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Altered extracellular amino acid concentrations following concussion or mild traumatic brain injury can result in delayed neuronal damage through overactivation of NMDA glutamatergic receptors. However, the consequences of repeated concussions prior to complete recovery are not well understood. In this study, we utilized in vivo cerebral microdialysis and a weight-drop model to investigate the acute neurochemical response to single and repeated concussions in adult rats that were fully conscious. A microdialysis probe was inserted into the hippocampus and remained in place during impact. Primary outcomes included concentrations of glutamate, GABA, taurine, glycine, glutamine, and serine, while secondary outcomes were righting times and excitotoxic indices. Compared to sham injury, the first concussion resulted in significant increases in glutamate, GABA, taurine, and glycine levels, longer righting times, and higher excitotoxic indices. Following the second concussion, righting times were significantly longer, suggesting cumulative effects of repeated concussion while only partial increases were observed in glutamate and taurine levels. GABA and glycine levels, and excitotoxic indices were comparable to sham injury. These findings suggest that single and repeated concussions may induce acute increases in several amino acids, while repeated concussions could exacerbate neurological symptoms despite less pronounced neurochemical changes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Animal models show that cocaine sensitization, a behavioral marker of addiction, is more significant in intact gonadal female than male rats and ovariectomy suppress this behavior in female rats. However, few studies explore changes in neurotransmission related to this phenomenon. Here we investigated the in vivo changes on GABA, glutamate, and taurine levels in the medial prefrontal cortex (mPFC) of gonadal intact or ovariectomized female rats after a cocaine challenge administration. Adult female rats were bilaterally ovariectomized (OVX), or sham-operated (SHAM) and randomly assigned to control (CTR), acute (ACT), or repeated (RPT) cocaine administration groups. In the challenge day, after eight days of daily cocaine (15 mg/kg) or saline administration and ten days of washout and stereotaxic surgery, RPT and ACT groups received cocaine, and the CTR group received saline. Horizontal locomotion was monitored concomitantly with microdialysate collection to determine extracellular GABA, glutamate, and taurine levels. Hormonal determination in blood samples confirmed the lower hormonal status of the OVX. Cocaine sensitization occurred in SHAM-RPT female rats after the challenge administration. Non-sensitized OVX-RPT rats showed a peak of GABA at 30 min after cocaine administration, with no change on glutamate and taurine levels. Therefore, elevated GABA levels in the mPFC and lower serum estrogen levels abolish cocaine sensitization behavior in ovariectomized female rats. We discuss some possible implications of these finding for future models of cocaine sensitization research lighting in the female hormonal influence., (Copyright © 2020. Published by Elsevier B.V.)

Amyloid-β peptide (Aβ) is considered a key protein in the pathogenesis of Alzheimer's disease because of its neurotoxicity, resulting in impaired synaptic function and memory. On the other hand, it was demonstrated that low (picomolar) concentrations of Aβ enhance synaptic plasticity and memory, suggesting that in the healthy brain, physiological Aβ concentrations are necessary for normal cognitive functions. In the present study, we found that Aβ (1-42) in concentrations of 10 pМ - 100nМ enhanced desensitization of the glycine-activated current in isolated CA3 pyramidal neurons and also reversibly suppressed its peak amplitude during short (600ms) co-application with agonist. The effect was most prominent at low glycine concentrations. When glycine receptors were activated by other receptor agonists - taurine and β-alanine, the changes of current kinetics and amplitudes induced by Aβ had a similar character. When Aβ (100 pM) was added to the bath solution, it caused, besides acceleration of desensitization, more pronounced reduction of peak current amplitude. This effect developed slowly, during a few minutes, and was more prominent at saturating concentrations of agonists. The results suggest that Aβ interacts with glycine receptors through three different mechanisms - by enhancing receptor desensitization, by rapid inhibition of the receptor, and also by means of a slowly developing inhibition of the amplitude of the current, possibly through intracellular mechanisms. The observed changes in the activity of glycine receptors induced by Aβ can lead to suppression of the tonic inhibition of hippocampal neurons mediated by extrasynaptic glycine receptors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We utilized proton magnetic resonance spectroscopy to evaluate the metabolic profile of the hippocampus and anterior cingulate cortex of the developing rat brain from postnatal days 14-70. Measured metabolite concentrations were modeled using linear, exponential, or logarithmic functions and the time point at which the data reached plateau (i.e. when the portion of the data could be fit to horizontal line) was estimated and was interpreted as the time when the brain has reached maturity with respect to that metabolite. N-acetyl-aspartate and myo-inositol increased within the observed period. Gluthathione did not vary significantly, while taurine decreased initially and then stabilized. Phosphocreatine and total creatine had a tendency to increase towards the end of the experiment. Some differences between our data and the published literature were observed in the concentrations and dynamics of phosphocreatine, myo-inositol, and GABA in the hippocampus and creatine, GABA, glutamine, choline and N-acetyl-aspartate in the cortex. Such differences may be attributed to experimental conditions, analysis approaches and animal species. The latter is supported by differences between in-house rat colony and rats from Charles River Labs. Spectroscopy provides a valuable tool for non-invasive brain neurochemical profiling for use in developmental neurobiology research. Special attention needs to be paid to important sources of variation like animal strain and commercial source., (Published by Elsevier B.V.)

In order to identify the neurochemical alterations in motor associated subcortical nuclei, and enhance our understanding of neurophysiology of progressive reduction in fine motor control with aging, the metabolic changes in striatum and cerebellar cortex in SD rats along with aging were investigated using a metabonomic approach based on high resolution "magic angle spinning" 1H-NMR spectroscopy and partial least squares-discriminant analysis. It was found that there were increased myo-inositol and lactate, and decreased taurine in these two brain regions of old rats. The above changes may be a marker for alterations of neuronal cells, which reduce fine motor control. Besides, some of the metabolites are gender-related and region-specific. Old female rats had decreased glutamate and increased creatine in striatum, while old male rats had increased choline in striatum, and increased GABA in cerebellar cortex, respectively. However, further analyses showed that most of the metabolites in male rats were not distinctively different with those of female ones except choline, which was in a relative lower level in striatum of male rats. All this results suggest that energy metabolism is an important indication of age-related change, which is not only in male, but also in female rats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Taurine, one of the most abundant endogenous amino acids in the mammalian central nervous system (CNS), is involved in neural development and many physiological functions. In this study, the interaction between taurine and GABA(A)/glycine receptors was investigated in young rat (P13-P15) anteroventral cochlear nucleus (AVCN) neurons using the whole-cell patch-clamp method. We found that taurine at low (0.1mM) and high (1mM) concentrations activated both GABA(A) and glycine receptors, but not AMPA and NMDA receptors. The reversal potentials of taurine-, GABA- or glycine-evoked currents were close to the expected chloride equilibrium potential, indicating that receptors activated by these agonists were mediating chloride conductance. Moreover, our results showed that the currents activated by co-application of GABA and glycine were cross-inhibitive. Sequential application of GABA and glycine or vice versa also reduced the glycine or GABA evoked currents. There was no cross-inhibition when taurine and GABA or taurine and glycine were applied simultaneously, but the response was larger than that evoked by GABA or glycine alone. These results suggest that taurine can serve as a neuromodulator to strengthen GABAergic and glycinergic neurotransmission in the rat AVCN., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Incidence of status epilepticus (SE) is higher in children than in adults and SE can be induced in developing rats. The cerebellum can be affected after SE; however, consequences of cerebellar amino acid transmission have been poorly studied. The goal of this study was to determine amino acid tissue concentration and GABA(A) receptor binding in the immature rat cerebellum after an episode of SE. Thirteen-day-old (P13) rat pups received intraperitoneal injections of lithium chloride (3 mEq/kg). Twenty hours later, on P14, SE was induced by subcutaneous injection of pilocarpine hydrochloride (60 mg/kg). Control animals were given an equal volume of saline subcutaneously. Animals were killed 24h after SE induction, the cerebellum was quickly removed, and the vermis and hemispheres were rapidly dissected out on ice. Amino acid tissue concentrations in the vermis and hemispheres were evaluated by HPLC and fluorescent detection. GABA(A) receptor binding in the medial vermis was analyzed by in vitro autoradiography. SE increased the tissue levels of the inhibitory amino acids taurine (80%) and alanine (91%), as well as glutamine (168%) in the cerebellar hemisphere; no changes were observed in the vermis. SE did not modify GABA(A) receptor binding in any cerebellar lobule from the vermis. Our data demonstrate that SE produces region-specific changes in amino acid concentrations in the developing cerebellum., (Copyright © 2011. Published by Elsevier B.V.)

