Long-lasting effects of audiogenic seizures on neurotransmitter amino acids in Rb mice

The existence of long-lasting (15–18 h) alterations of neurotrasmitter amino acid levels following a single or repeated acoustic stimulations in audiogenic seizure-prone Rb1 and Rb2 mice and suizure-resistant Rb3 mice were investigated. The levels of glutamate, aspartate, glycine, taurine, and of some of their precursors: glutamine and serine were determined. Fourteen brain areas were examined. Alterations were found only in 6 brain areas (pons, olfactory bulbs, superior colliculus, inferior colliculus, olfactory tubercles and raphe). Most frequent occuring changes were observed in pons and olfactory tubercles. These changes concerned mainly the excitatory amino acids, glutamate, and aspartate. Alterations of taurine, glycine and serine were also recorded.Abbreviations GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspertate - Glu glutamate - Gln glutamine - Ser serine - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Th thalamus - Hi hippocampus - A amygdala - SC superior colliculus - IC inferior colliculus - FC frontal cortex - C cerebellum - P pons medulla - Ra raphe - AA neurotransmitter amino acids - I inhibitory - E excitatory - SSL steady-state level Plesant memories of Lawrence Austin's sojourn in my group at Strasbourg gather upon me when I dedicate this article on this occasion for the contribution that Lawrence Austin has made for the cause of neurochemical researchers.     

Age-Dependent Changes in Brain GABA Turnover Rates in Two Inbred Strains of Mice

gamma-Aminobutyric acid (GABA) steady-state levels and turnover rates have been determined in 15 brain areas of 21-day- and 3-month-old DBA/2J (DBA) and C57B1/6J (C57) mice. These two inbred strains differ by their susceptibility to audiogenic seizures; moreover, the involvement of GABAergic neurotransmission has been suggested in the control of this behavior. Turnover rates are generally higher at 21 days than at 3 months of age. There are few significant differences in the GABA steady-state levels between 21-day-old seizure-prone DBA mice when compared with seizure-resistant C57 mice. In the DBA mice, the steady-state level is higher in the olfactory bulbs and lower in the posterior colliculus and the olfactory tubercles than in the C57 mice. Although there are some significant differences in GABA turnover rates and steady-state levels, intra or inter strains, it is difficult to correlate directly these differences with seizure susceptibility.     

Amino acid neurotransmitter alterations in three sublines of Rb mice differing by their susceptibility to audiogenic seizures

The levels of inhibitory amino acids (Tau, Gly), or excitatory amino acids (Glu, Asp) and Gln, precursor of GABA, have been determined, under resting conditions, in 17 brain areas of 3 sublines of inbred Rb mice displaying different responses to an acoustic stimulus. Rb1 mice were clonictonic seizure-prone, Rb2 mice were clonic seizure-prone and Rb3 mice were seizure resistant. Profile of distribution in the brain of each one of these amino acids differed. Maximum to minimum level ratio was higher for Tau (3.8) than for Glu or Asp or Gln (2). The level of Gly was similar in 13 out of the 17 areas examined. Multiple inter-subline differences were recorded for each amino acid. These differences have been analyzed considering the seizure susceptibility or severity of the three Rb sublines. Common lower levels (approximately –20%: Rb1/Rb3, Rb2/Rb3) of Gln in Temporal Cortex may be implicated in seizure susceptibility. Seirure severity (Rb1/Rb2) seems to correlate, in some areas, with additional lower amounts of GABA already reported and, to a lower extent, of Asp (–19% in striatum, inferior colliculus and cerebellum), of Tau and Gly; a tendency for a rise in Gln content was observed in certain others (10–20% in olfactory bulb, thalamus, hypothalamus, substantia nigra, and frontal, temporal and occipital cortex). The data and correlations recorded provide guidelines for further investigations for synaptosomal and metabolic alterations in the three sublines of the same strain of Rb mice.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspartate - Glu glutamate - Gln glutamine - GEPR genetically epilepsy-prone rat - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Hi hippocampus - Th thalamus - A amygdala - SC superior colliculus - IC interior colliculus - SN substantia nigra - FCx frontal cortex - TCx temporal cortex - OCx occipital cortex - C cerebellum - P pons - Ra raphe     

