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.     

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.     

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     

The activation of phosphatidylinositol turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors

In the rat cerebral cortex, the comparative effects of various muscarinic agonists on the release of [³H]dopamine ([³H]DA), [³H]acetylcholine ([³H]ACh), and [³H]5-hydroxytryptamine ([³H]5-HT) from superfused nerve endings and on phosphatidylinositol (PI) turnover were studied. Acetylcholine (ACh) was found to be the most potent among the agonists tested on all three release systems examined, and also on the activation of PI turnover. Oxotremorine and bethanechol were very weak agonists when tested as stimulators of PI turnover. However, oxotremorine was very effective as a release modulator, while bethanechol was completely ineffective. Our data suggest that the activation of PI turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

Stimulation of synaptosomal uptake of neurotransmitter amino acids by insulin: possible role of insulin as a neuromodulator

Insulin stimulates in a dose-dependent manner (concentration range of 0.1 - 10 microM) the synaptosomal uptake of amino acids characterized by high-affinity, Na+-dependent, veratridine-sensitive transport systems. This stimulation is observed in synaptosomes prepared from each of several regions of the adult rat brain. Both the initial rate of amino acid uptake and the overall capacity for amino acid accumulation are increased. Since these transport systems have been associated with the neurotransmitter role of the amino acids, we postulate that insulin can modulate neurotransmission in the rat central nervous system by increasing the efficiency of neuroactive amino acid reuptake.     

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.     

Transmitter release in hippocampal slices from rats with limbic seizures produced by systemic administration of kainic acid

The systemic injection of kainic acid (KA) has been shown to destroy neurons in the hippocampus and to induce limbic-type seizure activity. However, little is known on the neurochemical events that are associated with this convulsant effect. In the present work we studied the spontaneous and the K⁺-stimulated release of labeled -aminobutyric acid (GABA), glutamate, serotonin and dopamine, in hippocampal slices of KA-treated rats, at the moment of clinical seizures (2 h) and 72 h later. At the onset of convulsions we found a 40–45% decrease in the K⁺-stimulated release of GABA. The release of the other neurotransmitters was not significantly affected by KA treatment. After 72 h GABA release was still reduced by 30–40%. It is concluded that the epileptogenic effect of KA in the hippocampus is probably related to a diminished inhibitory GABAergic neurotransmission.     

Effect of arachidonic acid on [3H]d-aspartate outflow in the rat hippocampus

The aim of this study was to investigate the effect of arachidonic acid on [³H]d-aspartate outflow in rat hippocampus synaptosomes and slices. Arachidonic acid 1) increased basal outflow of [³H]d-aspartate in both synaptosomes and slices, and 2) increased K⁺-evoked overflow in slices but not in synaptosomes. The latter effect was dependent (at least in part) on arachidonic acid metabolism, most likely mediated by lipo-oxygenase metabolites and free radical production. It was prevented by nordihydroguaiaretic acid but not by indomethacin, and was significantly reduced by free radical scavengers (superoxide-desmutase and catalase). This effect was dependent upon stimulation since it could not be observed after a continuous perfusion of arachidonic acid in the absence of stimulation. Furthermore, it was long-lasting since a 30 min perfusion of arachidonic acid was sufficient to exert a significant effect on a stimulation following termination of the application.     

The role of ovarian steroid hormones in the regulation of basal and stress induced absence seizures

Ovarian hormones play an important role in the regulation of absence seizures in patients as well as in animal models. The present study examined whether chronic progesterone exposure would induce tolerance for the occurrence of absence seizures and whether reduction in gonadal steroids (via ovariectomy) would alter the number of basal and stress induced absence seizures in WAG/Rij rats, a genetic model for absence epilepsy.

Methods

In Experiment 1, female WAG/Rij rats equipped with EEG electrodes received progesterone (P) (20 mg/kg) or cyclodextrin (CD, solvent) i.p. injections once a day for 3 days while a third group received CD injections on Days 1 and 2 and P on Day 3. The EEG was recorded on the day preceding the injections and at each day after injections. In Experiment 2, female WAG/Rij rats equipped with EEG electrodes, were ovariectomized (OVX) or sham operated. EEG recordings were made before and at the 4th, 8th, 10th, 20th, and 35th day after surgery. Rats were then exposed to three series of 10 foot-shocks (FS, 1.5 mA, 1 s) over 3 days. The EEG was recorded 1 h before and 2 h after each FS series.