Considering the contribution of hippocampal formation and glutamate-mediated signalling to epileptogenesis, we investigated the effects of group III metabotropic glutamate receptor (mGluR)-selective ligands on the kindling of seizures. We also examined the concentration of the amino acid glutamate, GABA, alanine and taurine in the hippocampus of rats using a microdialysis technique. Pentylenetetrazol (PTZ), a non-competitive antagonist of the GABA(A) receptor, was administered intraperitoneally at 35 mg/kg body weight to induce seizures. It was determined that the kindling of PTZ-induced seizures could be attenuated by post intracerebroventricular administration of 100 nmol of the group III mGluR antagonist CPPG [(RS)-a-cyclopropyl-4-phosphonophenylglycine]. There were significant differences in tested parameters during the final stages of the kindling procedure. The group III mGluR agonist L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid at 100 nmol, i.c.v.] did not significantly affect the kindling of seizures in comparison to control rats, although there was acceleration of the process as compared to CPPG treated animals. We demonstrated that the baseline concentrations of glutamate, GABA, alanine, taurine, and the glutamine/GABA ratio were elevated in the hippocampus of fully kindled rats. Intracerebroventricular administration of a single dose of CPPG increased the concentrations of glutamate in the hippocampus of control, non-kindled rats. Intracerebroventricular administration of L-AP4 did not affect the hippocampal amino acid concentration in either animal group. Overall, these data suggest that there is a shift in the balance between neurotransmitters towards increased production of excitatory amino acids, and this may be mediated by group III mGluRs during seizure kindling.

We have previously shown that the inhibitory neuromodulator taurine attenuates the release of preloaded D-[3H]aspartate from murine corticostriatal slices evoked by ischemic conditions or by application of the sodium channel agonist veratridine. The release of D-[3H]aspartate (a non-metabolized analog of glutamate) was used as an index of glutamate release. The aim of the present study was to reveal the molecular mechanisms responsible for this inhibitory effect of taurine. It was shown that 10 mM taurine suppresses D-[3H]aspartate release evoked by 0.1 mM veratridine, but does not affect the high-K+ -(50 mM) or ouabain- (0.1 mM) evoked release. Taurine had no effect in Ca2+ -free medium when the synaptic exocytosis of D-[3H]aspartate was inhibited. Nor did it suppress the release from slices preloaded with the competitive glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THBA), which inhibits D-[3H]aspartate release mediated by the reverse action of glutamate transporters. Omission of Cl- from the incubation medium reduced the effect of taurine, signifying the involvement of a Cl- channel. The glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline did not block the taurine effect, although picrotoxin, a less specific blocker of agonist-gated chloride channels, completely prevented the effect of taurine on veratridine-induced D-[3H]aspartate release. The respiratory chain blocker rotenone or mitochondrial protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in combination with the mitochondrial ATPase inhibitor oligomycin, which inhibits the mitochondrial Ca2+ uniporter, also reduced the effect of taurine. The results obtained in the present study show that taurine acts specifically on the release of preloaded D-[3H]aspartate evoked by veratridine, but not on that evoked by other depolarizing agents, and affects the release mediated both by synaptic exocytosis and the reverse action of glutamate transporter. Taurine may attenuate D-[3H]aspartate release by regulation of mitochondrial Ca2+ sequestration and by activation of a chloride channel, but not that governed by GABA(A) or strychnine-sensitive glycine receptors.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate brain. GABA activates both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors in mammals. Whether non-mammalian vertebrates possess receptors with similar characteristics is not well understood. We used a mammalian GABA(B)-specific antagonist to determine the pharmacology of putative receptors in the brain of an anuran amphibian, the male bullfrog (Rana catesbeiana). Receptor binding assays with the antagonist [(3)H]CGP54626 revealed a single class of high affinity binding sites (with a K(D) of 2.97 nM and a B(max) of 2619 fmol/mg protein). Binding was time- and temperature-dependent, saturable and specific. Specific binding of [(3)H]CGP54626 was inhibited by several mammalian GABA(B) receptor agonists and antagonists. The rank order potency of agonists was: GABA = SKF97541 > (R)-Baclofen > 3-APPA. The rank order for antagonists was: CGP54626 = CGP55845 > CGP52432 > CGP35348. The GABA(A) receptor ligands muscimol and SR95531 had very low affinity for [(3)H]CGP54626 binding sites, while bicuculline compounds had no affinity. Binding of GABA was positively modulated by CGP7930. Taurine did not allosterically modulate GABA binding but did inhibit [(3)H]CGP54626 binding in a linear fashion. Bullfrog brain thus possesses binding sites with significant similarity to mammalian GABA(B) receptors. These receptors differ from mammalian receptors, however, in dissociation kinetics, ligand specificity and allosteric modulation.

The purpose of this study was to examine whether the efflux transport system for taurine from brain to blood is present at the blood-brain barrier (BBB) by using the brain efflux index (BEI) method and to determine whether the taurine transport system is regulated after central nervous system cell damage by tumor necrosis factor-alpha (TNF-alpha) in vivo. [(3)H]Taurine was microinjected into the parietal cortex area 2 of the rat brain, and was eliminated from the brain with an efflux transport rate of 1.22 x 10(-2) min(-1), and the process is saturable with a K(m) of 39.1 microM. This process was significantly inhibited by taurine transporter (TAUT) inhibitors, such as unlabeled taurine, beta-alanine, betaine, nipecotic acid and gamma-aminobutyric acid (GABA). In addition, the effect of tumor necrosis factor-alpha on [(3)H]taurine transport was investigated. [(3)H]Taurine uptake was increased and its efflux was reduced by pretreatment with tumor necrosis factor-alpha. Also, [(3)H]taurine efflux was reduced by tumor necrosis factor-alpha in a time- and dose-dependent manner. In conclusion, there is the efflux pump for taurine at the blood-brain barrier to reduce taurine concentration in the brain interstitial fluid, and this process was carrier mediated. In addition, the transport system for taurine through the blood-brain barrier was found to be regulated by tumor necrosis factor-alpha in vivo.