Long lasting effects of audiogenic seizures on synaptosomal neurotransmitter amino acids in Rb mice

Long lasting alterations of synaptosomal amino acid neurotransmitters following a single or several audiogenic seizures and/or acoustic stimulations were investigated in six brain areas-olfactory bulbs (OB), amygdala (A), hippocampus (Hi), cerebellum (C), inferior colliculus (IC), ponsmedulla (P)- of three sublines of Rb mice: audiogenic seizure-prone Rb1 and Rb2, seizure-resistant Rb3. Changes in the synaptosomal levels of aspartate (Asp), glutamate (Glu), taurine (Tau), 4-amino butyrate (GABA), glycine (Gly) and some closely related precursors, serine (Ser) and glutamine (Gln), were recorded 15–18 hours after a single or multiple acoustic stimulations. Changes were more frequent, or larger, after polystimulation. Some alterations appeared to be attributable to an effect of the acoustic stress.In both seizure-prone sublines, after a single or repeated seizures, an increase in synaptosomal Asp was observed in IC. Decreases in Asp and Tau in OB and Ser in A, an increase in Gln in IC were only observed after repeated seizures, in Rb1 and Rb2 mice.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Ser serine - Asp aspartate - Glu glutamate - Gln glutamine - OB olfactory bulbs - A amygdala - Hi hippocampus - C cerebellum - IC interior colliculus - P pons Professeur Paul Mandel passed away on 6th October, 1992Special issue dedicated to Dr. Bernard W. Agranoff.     

Involvement of synaptosomal neurotransmitter amino acids in audiogenic seizure-susceptibility and-severity of Rb mice

The involvement of synaptosomal neurotransmitter amino-acids in seizure susceptibility and seizure severity was explored. The amino-acid contents of brain synaptosomes were determined in three sublines of Rb mice differing in their response to an acoustic stimulus: Rb1, clonic-tonic seizure-prone, Rb2, clonic seizure-prone, and Rb3, seizure-resistant. Synaptosomes were prepared from 6 brain areas considered to be involved in seizure activity: olfactory bulbs, amygdala, inferior colliculus, hippocampus, cerebellum, pons-medulla. The steady-state levels of GABA and glycine (Gly), inhibitory amino-acids, of taurine (Tau), an inhibitory neurotransmitter of neuromodulator, of aspartate (Asp) and glutamate (Glu), excitatory amino-acids, as well as of serine (Ser) and glutamine (Gln), two precursors of neurotransmitter amino-acids, were determined by HPLC. Low levels of Tau, GABA, and Ser in hippocampus, Gly in amygdala, Glu in hippocampus, inferior colliculus and pons, Gln and Asp in inferior colliculus appeared to correlate with seizure-susceptibility. GABA and Asp in olfactory bulb, Gln in amygdala, hippocampus and pons, ser in olfactory bulb and pons, appeared to be associated either with seizure-severity or-diversity. A strong involvement of hippocampus (Tau, GABA, Ser, Glu, and Gln) and inferior colliculus (Asp, Glu, Gln) in audiogenic seizure-susceptibility, and of olfactory bulb (GABA, Asp) in seizure-severity and/or-diversity is suggested.Special issue dedicated to Dr. Alan N. Davison.     

Brain γ-Aminobutyric Acid Turnover Rates After Spontaneous Chronic Ethanol Intake and Withdrawal in Discrete Brain Areas of C57 Mice