Results

Tolerance developed after a single P injection and the effect of P on SWDs was facilitated by two preceding control injections. No differences were found between OVX and sham-operated females in the occurrence of SWDs either in resting conditions or after acute FS exposure. However, OVX females showed a more prominent day-to-day aggravation in SWDs after repeated FS administration.

Conclusions

The data suggest an important interaction between hormones of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes in seizure control. On the one hand, stress interferes with and facilitates the acute effects of progesterone on the occurrence of SWDs and, on the other hand, rats with an intact hypothalamo-pituitary-gonadal axis can better regulate the stress response and develop tolerance to the stressor.     

The effect of acetyldexphenmetrazine and dexphenmetrazine on the susceptibility to audiogenic seizures in mice.

The antiepileptic effect of dexphenmetrazine (DP) and acetyldexphenmetrazine (ADP) was tested on audiogenic seizures in a 100% susceptible strain of mice. DP had no antiepileptic effect, however, it markedly suppressed the postparoxysmal motor inhibition. ADP had a distinct anticonvulsive effect--it suppressed the convulsive component of the seizure, leaving its running component unaffected. The results are compared with the effect of both drugs on electrographic epileptic phenomena in the turtle brain (Servít and Strejcková 1976).     

Remote effects of early postnatal pituitary hormone melatonin injection on audiogenic seizures in Krushinsky-Molodkina rats

Rats Krushinsky-Molodkina inbred strain (KM) genetically prone to audiogenic seizures were injected with pineal hormone melatonin (50 mg/kg, s.c.) within the period 7th to the 14th posnatal days (PND). The remote effects of this injection adult KM rats consisted in a decrease in the latency and increase in severity of myoclonic seizures produced by audiogenic kindling (20 sound stimuli, 100 dB and 12-15 kHz). As compared to the control, in the cortex and hippocampus of rats of melatonin group, we also found a significant reduction of both total and functional activity of Ca2+/calmodulin-dependent protein kinase II (CAMK II) after audiogenic kindling. On the contrary, melatonin administration within the 1st to 7th PND and the 14th to the 21st PND resulted in a decrease in seizure activity. In the first case, both the total (cortical) and functional (hippocampal) CAMK II activities in melatonin-injected rats were increased as compared to control, whereas in the second case, only a slight increase in Ca2+-independent CAMK II activity in the hippocampus of melatonin-injected rats was observed. Probably, the melatonin administration in the period of early postnatal development changes the features of expression and/or regulation of CAMK II activity, and this could be one of the mechanisms of audiogenic seizure modulation in KM rats.     

Lethal and sublethal effects of long‐lasting insecticide‐treated nets on the invasive bug Halyomorpha halys

Halyomorpha halys is a polyphagous insect species with an original eastern Asiatic distribution, which was recently and accidentally introduced in the USA and Europe, where it became a serious agricultural pest. Chemicals have been widely used for its control leading often to failure of IPM programmes. Several approaches aimed at pest monitoring and control are currently under investigation, for example trapping, screening, border management and biological control. In the present work, we investigated the lethal and sublethal effects of the use of a long‐lasting insecticide‐treated net (LLIN with α‐cypermethrin), focusing on the perspective to control H. halys in an integrated approach. All experiments were carried out in the laboratory either in small arenas, at five exposure times (5, 15, 30, 45 and 60 min) or in large cages at 8 hr exposure. In small arenas, the LLIN induced sublethal effects and/or effectively killed the adults. A higher adult mortality was observed after longer exposure times (LT₉₀ was 51.64 min for females and 40.83 min for males). However, several specimens recovered from sublethal effects, with higher recovery rates after shorter exposure times. In the cage experiments, a significantly higher mortality (65% males and 75% of females) was recorded compared with controls. LLINs are physical barriers that can improve crop protection due to their insecticidal activity, and can be reasonably applied in various attract‐and‐kill systems both in glasshouses and in the field.     