The principal cause of mortality in patients with acute liver failure (ALF) is brain herniation resulting from intracranial hypertension caused by a progressive increase of brain water. In the present study, ex vivo high-resolution 1H-NMR spectroscopy was used to investigate the effects of ALF, with or without superimposed hypothermia, on brain organic osmolyte concentrations in relation to the severity of encephalopathy and brain edema in rats with ALF due to hepatic devascularization. In normothermic ALF rats, glutamine concentrations in frontal cortex increased more than fourfold at precoma stages, i.e. prior to the onset of severe encephalopathy, but showed no further increase at coma stages. In parallel with glutamine accumulation, the brain organic osmolytes myo-inositol and taurine were significantly decreased in frontal cortex to 63% and 67% of control values, respectively, at precoma stages (p<0.01), and to 58% and 67%, respectively, at coma stages of encephalopathy (p<0.01). Hypothermia, which prevented brain edema and encephalopathy in ALF rats, significantly attenuated the depletion of myo-inositol and taurine. Brain glutamine concentrations, on the other hand, did not respond to hypothermia. These findings demonstrate that experimental ALF results in selective changes in brain organic osmolytes as a function of the degree of encephalopathy which are associated with brain edema, and provides a further rationale for the continued use of hypothermia in the management of this condition.

Morphological changes of the hippocampus were investigated in mice exhibiting signs of intoxication following short-term exposure to 6% ethanol. These alterations were examined by a double immunofluorescent study using antibodies to taurine and anti-glial fibrillary acidic protein (GFAP) antibody. Antibody-labeled taurine was localized mainly in the astrocytes and endothelial cells of control mice. Ethanol administration resulted in a significant increase in the accumulation of taurine and GFAP immunoreactivity (IR) in the stratum lacunosum-moleculare (sl-m) of the hippocampus. Specifically, the cell bodies of taurine-positive astrocytes were hypertrophied, their processes were elongated in the pericapillary region, and some colocalized with GFAP-IR cells. Furthermore, quantitative analysis revealed that the merged area in ethanol-treated mice was twice that (71.6% vs. 35.8%) of control mice. Since taurine is involved in various neuroprotective functions, the present observations suggest that the expression of taurine IR in reactive astrocytes after ethanol exposure might play an important role in neuroprotective processes.

Osmotically swollen rat cerebral astrocytes develop an increased anion conductance which can mediate chloride and taurine release. We used whole cell patch clamp to study mechanisms that modulate this conductance. Astrocyte chloride conductance increased within 4 min of exposure to 200 mOsm medium and was 670+/-123% of its initial value after 15 min (mean+/-S.E.M.). This conductance was substantially reduced in 0.1 mM extracellular calcium with 20 mM BAPTA added to the electrode solution and was completely inhibited with calcium-free perfusion solution containing 1 mM EDTA (n=4). The conductance increase in 200 mOsm medium also was inhibited in a dose-dependent manner by nimodipine with a calculated K(i) of 0.31+/-0.4 microM and mean+/-S.E.M. inhibition of 84.4+/-4% at 100 microM nimodipine. In the presence of 100 microM W-7, a calmodulin antagonist, the mean+/-S.E.M. conductance increase after 15 min was 223+/-40% of the initial value while 300 microM W-7 or 100 microM trifluoperazine inhibited the conductance increase completely (n=6). With taurine as the major anion in electrode and perfusion solutions, a significant conductance increase was observed in 200 mOsm medium. This conductance increase was inhibited by 300 microM W-7 or 100 microM nimodipine. We conclude extracellular calcium influx via L-type calcium channels leads to increased astrocyte anion conductance in 200 mOsm conditions via calmodulin-dependent activation of anion channels. Efflux of anionic taurine from swollen astrocytes also may be affected by calcium influx through a similar calcium/calmodulin-dependent process.

The effects of oral ENA713 and CHF2819 (0.5, 1.5 and 4.5 mg/kg), two novel acetylcholinesterase inhibitors, on extracellular concentrations of amino acids in rat hippocampus, were evaluated using in vivo microdialysis. ENA713, at 4.5 mg/kg, but not CHF2819, significantly decreased glutamate, taurine, arginine and citrulline levels, without affecting aspartate concentrations. These results suggest that the modulation of amino acidergic transmission could represent an additional mechanism of action in Alzheimer's disease for some acetylcholinesterase inhibitors.

Lubeluzole is a newly designed neuroprotectant which has proved effective in the treatment of experimental stroke in rats, mainly by inhibition of the glutamate-activated NO pathway, but also by counteracting osmotic stress by a mechanism associated with the release of the osmotically active amino acid taurine (Tau). Here we show that lubeluzole administered i.p. decreases by 25% the high (50 mM) K+-evoked accumulation of Tau in striatal microdialysates of healthy rats and by 34% in rats with thioacetamide-induced hepatic failure, where the increased extracellular accumulation of Tau signifies ongoing hepatic encephalopathy. Lubeluzole does not affect the nonstimulated accumulation of Tau in either group of rats. The results indicate that lubeluzole may be effective in ameliorating ionic or osmotic stress in a range of pathological conditions involving the rise of extracellular K+, and also in decreasing the vulnerability to stress in rats with hepatic failure.

The amino acid taurine serves many functions in the nervous system serving as inhibitory neurotransmitter/neuromodulator, neurotrophin, antioxidant, and osmolyte. Taurine levels are increased following brain injury and glucocorticoid administration. Thus, the purpose of this study was to examine spinal taurine concentrations following spinal cord injury (SCI) and methylprednisolone (MP) treatment of SCI. A total of 44 adult male Sprague-Dawley rats were divided into control and lesion groups. Control rats received a T6 vertebral laminectomy while lesioned rats received a laminectomy followed by complete spinal transection. Half of the animals in each group received MP intravenously following sham-operation or SCI. Rats survived for 7 or 30 days and concentrations of taurine in spinal gray and white matter, in spinal segments both near and distant from the injury epicenter, were resolved by HPLC analysis. Taurine levels were increased 7 and 30 days following transection in spinal segments immediately adjacent to the lesion and were further elevated by MP treatment. No increases were seen in far rostral/caudal segments, and MP treatment alone had no effect on spinal taurine levels. These findings demonstrate that spinal injury results in increased taurine concentrations in spinal segments undergoing the greatest degree of cellular reactivity and tissue reorganization and that MP therapy potentiates these increases. These findings are significant in that they further characterize the effects of acute MP therapy in spinal tissue. Since taurine is thought to be involved in neuroprotection and/or regeneration following injury, the potentiation of taurine levels by MP treatment may relate to its therapeutic properties.