The effect of 4 weeks of spontaneous chronic ethanol intake in drinking water and then ethanol withdrawal on the gamma-aminobutyric acid (GABA) steady-state levels and turnover rates was investigated in 15 brain areas of C57 Bl/6J alcohol-preferring mice. These mice did not display typical ethanol withdrawal convulsions. There was no statistically significant difference in the brain GABA steady-state levels among the control group, chronic ethanol-treated mice, and mice after ethanol withdrawal. In contrast, chronic ethanol treatment induced significant variations in GABA turnover rate, as measured by gabaculine-induced accumulation of GABA, in eight of 15 areas examined versus a decrease in seven brain areas [cerebellum (-29%), amygdala (-28%), olfactory tubercles (-24%), septum (-24%), striatum (-53%), frontal cortex (-21%), and hippocampus (-24%)]; an increase in turnover rate in the posterior colliculus (100%) was observed. At 26 h after ethanol withdrawal, in the seven areas in which GABA turnover rate decreased after spontaneous chronic ethanol intake, a return to the initial control value was observed; in the posterior colliculus, the turnover rate did not change, remaining higher than the control value. This persisting alteration of GABA turnover rate may be related to the absence of the ethanol withdrawal syndrome in the C57 mouse strain.     

Long lasting effect of a single audiogenic seizure on GABA turnover rates and steady-state levels

GABA turnover rates (TOR) and steady-state levels (SSL) were determined, 16–18 h after a single acoustic stimulation, in 15 brain areas of 3 mouse sublines. Each subline differs in its response to an acoustic stimulation (Rb1 mice are clonic-tonic seizure-prone, Rb2: clonic seizure-prone, Rb3: seizure-resistant). TOR and SSL were compared to those of unstimulated control mice and to those of repeatedly stimulated mice of the same subline. Following a single acoustic stimulation long-lasting alterations of GABA metabolism, mainly large alterations of GABA TOR, are observed. Most of the effects elicited after repeated stimulations, either on SSL or TOR, are not those of the last stimulation and repeated seizures (and/or stimulations) strengthen the effect of a single one. It appears that, for each of the Rb sublines, a specific and quite simple profile of the alterations of GABA metabolism in response to a single or repeated audiogenic seizures (and/or stimulations) can be given. The global analysis through the correlation of GABA TOR and SSL gives an indication that the alterations of the parameters of the correlation observed are to be allocated to the audiogenic seizures. Furthermore the tonic and clonic components of the audiogenic seizures can be distinguished.     

Correlation between the steady state and turnover of gamma-aminobutyric acid during brain maturation in mice

In 2 inbred strains of mice (C57Bl/6J, DBA/2J) in 15 areas, the in vivo GABA turnover rates are significantly correlated with the GABA steady-state levels in 21 day-old mice. In 3 month-old mice the correlation stands only in some areas, the same ones in the 2 strains: olfactory bulbs, frontal cortex, septum, amygdala, hypothalamus, hippocampus, cerebellum. Moreover, the turnover rates decrease sharply with age.     

Effect of repeated convulsive seizures on brain GABA levels

Repeated audiogenic seizures (4 times a day for 14 days), in genetically selected sensitive mice, induce a significant decrease in GABA level in the following brain areas: nucleus caudatus, posterior colliculus, occipital and frontal cortex, cerebellum, substantia nigra, hippocampus, amygdala, and temporal cortex. No variations were observed in olfactory bulbs, pons medulla, hypothalamus, thalamus, or cochlear area.     

Brain monoamines following castration of aggressive muricidal rats

The effect of castration on the levels of brain monoamines and their metabolites has been investigated in rats which became or did not become muricidal following long-term isolation. Fourteen brain areas were explored: olfactory bulbs (OB), olfactory tubercles (OT), septum (Se), striatum (Sr), amygdala (A), thalamus (Th), hypothalamus (Hy), hippocampus (Hi), superior colliculus (SC), inferior colliculus (IC), raphe (Ra), pons-medulla (PM), frontal cortex (FC), temporal cortex (TC) and parietal cortex (PC). Except in the raphe of non muricidal rats and in the striatum of muricidal animals, all other areas examined demonstrate some changes of monoamines neurotransmitter or their metabolites after castration. The strongest changes, always increases, were found in the thalamus. In several brain areas, the changes occurring after castration, differ quantitatively and qualitatively in muricidal and non-muricidal rats.Special issue dedicated to Dr. Claude Baxter.Prof. P. Mandel passed away on October 6th, 1992.     