Effect of Low pH on Glutamate Uptake and Release in Isolated Presynaptic Endings from Rat Brain

The effect of acidification of the incubation medium on the membrane potential and glutamate uptake and release was studied in isolated presynaptic neuronal endings (synaptosomes) from rat brain. Using the fluorescent probe diS-C₃-(5), a rapid depolarization of plasma membrane was detected at pH 6.0, most probably as a result of the inhibition of the sodium pump and potassium channel blockade. The membrane potential decrease did not result in increase of basal efflux of glutamate. Glutamate release following K⁺-induced depolarization was decreased upon lowering pH to 6.0. Acidosis inhibited mainly calcium-dependent (vesicular) release of glutamate and did not significantly reduce [¹⁴C]glutamate uptake. This inhibition of glutamate release but not of glutamate uptake may be a mechanism of the protective effect of acidosis during brain ischemia.     

中药天年饮对衰老大鼠脑单胺类神经递质含量的影响

目的：观察中药天年饮（Tiannianyin,TNY-traditional chinese medicine）对D-半乳糖衰老大鼠脑单胺类神经递质去甲肾上腺素（NE）、多巴胺（DA）、5-羟色胺（5-HT）含量的影响。方法：选用成年雄性SD大鼠40只．随机分为4组．每组均为10只：正常组、衰老模型组、TNY用药组、阴性对照组。Ⅱ半乳糖连续腹腔注射制作亚急性衰老的大鼠模型．采用高效液相色谱-电化学法检测各组大鼠下丘脑、海马NE、DA、5-HT的含量。结果：D-半乳糖衰老大鼠下丘脑、海马NE、DA、5-HT的含量明显降低（与正常大鼠相比P〈0．01）：TNY可明显提高脑单胺类神经递质的含量（用药组与模型组相比P〈0．05）。结论：TNY可有效调整中枢神经递质的合成,具有良好延缓衰老的作用。     

Effect of CGP 36742 on the extracellular level of neurotransmitter amino acids in the thalamus.

We have evaluated the effect of the brain penetrating GABAb antagonist, CGP 36742 on GABAb receptors using in vivo microdialysis in the ventrobasal thalamus of freely moving rat. When a solution of 1 mM CGP 36742 in ACSF was dialyzed into the ventrobasal thalamus, 2-3-fold increases of extracellular Glu, Asp and Gly running parallel with significant decreases of contralateral extracellular Asp and Gly were observed. Unilateral applications of Glu receptor antagonists (0.5 mM MK801, 0.1 mM CNQX) evoked 2-3-fold decreases of CGP 36742-specific elevations of extracellular Asp, Glu and Gly. Administration of CNQX and MK801 in the absence of CGP 36742 did not alter the extracellular Glu and Gly concentrations whereas extracellular Asp concentrations diminished by 42-45% at both sides. By contrast, no changes of extracellular Gly accompanied the 5-10-fold enhancements of extracellular Asp and Glu, observed during application of the Glu uptake inhibitor, tPDC (1mM). Suspensions of resealed plasmalemma fragments from the rat thalamus were mixed rapidly with the membrane impermeant form of the fluorescence indicator, bis-fura-2 and the changes in fluorescence intensity in response to CGP 36742 (0.5 mM), and the GABAb agonist, baclofen (0.1 mM), were monitored on the time scale of 0.04 ms(-10)s. Progress of CGP 36742-mediated influx, and baclofen-mediated efflux of Ca++ ion, antagonized by CGP 36742, was observed in the 1 ms(-10s) period of time. These data support the hypothesis that background ventrobasal activities and thalamocortical signaling are under the control of inhibitory GABAb receptors in the ventrobasal thalamus.     