Brain hypothermia has been reported to protect against ischemic damages in adult animals. Our goal in this study was to examine whether brain hypothermia attenuates ischemic neuronal damages in the hippocampus of aged animals. We also determined effects of hypothermia on ischemia-induced releases of amino acids in the hippocampus. Temperature in the hippocampus of aged rats (19-23 months) was maintained at 36 degrees C (normothermia), 33 degrees C (mild hypothermia) or 30 degrees C (moderately hypothermia) using a thermoregulator during 20 min of transient forebrain ischemia. Cerebral ischemia increased extracellular concentrations of glutamate and aspartate by 6- and 5-fold, respectively, in the normothermic group. Mild and moderate hypothermia, however, markedly inhibited the rise of these amino acids to less than 2-fold. Elevation of extracellular taurine, a putative inhibitory amino acid, was 16-fold in the normothermic rats. Mild hypothermia attenuated ischemia-induced increase in taurine (10-fold), and moderate hypothermia inhibited the increase. Ischemic damages, evaluated by histopathological grading of hippocampal CA1 area 7 days after ischemia, was significantly ameliorated in the mild (1.3+/-0.5, mean+/-S.E.M.) and moderate hypothermic rats (0.8+/-0.3) compared with the normothermic ones (3.4+/-0.4). These results suggest that brain hypothermia protects against ischemic neuronal damages even in the aged animals, and the protection is associated with inhibition of excessive effluxes of both excitatory and inhibitory amino acids.

Insulin has been demonstrated to be neuroprotective in brain and spinal cord ischemia. The mechanism of neuroprotection may involve alterations in metabolism, protein synthesis or uptake of GABA by astrocytes. Conversely, hyperglycemia increases the extent of neurologic damage observed during ischemia/reperfusion. Diabetic patients are 2-4 times more likely to suffer a stroke as normoglycemic patients and they also have worsened neurologic outcome. Determining if insulin, which many diabetics already use as therapy, can be neuroprotective, would be a possible means of alleviating the detrimental outcome from diabetic stroke. This study looked at the relationship between topically administered insulin (1 mIU insulin/ml and 100 mIU insulin/ml) during a four vessel occlusion model of global ischemia and the release of amino acids, especially glutamate, from the cortex in streptozotocin (STZ)-treated rats. The rats were utilized either 5-7 days (ASTZ) or 4-6 weeks (CSTZ) after a single STZ injection. In the ASTZ animals both doses of insulin increased the amount of the excitotoxic amino acids, aspartate and glutamate, released during reperfusion and the higher dose also increased the levels of taurine and GABA during reperfusion. In the CSTZ animals, both doses of insulin increased the amount of excitotoxic amino acids during reperfusion and the lower dose increased GABA levels released during reperfusion. The differences between the ACTZ and CSTZ animals may be due to metabolic differences in the utilization of glucose. Insulin may act as a neuroprotectant by increasing extracellular GABA resulting in neuroinhibition.

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na(+)-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using DL-threo-beta-benzyloxyaspartate (DL-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1-3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus DL-TBOA (100 microM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of DL-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of DL-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na(+), and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na(+) uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na(+)-dependent, glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

Large concentrations of the beta-amino acid, taurine, can be found in many forebrain areas such as the basolateral amygdala, a portion of the limbic forebrain intimately associated with the regulation of fear/anxiety-like behaviors. In addition to its cytoprotective and osmoregulatory roles, taurine may also serve as an agonist at GABA(A)- and strychnine-sensitive glycine receptors. In this latter context, the present study demonstrates that application of taurine to acutely isolated neurons from the basolateral amygdala of adult rats causes significant alterations in resting membrane current, as measured by whole-cell patch clamp electrophysiology. Using standard pharmacological approaches, we find that currents gated by concentrations of taurine

In an evaluation of the contribution of swelling-induced amino acid release, through the regulatory volume decrease (RVD) process, to cerebral ischemic injury, studies of the role of phospholipases and protein kinases in the response to hyposmotic stress were undertaken using an in vivo rat cortical cup model. Hyposmotic stress induced significant releases of aspartate, glutamate, glycine, phosphoethanolamine, taurine and GABA from the rat cerebral cortex. Taurine release was most affected, exhibiting a greater than 9-fold increase during the hyposmotic stimulus. The phospholipase A2 (PLA2) inhibitors 4-bromophenacyl bromide (1 microM) and 7,7-dimethyleicosadienoic acid (5 microM) had no significant effects on hyposmotically induced amino acid release. AACOCF3 (50 microM), an inhibitor of cytosolic PLA2 decreased taurine release to 84% of DMSO controls. The release of the other amino acids was not affected. The phospholipase C inhibitor U73122 (5 microM) had no significant effects on amino acid release. The protein kinase C (PKC) inhibitor chelerythrine (5 microM) significantly reduced hyposmotically induced taurine release to 72% of saline controls but had no significant effects on the other amino acids. Stimulation of PKC with phorbol 12-myristate, 13-acetate (10 microM) did not significantly change taurine, glutamate, glycine or phosphethanolamine release. The releases of aspartate and GABA were enhanced 2 to 3 fold. Phorbol 12,13-didecanoate (10 microM), another potent stimulator of PKC, significantly increased taurine release to 122% of DMSO controls. The releases of aspartate, glutamate and glycine were enhanced 2.5 to 3.5 fold. Similarly, stimulation of protein kinase A with forskolin (100 microM) significantly increased taurine, aspartate, and glycine release 1.5- to 2-fold compared to DMSO controls. In summary, phospholipases may play a minor role in volume regulation. These studies also support the hypothesis that protein kinases play a modulatory role in the RVD response. The results show that although RVD may play a role, additional mechanisms, including phospholipase activation, must be involved in the ischemia-evoked release of excitotoxic amino acids.

Postmortem alterations in the neuronal cytoskeleton resemble some aspects of the cytoskeletal disruption associated with neurodegenerative disorders, and are also similar to those observed following ischemia and produced by excitotoxins in vivo and in vitro. This suggests the involvement of excitotoxic mechanisms during the postmortem interval. The purpose of this study was to determine if extracellular levels of glutamate are elevated postmortem. Extracellular levels of GABA and taurine were also monitored using in vivo microdialysis. These three amino acids were analyzed using high-performance liquid chromatography. When postmortem rat brain temperature cooled rapidly to near room temperature, dialysate concentrations of glutamate were not increased in the hippocampal CA1 region during a 2-h postmortem interval, although increased extracellular levels of GABA and taurine were observed. In contrast, maintenance of brain temperature at 37 degrees C resulted in a 12-to-40 fold elevation in extracellular glutamate levels 20-120 min postmortem. In addition, the elevation in dialysate taurine concentration was greater than that observed in rats in which postmortem brain temperature was not maintained. Excitatory amino acid antagonists, NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylohepten-5, 10-imine hydrogen maleate blocked the additional elevation in taurine associated with maintaining brain at 37 degrees C, but had less robust effects against glutamate and GABA release. The results indicate that extracellular concentrations of glutamate, taurine and GABA increase in postmortem rat brain when physiologic temperatures are maintained, but that these increases are blunted when brain temperature decreases. After death, the human brain cools much more slowly than does the rat brain. Therefore, extracellular glutamate levels are likely to increase in the postmortem human brain and may contribute to excitotoxic neuronal damage occurring in the interval between death and autopsy., (Copyright 1999 Elsevier Science B.V.)

