pericranial temperatures were adjusted to 38 degrees C, 34 degrees C, 31 degrees C, or 25 degrees C in halothane-anesthetized rats. The cortical DC voltage was recorded from a glass microelectrode while the cortical concentrations of glutamate, aspartate, glycine, and gamma-aminobutyric acid (GABA) were measured by microdialysis. A cardiac arrest was induced with intravenous KCl, and the times until electroencephalograph isoelectricity and terminal depolarization were recorded. Dialysate concentrations of the four compounds were measured at 10, 20, and 30 minutes after depolarization. The times to isoelectricity and depolarization varied inversely with temperature; depolarization time increased from 70 +/- 9 seconds at 38 degrees C (mean +/- SD) to 294 +/- 34 seconds at 25 degrees C. The dialysate concentrations of all four compounds increased during ischemia, and the rate of increase was inhibited by cooling. After 30 minutes of ischemia, glutamate concentration in 38 degrees C animals was 58.4 +/- 31.8 mumol/L; this decreased to 15.9 +/- 8.4 mumol/L at 25 degrees C. The magnitude of the effects of temperature on amino acid release differed with the compound measured. For glutamate, the calculated Q10 was 3.63. Corresponding values for aspartate and glycine were 3.68 and 1.95, respectively. By contrast, Q10 for GABA release was 6.31, indicating greater sensitivity to cooling. These results suggest that effects of hypothermia on EAA concentrations during cerebral ischemia may be the result of both a delay until initial EAA release as well as a direct effect of temperature on the rate of amino acid release. The observed temperature effects are more consistent with carrier-mediated processes controlling EAA release.

A comparison between the stimulated and paroxysmal release of endogenous amino acids from rat cerebellar, striatal and hippocampal slices: a manifestation of spreading depression?

Spreading depression, which can be evoked by a variety of stimuli both in vitro and in vivo, is associated with profound changes in extracellular ion concentrations and enhanced release of neurotransmitter amino acids. We have observed a transient spontaneous release of amino acids in slice preparations obtained from rat cerebellum, striatum and hippocampus; this phenomenon has similar properties to stimulus-evoked spreading depression. Aspartate, glutamate, glutamine, serine, glycine and γ-aminobutyric acid (GABA) release were potentiated during these episodes in all three brain regions, with a variable effect upon taurine release. When compared to glutamate release, a consistently high release of aspartate, glycine and serine was observed. Amino acid release, evoked by whole slice depolarization using veratridine (10–25 μM) or elevated potassium (35–60 mM) consistently enhanced glutamate release, and to a lesser extent aspartate release, but had negligible effect upon the other amino acids. Thus, the release profiles for spontaneous and depolarization-evoked release are markedly different. We suggest that the spontaneous release observed in brain slices represents a spreading depression-like phenomenon; the putative roles of the amino acids are discussed.

Opisthotonos following propofol: A nonepileptic perspective and treatment strategy

In this report of opisthotonos during recovery from propofol anaesthesia, we relate clinical observations with scientific considerations, and propose a strategy for treatment of this rare side effect. Following a brief operative procedure, a healthy 29-yr-old woman developed recurrent opisthotonos while recovering from anaesthesia with alfentanil, propofol, and nitrous oxide. In contrast to accumulating reports, the patient remained conscious during each episode of back extension and retrocollis. The preservation of consciousness and similarities to strychnine-induced opisthotonos suggest to us that the mechanism may have a brainstem and spinal origin. Recent investigations show that propofol potentiates the inhibitory transmitters glycine and gamma-aminobutyric acid (GABA) which would enhance spinal inhibition during anaesthesia. Postanaesthetic opisthotonos, however, may be due to a propofol-induced tolerance to inhibitory transmitters. This rebound phenomenon would lead to an acute, enduring refractoriness in inhibitory pathways of the brainstem and spinal cord, resulting in increased activity of extensor motoneurons. We recommend a therapeutic strategy that restores inhibition by glycine and GABA at multiple sites; the preferred therapeutic agents would be diazepam and physostigmine. The episodes are usually short-lived, but two of the reviewed 17 patients developed recurrent retrocollis for four and 23 days following antiepileptic drug therapy. Since high doses of phenytoin and carbamazepine can result in opisthotonos, we recommend that anticonvulsants be reserved for postanaesthetic patients with electroencephalographic evidence of seizure activity.

An Experimental Model Combining Microdialysis with Electrophysiology, Histology, and Neurochemistry for Exploring Mechanisms of Secondary Damage in Spinal Cord Injury: Effects of Potassium

An experimental model for characterizing effects of agents suspected to cause damage secondary to spinal cord injury is presented. Microdialysis was used to administer candidate damaging agents and to sample the release of other substances in response to administered agents. Damage was assessed by monitoring the amplitudes of evoked potentials during candidate administration and by postmortem histologic examination. This approach enabled the correlation of electrophysiologic, histologic, and neurochemical parameters in the same experiment. Potassium was chosen as a candidate damaging agent, because (1) in high extracellular concentrations it destroys neurons and (2) its extracellular concentration rises substantially following impact injury to the spinal cord. A technique using parallel administering and collecting microdialysis fibers was developed to estimate the in vivo concentration of K+ outside the administering fiber. Administration of 100 mM KCl, 50 mM K2HPO4, or 100 mM potassium glucuronate in artificial cerebrospinal fluid blocked electrical conduction and destroyed cell bodies. Release of the neurotransmitter amino acids aspartate, glutamate, glycine, and gamma-aminobutyric acid (GABA) and the possible neurotransmitter taurine were dramatically increased by administration of 100 mM KCl. Levels of non-neurotransmitter amino acids increased to lesser degrees. Impairment of conduction, destruction of cell bodies, and release of excitotoxins were all consistent with elevated K+ serving as a secondary damaging agent in spinal cord injury.