Effects of N-uronoyl derivatives of neurotransmitter amino acids on rats under conditions of behavioral tests

Under conditions of a few behavioral tests on rats, we examined the psychotropic activity of novel N-uronoyl derivatives of amino acids: (i) N-(1,2:3,4-di-O-isopropylidene-α-D-galactopyranuronoyl-β-alanine (DAGU-Ala), (ii) DAGU-glycyl-D,L-glutamic acid (DAGU-Gly-Glu), and (iii) DAGU-glycyl-glycine (DAGU-Gly-Gly) injected i.p. in a 50 mg/kg dose. In an open-field test, DAGU-Gly-Glu and DAGU-GLy-Gly showed antistress properties (they decreased the intensity of locomotion, decreased the number of defecation acts, and intensified the research activity), while DAGU-Ala possessed a psychostimulating effect (intensification of locomotor and research activity and an increase in the number of defecations). In the “black-and-white chamber” test, only DAGU-Gly-Gly exerted an anxiolytic effect; it somewhat increased the frequency and duration of emergings of animals from the dark section and duration of visits to the illuminated section of the chamber. DAGU-GLy-Glu manifested antidepressant properties; it increased the time of active swimming of the animal in the Porsolt test and decreased the duration of passive “hanging” of rats in the “suspension by the tail” test. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 52–61, January–February, 2007.     

复方中药对大鼠力竭运动与恢复过程中端脑神经递质含量的影响

目的：探讨复方中药对运动大鼠中枢神经递质含量的影响,进一步认识中药提高运动能力和促进运动性疲劳恢复的作用机理。方法：选8周龄大鼠64只,随机分成服药组和对照组,服药组灌服中药煎剂8周。然后,每组再分成4个亚组分别于不同状态下断头处死,测其中枢递质含量。结果：服药组大鼠力竭运动时间极显著长于对照组（P〈0．01）;安静时,除谷氨酸（GLU）含量服药组极显著高于对照组（P〈0．01）外、其余各指标无组间显著性差异;定量负荷后,服药组5-羟色胺（5-HT）、5-羟吲哚乙酸（5-HIAA）、弘氨基丁酸（GABA）、多巴胺（DA）含量和5-HT／5-HIAA显著低于对照组,GLU、GLU／GABA和DA／5-HT明显高于对照组;力竭即刻,服药组5-HT、GABA含量和5-HT／5-mAA显著低于对照组（P〈0．05）,GLU含量、DA／5-HT和GLU／GABA显著高于对照组（P〈0．05）;恢复12h。服药组5-HT含量和5-HT／5-HIAA极显著低于对照组（P〈0．01）,GLU、DA、GABA含量和DA／5-HT明显高于对照组（P〈0．05）。结论：在大鼠运动至力竭性的过程中,复方中药制剂有明显抑制5-HT、5-HIAA、DA、GABA生成和促进GLU中枢递质合成的作用,其综合效应表现为兴奋性递质相对显著增多,使中枢神经兴奋性增强、明显延长大鼠运动时间和促进中枢疲劳的恢复。     

Preweaning enrichment has no lasting effects on adult hippocampal neurogenesis in four-month-old mice

Since both living in an enriched environment and physical activity stimulate hippocampal neurogenesis in adult mice, we endeavored to examine whether preweaning enrichment, a sensory enrichment paradigm with very limited physical activity, had similar effects on neurogenesis later in life. Mice were removed from the dams for periods of increasing length from postnatal day 7 to 21, and exposed to a variety of sensory stimuli. At the age of 4 months, significant differences could be found between previously enriched and non-enriched animals when spontaneous activity was monitored. Enriched mice moved longer distances, and spent more time in a defined center zone of the open field. Adult neurogenesis was examined by labeling proliferating cells in the dentate gyrus with bromodeoxyuridine (BrdU). Cell proliferation, survival of the newborn cells, and net neurogenesis were similar in both groups. Volumetric measurements and stereological assessment of total granule cell counts revealed no difference in size of the dentate gyrus between both groups. Thus, in contrast to postweaning enrichment, preweaning enrichment had no lasting measurable effect on adult neurogenesis. One of the parameters responsible for this effect might be the lack of physical activity in preweaning enrichment. As physical activity is an integral part of postweaning enrichment, it might be a necessary factor to elicit a neurogenic response to environmental stimuli. The result could also imply that baseline adult hippocampal neurogenesis is independent of the changes induced by preweaning enrichment and might not contribute to the sustained types of plasticity seen in enriched animals.