Demonstration of the correlation of the degree of synthesis and 4-aminobutyric acid levels in the central nervous system; effect of repeated convulsive seizures

GABA turnover rates have been determined in 15 brain areas in five inbred strains of Mice or sublines (DBA/2J, C57/6J, Swiss Rb1, Swiss Rb2, Swiss Rb3). GABA turnover rates and levels are correlated (2 P less than 0.05). After repeated seizures (twice a day for 15 days), induced by an acoustic stimulus in Swiss Rb1 Mice selected for audiogenic seizures, this correlation is no longer observed.     

Development of a synaptosomal model to determine drug-induced in vivo changes in GABA-levels of nerve endings in 11 brain regions of the rat

An experimental procedure was developed which allowed the simultaneous measurement of GABA in synaptosomes from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Post-mortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. Experiments with addition of aminooxyacetic acid (AOOA, 1 mM) to the homogenizing medium indicated that GABA was not being degraded during synaptosome preparation. The presence of exogenous GABA (1 mM) did not alter the GABA levels in the organelles, indicating that no significant redistribution of GABA occurred during subcellular fractionation. On the basis of these findings, it was suggested that synaptosomal fractions could be used as a model to monitor indirectly the drug-induced changes in GABA levels of nerve endings in discrete brain areas of the intact animal. In vivo experiments with AOAA (30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosmal GABA levels in different brain regions. Although AOAA was more potent than VPA in increasing GABA in whole tissue of most brain regions, significant increases of synaptosomal GABA levels after AOAA were only determined in olfactory bulbs and frontal cerebral cortex. In contrast, VPA induced significant synaptosomal GABA increases in olfactory bulbs, hypothalamus, superior and inferior colliculus, substantia nigra, and cerebellum. The data indicate that the synaptosomal model can provide useful information on the in vivo effects of drugs on GABA levels in nerve terminals and their ability to exert this effect in specific brain areas.     

Cocaine

Cocaine HCl (0, 10, or 50 mg/kg) was injected into adult male ICR mice ip. Thirty minutes later, the brains were removed, and nine regions were isolated: olfactory bulbs, olfactory tubercles, prefrontal cortex, septum, striatum, amygdala, hypothalamus, hippocampus, and thalamus. Using high-performance liquid chromatography, concentrations of norepinephrine, dopamine, serotonin, and their major metabolites and the metabolite/neurotransmitter ratios were determined as an indicator of utilization. Serotonergic systems responded most dramatically. 5HIAA/5-HT decreases were seen in all the brain regions, except the septum, hippocampus, and olfactory bulbs. In most instances, the alterations were dose-dependent. The most profound changes were seen in the amygdala, prefrontal cortex, hypothalamus, and thalamus. For noradrenergic systems, significant responses were seen only in the amygdala, prefrontal cortex, and hypothalamus, but then only at the lower dose. The dopaminergic responses were more complex and not always dose-dependent. The DOPAC/DA ratio was decreased only in the amygdala and striatum at the lower dose, and the olfactory tubercles at the higher dose. It was increased in the septum. The HVA/DA ratios were decreased in the amygdala, prefrontal cortex, and hypothalamus, but only at the lower dose (like MHPG/NE). The 3MT/DA ratio was decreased in the thalamus at the lower dose and in the olfactory tubercles at the higher dose, whereas it was increased in the prefrontal cortex at the lower dose. The HVA and DOPAC routes of degradation were both utilized only by the amygdala. Thus, cocaine produced its most comprehensive effects in this nucleus, as well as the greatest absolute percentage changes for all three of the monoamine systems studied.     

Effect of propyl-2-pentene-2-oic acid on GABA levels in different regions of mouse brain]

An elevation in cerebral GABA level (65%) is observed after administration of an anticonvulsant, sodium propyl 2-pentene-2 oate, a branched chain fatty acid, comformationally restricted GABA analogue, competitive inhibitor of GABA-T in regard to GABA. The concentration of GABA increases in some regions i.e. substantia nigra, frontal and temporal cortex, cerebellum and olfactory bulbs. The GABA level remains unchanged in caudate nucleus, hippocampus and occipital cortex. Results are discussed comparatively to the effect of sodium n-dipropylacetate.     