The contributions of sodium and chloride ions and of osmotic stresses to the ischemia-evoked efflux of excitotoxic and other amino acids were explored using a rat four vessel occlusion model. Replacement of Na+ with choline or N-methyl-D-glucamine (NMDG) and of Cl- with sulfate or gluconate was used to evaluate the contribution that these ions make to amino acid efflux. The contribution of ischemia-evoked swelling to amino acid release was studied by applying mannitol or sucrose to minimize the cell volume increases and the compensatory regulatory volume decrease evoked efflux of amino acids. Aliquots of artificial cerebrospinal fluid (aCSF), appropriately adjusted for ion replacement or 150 mM mannitol or sucrose, were pipetted into cortical cups 35 min prior to ischemia and perfusate samples were obtained prior to, during and following ischemia (20 min) and reperfusion (40 min). Replacement of Na+ by NMDG depressed basal (normoxic) efflux of most amino acids, with choline substitution having little effect. During ischemia NMDG substitution increased glutamate and GABA efflux and choline enhanced the release of aspartate, glutamate, GABA and taurine. A reduction in extracellular Na+ would facilitate reversal of Na+-dependent transporters with extrusion of amino acids. Another possible explanation for the elevated release is that the absence of Na+ would inhibit the Ca2+/Na+ counter transport system, with a deleterious accumulation of intracellular Ca2+. Chloride replacement with sulfate or gluconate enhanced the efflux of aspartate, glutamate, GABA and taurine during ischemia. Removal of Cl- would depolarize cells, and block the Cl--dependent action of inhibitory amino acid transmitters, with both actions enhancing the ischemic injury and, consequently, amino acid release. Exposure to hyperosmotic mannitol (150 mM) aCSF enhanced ischemia-evoked release of some amino acids (taurine, GABA) and decreased that of aspartate and phosphoethanolamine. Sucrose aCSF enhanced the ischemia-evoked release of most amino acids. A potential explanation for these observations is that both agents may be able to rapidly penetrate the plasma membranes of ischemic neurons, actually contributing to the release of other osmolytes. The unanticipated nature of many of the observations made during these manipulations of the aCSF serves to accentuate the complex nature of the mechanisms responsible for the ischemia-evoked amino acid efflux into the interstitial spaces., (Copyright 1999 Elsevier Science B.V.)

The glutamate concentrations in dialysate samples obtained from microdialysis probe implanted in the cortex were assayed during artificially induced spreading depression (SD) and SD with hypoperfusion. The glutamate concentrations did not differ even after SD induction with hypoperfusion (all p>0.05 cf. control), whereas anoxic depolarization caused significantly high glutamate release. Prolonged SD in hypoperfused area did not expose cerebral neurons to high glutamate concentrations., (Copyright 1999 Elsevier Science B.V.)

Taurine, a regulatory amino acid of various biochemical processes in the retina, requires an efficient uptake system to maintain the high physiological concentration of taurine in the retina. Taurine uptake was characterized in both whole retinal preparations and in isolated rod outer segments (ROS) in terms of uptake kinetics and possible protein kinase C (PKC)-dependent regulation. Two uptake systems, a high- and a low-affinity system, were found in whole retinal preparations while only the high-affinity system was found in the isolated ROS. All the uptake systems characterized were inhibited by guanidinoethane sulfonate (GES), a well-known competitive inhibitor of taurine uptake. Stimulation and inhibition of PKC activity with phorbol myristate acetate and with staurosporine, respectively, produced no significant effect on taurine uptake. On the other hand, chelerythrine (CHT), a documented potent PKC inhibitor, was found to cause significant inhibition of the two taurine uptake systems, presumably through a PKC-independent mechanism. The data demonstrate that CHT may be a useful tool in studying taurine uptake in the retina and specifically in the ROS., (Copyright 1999 Elsevier Science B.V.)

Swelling of neonatal rat primary astrocyte cultures by hypotonic media leads to regulatory volume decrease (RVD) and the resumption of resting cell volume. RVD is associated with activation of conductive K+ and Cl- channels, allowing for the escape of KCl, as well as the release of osmoregulators, such as taurine and myoinositol. As we have previously shown [D. Vitarella, H.K. Kimelberg, M. Aschner, Inhibition of RVD in swollen rat primary astrocyte cultures by methylmercury (MeHg) is due to increase amiloride-sensitive Na+ uptake, Brain Res. 732 (1996) 169-178.], MeHg, when added to hypotonic buffer inhibits RVD, primarily due to increased cellular permeability to Na+ via the Na+/H+ antiporter. The present study was, therefore, undertaken to assess the ability of cation-anion cotransport blockers to reverse the inhibitory effect of MeHg on RVD in swollen astrocytes, and to further characterize MeHg-induced changes in astrocytic osmoregulatory release processes. The studies demonstrate the following: (1) MeHg-induced inhibition of RVD is partially inhibited by the Na+/H+ antiporter blocker, amiloride, but not SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), furosemide or bumetanide; (2) exposure of swollen astrocytes to MeHg is associated with specific effects on osmoregulatory release, leading to significant inhibition of taurine release and a significant increase in potassium and myoinositol release compared with release in hypotonic conditions., (Copyright 1998 Elsevier Science B.V.)

Taurine release in the hippocampus is markedly potentiated in various cell-damaging conditions, including ischemia and excitotoxic damage produced by glutamate. The increase in the levels of taurine may provide an important protective mechanism against excitotoxicity. The mechanisms of the enhanced release were now studied in mouse hippocampal slices using a superfusion system. The basal release of [3H]taurine was significantly increased in Na+-deficient media in normal conditions, whereas the ischemia-evoked release was decreased, indicating the participation of Na+-dependent transport processes. The involvement of taurine transport carriers in the release was confirmed with the structural analogs, hypotaurine and beta-alanine. These amino acids potentiated the release by trans-stimulation in normoxia. In Na+-free conditions, this heteroexchange was not discernible, the carriers not being functional without Na+. In ischemia, the marked potentiation of taurine release by hypotaurine and beta-alanine further indicates that the Na+-requiring transporters also operate in ischemia. The effects of membrane disruption on taurine release due to activation of phospholipases were estimated using phospholipase and protein kinase inhibitors, which had no marked effects on hippocampal taurine release. The chloride channel blockers, 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonate (SITS) and diisothiocyanostilbene-2,2'-disulphonate (DIDS), reduced the ischemia-induced release, suggesting that taurine diffusion through an anion channel is partially responsible for the enhanced release in ischemia., (Copyright 1998 Elsevier Science B.V.)

Metallothionein (MT) proteins have been postulated to play a role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, and MT-1 and MT-2 are astrocyte-specific MT isoforms, we investigated the ability of MTs to attenuate MeHg-induced cytotoxicity. The toxic effects of MeHg on astrocytes were investigated in a model of regulatory volume decrease (RVD) in which the cells are swollen by exposure to a hypotonic buffer. Preexposure to CdCl2 (1 microM) for 72, 96 or 120 h, prior to acute exposure to hypotonic buffer and MeHg (10 microM) led to a time-dependent increase in the intracellular levels of astrocyte MT proteins. The acute MeHg-induced inhibition of RVD was significantly, and almost fully reversed by preexposure to CdCl2. This reversal was time-dependent, 120-h preexposure to CdCl2 producing the greatest reversibility. Furthermore, the ability of astrocytes to efficiently volume regulate in the presence of MeHg-containing hypotonic buffer was highly correlated (r = 0.99) with the intracellular levels of MT proteins. The release of [3H]taurine, an osmolyte involved in the RVD process was also measured. The inhibitory effect of MeHg on [3H]taurine in swollen cells was significantly, and fully reversed by CdCl2 preexposure. The study suggests that astrocytes induced to express high levels of MT proteins are resistant to the acute inhibitory effect of MeHg on RVD.

The extracellular changes of amino acids (glutamate, taurine and GABA) in the nucleus accumbens of freely moving rats were estimated using the microdialysis technique following acute and chronic ethanol injections (1, 2, and 3 g/kg body weight). Compared to baseline values, taurine increased by 154% +/- 73%, 142% +/- 40% and 162% +/- 75% 20 min after the acute injection of respectively, 1, 2, and 3 g/kg body weight ethanol, while 40 min after ethanol injection, taurine had increased by 124% +/- 36%, 146% +/- 54% and 168% +/- 98%. No changes in either glutamate or GABA were detected at any time points assayed. In the rats which had received chronic ethanol administration prior to a further acute ethanol injection (1, 2, and 3 g/kg body weight), taurine increased by 138% +/- 73%. 144% +/- 39% and 180% +/- 85% 20 min after the ethanol injection at 40 min post ethanol injection taurine had increased by 134% +/- 44%, 160% +/- 56% and 158% +/- 45%, compared to the basal baseline value. No significant changes were observed in either glutamate or GABA microdialysate content in these chronic studies. The biological role played by taurine after acute ethanol injection in the nucleus accumbens remains unclear but may be associated with a yet, undefined mechanism, in reducing the cytotoxicity of ethanol.

Primary astrocyte cultures from neonatal rats were swollen by exposure to hypotonic buffer with and without 10 microM methylmercury (MeHg). We investigated the effects of MeHg on K+ (using 86Rb), taurine, D-aspartate (a non metabolizable analogue of glutamate) and Na+ fluxes during regulatory volume decrease (RVD), with an electrical impedance method for determination of cell volume, coupled with on-line measurements of efflux of radioactive ions and amino acids. Addition of 10 microM MeHg completely inhibited RVD in swollen astrocytes, increased the uptake of 22Na+, increased 86Rb release, and decreased 3H-taurine release. There was no effect on the rate of release of 3H-D-aspartate from swollen astrocytes. 0.5 mM amiloride completely inhibited MeHg-induced increased Na+ influx during RVD, while 1 mM furosemide had no effect. When Na+ in the hypotonic buffer was replaced with N-methyl-D-glucamine (NMDG), RVD in the presence of MeHg was indistinguishable from controls. These results indicate that MeHg increases cellular permeability to ions such as Na+ and K+, and that an increase in Na+ permeability via Na+/H+ exchange, offsetting K+ loss, is the primary mechanism in its inhibition of RVD in swollen astrocytes.

The putative osmoregulatory agent, taurine, is lost from the brain during hypo-osmotic stress or ischemia, but the regulatory mechanisms involved in this loss have not been fully elucidated. In this study, we have examined taurine transport by the isolated rat choroid plexus, one element of the brain-blood interface, and examined how it may be regulated as part of brain volume regulation. Choroid plexus taurine uptake was Na- and Cl-dependent with a Vmax and Km of 6.5 +/- 0.3 pmol/mg/min and 232 +/- 33 microM. The latter is substantially greater than the normal CSF taurine concentration and this may be important in removing taurine released into the CSF during parenchymal cell swelling. Taurine uptake also appears calmodulin dependent as it was reduced by 84 and 91% in the presence of 25 microM trifluoperazine and 100 microM W-7, two calmodulin inhibitors. Taurine efflux from choroid plexus was stimulated by trifluoperazine, taurine, and hypo-osmotic stress. The latter two effects were reduced by niflumic acid, suggesting that taurine and hypo-osmotic stress act on the same pathway. The stimulation of efflux by hypo-osmotic stress decreased with time, whereas the effect of external taurine was sustained. If this efflux pathway is involved in the movement of taurine from choroid plexus to blood, these results suggest that changes in extracellular taurine may be more important than the direct effect of hypo-osmolality in the long-term loss of taurine from the brain.

L-Cysteine produces excitotoxic brain damage but its chemical structure differs from that of other excitotoxins. Although it is an NMDAmimetic, its mode of action is complex and may encompass antiexcitotoxic components. The purpose of the present study was to investigate whether cysteine kills neurons by potentiating the effects of glutamate and/or by releasing glutamate. In primary cultures of cortical neurons, 24 h of exposure to glutamate caused a concentration-dependent, dizocilpine-sensitive cell death as measured by release of lactate dehydrogenase. Cysteine was also toxic but higher concentrations were required. In addition, N-acetylcysteine produced mild toxicity at 1 mM. There was no general potentiation between either glutamate and cysteine or glutamate and N-acetylcysteine although some combinations acted synergistically. In no case did the thiols inhibit glutamate toxicity. The interaction between glutamate and cysteine toxicity was also assessed in the immature rat arcuate nucleus in vivo. When given at a dose (0.5 mg/g) that did not cause any toxicity per se, cysteine enhanced the toxicity of glutamate (0.3-0.8 mg/g). Cortical microdialysis was carried out in anesthetized rats (8-10 days old) administered a toxic dose of cysteine (1 mg/g). The levels of taurine were elevated 15-fold, phosphoethanolamine 3-fold and alanine 2-fold. Despite the observation that glutamine decreased markedly and rapidly, there was only a delayed doubling of glutamate concentrations. It is therefore unlikely that cysteine induces neurotoxicity by releasing glutamate. Taken together, the results suggest that there is a synergistic effect between cysteine and glutamate. Speculatively, this potentiation may be produced by reduction by cysteine of the redox site of the glutamate-activated NMDA receptor-ionophore complex.

The release of amino acids in the locus coeruleus (LC) of conscious, freely moving rats was studied in time periods of 3 min by use of push-pull superfusion under basal conditions and during application of various experimental stimuli known to influence the activity of the LC-noradrenergic system. Tail pinch for 3 min led immediately to a pronounced tetrodotoxin-sensitive increase in the release rates of the excitatory amino acids (EAA) glutamate (Glu) and aspartate (Asp) and to moderate increases in GABA and taurine (Tau) outflow. Immobilization stress for 9 min elevated the release of the EAA Glu and Asp, as well as that of the inhibitory amino acid GABA to a similar extent. A fall of blood pressure (BP) by nitroprusside or haemorrhage slightly enhanced the release rates of Glu and Asp. Noradrenaline-induced rise in BP, as well as hypervolaemia increased the release rate of GABA, but did not influence the release rates of Glu, Asp, Tau and arginine (Arg). The results provide direct evidence that the amino acid release pattern in the LC of conscious rats differs in response to various stimuli, according to the modality of the stimulus. A functional significance of excitatory and inhibitory amino acids in the regulation of LC activity during stress and haemodynamic changes is suggested.