Use of Inhibitors of γ-Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid

The technique of estimating gamma-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats, but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/kg i.p., 5-30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation.     

tele-Methylhistamine Levels and Histamine Turnover in Nuclei of the Rat Hypothalamus and Amygdala

An HPLC method using fluorescence detection for the determination of tele-methylhistamine (t-MH) was improved to a sensitivity level which enabled the detection of 0.05 pmol of tissue t-MH. The t-MH contents and the histamine turnover rates in various nuclei of the rat hypothalamus and amygdala were subsequently measured. The histamine turnover rates were estimated from pargyline-induced t-MH accumulation. Both the t-MH levels and the histamine turnover rates were shown to be relatively high in the nuclei dorsomedialis and premammillaris ventralis of the hypothalamus, and also in the nucleus medialis of the amygdala. The steady-state t-MH levels in various nuclei of the hypothalamus and amygdala correlated well with the histamine turnover rates in these nuclei.     

Effects of insulin on rat brain noradrenaline

A single intracardial injection of streptozotocin produced a significant increase in rat hypothalamic noradrenaline while no changes were observed in the olfactory tubercles. The parenteral administration of a single dose of insulin decreased rat hypothalamic noradrenaline; the effect had a rapid onset and lasted for at least six hours. Similar noradrenaline reductions were observed in the olfactory tubercles but in this tissue the depletion started later and recovered earlier. In addition, in olfactory tubercles after insulin injection, tyrosine level and dopamine metabolism were increased. The results show that the increases in hypothalamic NA observed in streptozotocin diabetic rats are counteracted by insulin administration and possibly the consequence of changes in noradrenaline turnover.     

THE COMPARATIVE EFFECTS OF VARIOUS HYDRA-ZIDES ON γ-AMINOBUTYRIC ACID AND ITS METABOLISM

Abstract— The administration of different hydrazides to chicks (20-23 days post-hatching) in amounts giving similar latent periods before the onset of seizures produced (i) similar rates of decrease in content of cerebral GABA, (ii) considerable but dissimilar inhibitions of cerebral GAD activity, (iii) slight inhibitions of cerebral GABA-T activity. The results support the view that low GABA levels were involved in. the etiology of the seizures but seemed to rule out the possibility that a reduced turnover of GABA was responsible for the occurrence of the convulsions.     

Alterations in the Content of Amino Acid Neurotransmitters Before the Onset and During the Course of Methoxypyridoxine-Induced Seizures in Individual Rabbit Brain Regions

In rabbits, generalized seizures were induced by methoxypyridoxine, and changes in amino acid concentrations of 15 brain regions were investigated before seizure onset and during the course of sustained epileptiform activity. As previously reported, gamma-aminobutyric acid (GABA) concentration decreased preictally in most regions. At the same time, taurine level was elevated in the hypothalamus, thalamus, hippocampus, caudatum, and frontal cortex. After 90 min of seizures, it was significantly decreased in the hypothalamus, periaqueductal grey, substantia nigra, frontal cortex, and cerebellum. Glycine content was reduced preictally only in the substantia nigra; after seizure onset its concentration rose in all brain areas. Glutamate content in the frontal cortex decreased before seizure onset; after 1.5 h of seizures, its concentration in cerebellum, caudatum, and hippocampus was reduced. Aspartate level was decreased in most areas after sustained seizures; in putamen, however, it was elevated. In contrast, glutamine content increased preictally in the superior colliculus and in all brain areas by approximately 200% after 90 min of seizures. Alanine and valine content also rose markedly in most brain areas after prolonged seizures, and threonine showed the same tendency. The single brain regions were observed to respond to methoxypyridoxine in highly individualistic ways. For example, the glycine content of the substantia nigra, which is believed to utilize this amino acid as a neurotransmitter, decreased preictally. The potential importance of the superior colliculus in seizure induction is considered in view of the early rise in glutamine level. The antagonistic preictal behavior of taurine and GABA is discussed with respect to synthesis, uptake from the blood, and antiepileptic properties.