Cell volume, potassium content, and potassium influx were measured in rat cerebral astrocytes grown in primary culture following exposure to hypoosmotic medium containing either 3.2 mM or 50 mM potassium. Some solutions also contained 1 mM L-644,711, an anion transport inhibitor. L-644,711 inhibited volume regulation and taurine efflux induced by hypoosmotic exposure in medium containing either potassium concentration. L-644,711 also inhibited potassium uptake associated and not associated with the sodium/potassium pump. The correlation of reduced taurine efflux and volume decrease produced by L-644,711 exposure indicates the important role for this amino acid in hypoosmotic astrocyte volume regulation. However, the effects of L-644,711 on potassium transport indicate that multiple actions of this drug may be important factors in its effect on astrocyte volume regulatory mechanisms.

Mercuric chloride (MC) and methylmercury (MeHg) were found to increase amino acid release from astrocytes. This suggests interaction with sulfhydryl (-SH) groups which are controlled by glutathione [GSH] levels. In the present study, we evaluated the effects of alterations in intracellular glutathione concentrations [GSH]i on the outcome of MC and MeHg treatment. [GSH]i were increased in a time-dependent fashion by incubating the astrocytes with 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor. OTC attenuated the release of [2,3-3H]D-aspartic acid from astrocytes exposed to MC- (5 microM) and MeHg-(10 microM). MeHg-induced [3H]D-taurine release was also reduced by pretreatment of astrocytes with OTC. Treatment with BSO (50 microM) decreased [GSH]i in astrocytes, and increased [2,3-3H]D-aspartate release from MC- and MeHg-treated astrocytes, and [3H]D-taurine release from MeHg-treated cells. Neither OTC nor BSO when added to cultures in the absence of MC or MeHg had an effect on amino acid release by astrocytes. The current study underscores both the sensitivity of astrocytes to mercurials in terms of amino acid release and the relationship of these effects of astrocytic [GSH]i.

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), gamma-aminobutyric acid (GABA) and D-aspartate (D-ASP), an analog of L-glutamate (L-GLU), from cultured rat cortical astrocytes was studied. NH4Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na+ was removed, and was almost completely abolished when Cl- was omitted. In contrast, TAU basal release was enhanced upon removal of Na+ or Cl-. Ammonia inhibited the release of GABA and D-ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.

We have assessed by means of immunocytochemistry, the cellular distributions of the amino acid transmitters GABA, glycine and glutamate, and the free-radical scavenger taurine, in the retinae of adult rabbits at various times after death. Within 10 min of death, horizontal cells began to display immunoreactivity for GABA, whilst displaced amacrine cells began to display immunoreactivity for glycine. By 40 min postmortem, GABA was present in glial cells. Glutamate, which is not normally detectable in retinal glia, was detected in such glia by 20 min postmortem. By contrast immunocytochemically detectable glycine did not accumulate in glia. There was a gradual diminution of immunoreactivity for taurine in glial cells and photoreceptors. By 2 h postmortem, most immunoreactivity had disappeared from the retina. We conclude that amino acid transmitters show rapid changes in their distributions immediately after death, which may be related to changes in the patterns of transmitter release and uptake, and changes in degradation mechanisms. The rapid changes in cellular localisation of amino acid immunoreactivity illustrated in this study, indicate that the fixation of nervous tissues must be performed rapidly. Moreover, the massive loss of immunoreactivity by 2 h postmortem suggests that any assays for content of these transmitters at this, and subsequent time-points, will bear little resemblance to the values obtained at the time of death.

Untreated rats and rats given the A1 receptor adenosine agonist, R-phenylisopropyladenosine (R-PIA), were subjected to four vessel ischemia. The effect of R-PIA on hippocampal amino acid release, hippocampal neuronal damage, exploratory behavior, learning capacity and global neurological score were evaluated. R-PIA decreased by half the glutamate released during ischemia and improved the global neurological scores 3, 24, 48, 78 h and 7 days after ischemia. But R-PIA had no effect on taurine/GABA release (during ischemia), hippocampal neuronal damage (7 days post-ischemia), exploratory behavior (48 h post-ischemia) or learning capacity (7 days post-ischemia). Thus, a decrease in glutamate release by R-PIA is not systematically correlated with an improvement of histological damage or learning capacity. Reduced glutamate release is not therefore a sufficient criterion on which to evaluate the neuroprotective capacity of a drug.

The opioid receptor antagonist nalmefene improves cellular bioenergetics and attenuates the reduction in tissue glutamate levels after global cerebral ischemia/reperfusion. The latter finding suggests that nalmefene might inhibit glutamate release during ischemia. To test this hypothesis, we used microdialysis techniques to examine the effect of nalmefene pretreatment on extracellular excitatory amino acid levels during global cerebral ischemia in rats. Saline, (-)-nalmefene (20, 100 or 500 micrograms/kg) or the inactive nalmefene enantiomer (+)-nalmefene (100 micrograms/kg) were given 15 min prior to induction of ischemia using a multi-vessel occlusion model. Pretreatment with (-)-nalmefene decreased peak dialysate glutamate in a dose-dependent fashion as compared to saline-treated controls, whereas (+)-nalmefene had no effect. These results suggest that opioid receptors may modulate glutamate release during ischemia and that inhibition of excitatory amino acid release may contribute to the protective actions of opioid receptor antagonists in cerebral ischemia.

Continuous perfusion methods are widely used to monitor release of substances, particularly transmitters, from brain cell cultures growing as monolayers. However, if stimuli used to produce release also cause loss or lysis of cells, the appearance of label in the perfusate due to such effects will be indistinguishable from release. Using a perfusion method we have studied release of preloaded, radiolabelled amino acids from primary astrocyte cultures due to a variety of stimuli; hypotonic or high K+ media, activation of beta-receptors or swelling-induced release due to isosmotic ethanol. In this study primary astrocyte cultures were simultaneously labelled with Na2(51)CrO4 and allowed to take up radiolabelled D-aspartate or taurine. It was found that while all of the above methods caused release of radiolabelled amino acids none caused release of 51Cr into the perfusion fluid. In contrast, perfusion with 0.05% (v/v) Triton X-100 did lead to release of 51Cr. Thus a variety of means of inducing swelling or shape changes in astrocytes causes true release of radiolabelled amino acids and simultaneously monitoring 51Cr release seems a convenient means of distinguishing such release from cell loss or lysis.

[3H]Taurine uptake and release was characterized in astrocytes from rat cerebral cortex grown in normal and hyperosmotic culture conditions to investigate mechanisms of cell volume regulation and adaptation to states of altered osmolality. In high concentrations of taurine (1 mM), uptake was linear in both osmotic conditions for at least 30 min. The uptake rate in 1 mM taurine was not affected by exposure to hyperosmotic conditions. The mean +/- S.E.M. apparent binding constant for carrier-mediated taurine transport, Km, was not altered by hyperosmotic conditions (22.8 +/- 5.1 microM in iso-osmotic media, 21.3 +/- 11.9 microM in hyperosmotic media). However, maximal velocity of uptake, Vmax (mean +/- S.E.M.), of taurine was significantly lower in hyperosmotically treated astrocytes (0.175 +/- 0.035 nmol/mg protein.min) compared with the Vmax of iso-osmotically treated astrocytes (0.299 +/- 0.026 nmol/mg protein.min). The diffusional transport rate, Kdiff, was not affected by growth in hyperosmotic conditions (0.221 +/- 0.033 microliter/mg protein.min in iso-osmotic media, 0.295 +/- 0.043 microliter/mg protein.min in hyperosmotic media). Taurine release rate, expressed as a percent of the total cell content, was not affected by hyperosmotic exposure. However, astrocytes grown in hyperosmotic conditions contain nearly 60% more taurine than control cells. Thus, the absolute rate of taurine release (mean +/- S.E.M.) was significantly larger (P < 0.05) in hyperosmotic cells (0.1592 +/- 0.0082 nmol/mg protein.min) compared with control cells (0.0943 +/- 0.0096 nmol/mg protein.min). Quantitative analysis of these data indicate that maintenance of elevated taurine contents by cultured cerebral astrocytes exposed to hyperosmotic conditions is not due to alterations in rates of transport.

The localizations of taurine, gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) within the developing rat optic nerve were determined using immunocytochemical techniques on tissues from animals ranging in age from embryonic day 20 to postnatal 28 days. Mature nerves from 3-4-month-old adults were also examined. At the younger ages, taurine immunoreactivity was intense and localized specifically to the optic nerve axons, but by postnatal day 15 and thereafter its predominant localization was in macroglia. Some of these glia were astrocytes as indicated by the specific marker, GFAP. GABA immunoreactivity was present at the same time as taurine but was found only in macroglia. In mature nerves the patterns of taurine, GABA and GFAP distribution (within glia) were highly similar.

BW 1003C87, 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine ethane sulphonic acid, has been tested for its in vitro and in vivo effects on glutamate release in rat brain tissue, and for its cerebro-protective action in two rodent models of cerebral ischemia. In rat brain slices the release of glutamate evoked by veratrine is inhibited by BW 1003C87 (IC50 = 1.6 microM). In anaesthetised rats with microdialysis probes implanted in the dorsal hippocampus the increase in extracellular glutamate evoked by veratrine is markedly reduced by co-infusion of BW 1003C87, 100 microM. In anaesthetised rats with microdialysis probes implanted in the cortex and the caudate nucleus ipsilateral to a middle cerebral artery (MCA) occlusion the increase in dialysate glutamate concentration seen in the first 2 h following MCA occlusion is markedly attenuated by the prior administration of BW 1003C87, 20 mg/kg i.v. In rats subjected to 10 min of bilateral common carotid artery occlusion the loss of CA1 pyramidal neurons (assessed 7 days later) is reduced by administration of BW 1003C87 (20 mg/kg i.v., at the time of ischemia and 4 h later). The volume of cortex showing infarction 72 h after unilateral MCA occlusion is reduced by treatment with BW 1003C87 (20 mg/kg, i.v., beginning 5 min after occlusion). Inhibition of glutamate release may provide a therapeutic approach in cerebral ischemia as well as in epilepsy.

To understand the initial events in chemosensory transduction (i.e. binding of odorants to olfactory receptor sites), we have utilized a radioligand-receptor binding assay on a tissue preparation that is enriched in dendritic membrane from olfactory receptor cells in the spiny lobster Panulirus argus. Radioligands used were tritiated adenosine 5'-monophosphate (AMP) and taurine, which are two of the most excitatory compounds for spiny lobsters. Our results indicate the existence of two independent types of binding sites--a taurine binding site and an AMP binding site. For both the taurine and AMP binding sites, odorant binding is rapid, reversible, and saturable. KD values for the taurine and AMP binding sites are 2.3 and 2.0 microM, respectively, and Bmax values are 159 and 3.2 fmol/micrograms protein, respectively. The fact that the specificity, affinity, and independence of these two binding sites as defined in these biochemical studies are in agreement with those from electrophysiological studies suggests that these binding sites are olfactory receptor sites involved in sensory transduction.

The effect of repeated administration of either methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) or vehicle on the extracellular concentrations of glutamate (GLU), aspartate, taurine, dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), was studied in awake, freely moving rats using in vivo microdialysis. MA (7.5 mg/kg, i.p.) administered every 2 h for a total of 3 injections, increased the extracellular concentration of GLU in the anteromedial striatum. By contrast, neither vehicle nor MDMA (9.2 and 13.8 mg/kg) increased GLU efflux following repeated administration. Both MA and MDMA increased the extracellular concentration of DA in the striatum. However, the cumulative increase in DA was significantly greater in the MDMA treated animals as compared to the MA group. The concentrations of DA, serotonin (5-HT) and their metabolites were determined in the striatum 7 days following the repeated administration of MA, MDMA and vehicle. MA, but not MDMA or vehicle, decreased the concentration of DA in the striatum. Conversely, MDMA (13.8 mg/kg) decreased the concentration of 5-HT, whereas MA, MDMA (9.2 mg/kg) and vehicle had no effect on striatal 5-HT content. These data are suggestive that the long-term (7 day) DA neurotoxicity produced by the repeated administration of MA is mediated, in part, by a delayed increase in extracellular concentrations of GLU. In contrast, repeated administration of MDMA, at a dose which produced a long-term (7 day) depletion of striatal 5-HT content, had no effect on GLU efflux in the striatum.

Microdialysis sampling was used to measure the release of oxytocin (OXY) and monoamine and amino acid transmitters from the region of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST) during parturition and suckling in sheep. Results showed that OXY and gamma-aminobutyric acid release increased in both the MPOA and BNST during parturition and suckling. Noradrenaline (NA) release increased in both structures during parturition but not during suckling. Dopamine (DA) release increased in the MPOA and decreased in the BNST during both parturition and suckling. Aspartate release increased in the MPOA during parturition, and the BNST during suckling, and glutamate release increased in the MPOA and BNST at parturition and only in the BNST during suckling. No changes in the release of serotonin or taurine occurred in these structures during parturition or suckling. In a further experiment on 6 estrogen-primed sheep, OXY (10 micrograms/ml) was infused into the MPOA via bilaterally placed microdialysis probes. This treatment inhibited rejection behavior towards lambs, but did not activate positive maternal responses. These OXY infusions also stimulated release of NA. These results show that complex patterns of neurochemical release occur in two closely related areas of the brain, the BNST and MPOA, during parturition when maternal behavior is stimulated. However, while these patterns of release are similar in the two structures, particularly at birth when maternal behavior is stimulated, they are not identical during labor contractions and suckling. The release of oxytocin within the MPOA during parturition may be important for stimulating a reduction in aggression towards lambs, although this action might be mediated via the effect of OXY on NA release.