Neurotensin increases endogenous glutamate release in rat cortical slices

In the present study, the effects of the tridecapeptide neurotensin [NT(1-13)] and its fragments, NT(1-7) and NT(8-13), on endogenous glutamate release from rat cortical slices, were evaluated. NT(1-13) (100-1000 nM) slightly increased spontaneous glutamate release, while it was ineffective at 1 and 10 nM concentrations. Neither the biologically active NT fragment NT(8-13) nor the inactive one NT(1-7) affected basal glutamate release. NT(1-13) (1-1000 nM) enhanced potassium (35 mM)-evoked glutamate release displaying a bell-shaped concentration response curve. In addition NT(8-13) (10 nM) increased K+-evoked-glutamate release similarly to the parent peptide (10 nM), while the biologically inactive fragment NT(1-7) (10-100 nM) was ineffective. The effects of NT(1-13) and NT(8-13) were fully counteracted by the selective neurotensin receptor antagonist SR48692 (100 nM). These findings suggest that NT plays a role in regulating cortical glutamate transmission.     

Cyclic AMP-regulated GABA release at inhibitory synapses in rat cerebellar slices

We have investigated the effects of agents interfering with the cAMP pathway on the rate of miniature IPSCs in cerebellar slices. Noradrenaline and group II glutamate metabotropic receptor agonists respectively enhance and reduce the rate of miniature IPSCs, presumably because they respectively increase and decrease the presynaptic concentration of cAMP.     

Enhanced GABA release in cerebral cortical slices derived from rats with thioacetamide-induced hepatic encephalopathy

The release of newly loaded [³H]GABA was studied in slices of different brain regions derived from rats in which acute hepatic encephalopathy (HE) was induced with a hepatotoxin thioacetamide. HE increased both spontaneous and high (50 mM) ammonium chloride-evoked GABA release in cerebral cortical slices by 38% and 50%, respectively. No effects of HE were noted in cerebellar or striatal slices. An increased release of GABA in the cerebral cortex may contribute to the endogenous benzodiazepine-mediated enhancement of GABAergic tone, which is thought to be partly responsible for the pathophysiological mechanism of HE.     

GABA augments basal and electrically stimulated H-norepinephrine release in hypothalamic, preoptic area and cortical slices of female rats

These studies examined the regulation by GABA of norepinephrine release from hypothalamus, preoptic area and frontal cortex. Using superfused brain slicesfrom female rats, we show that 100 μM GABA enhances both basal and electrically stimulated release of ³H-norepinephrine in all three brain regions. The GABA_A agonist muscimol (100 μM) significantly augments ³H-norepinephrine release, but it is somewhat less effective than GABA. The GABA_B agonist baclofen has little or no effect on basal ³H-norepinephrine efflux. GABA also augments both the magnitude and duration of electrically evoked ³H-norepinephrine release in slices from all three brain regions. GABA facilitation of electrically stimulated ³H-norepinephrine release is mediated through GABA_A receptors as evidenced by its blockad by 10 μM bicuculline, a GABA_A antagonist, but not by 200 μM 2-OH-saclofen, a GABA_B antagonist. These data show that the inhibitory amino acid neurotransmitter GABA enhances both basal and evoked release of ³H-norepinephrine in brain slices from female rats. These effects are predominantly mediated by GABA_A receptors. GABA modulation of hypothalamic norepinephrine release may play a role in the regulation of gonadotropin secretion and reproductive behaviors such as lordosis.     

Inhibitory cholinergic control of endogenous GABA release from electrically stimulated cortical slices and K-depolarized synaptosomes

In the present study we characterize the optimal experimental conditions under which to investigate the cholinergic regulation of endogenous electrically evoked γ-aminobutyric acid (GABA) release from guinea pig cortical slices. Superfusion with the neuronal GABA reuptake inhibitor, SKF89976A (10 μM) caused cortical GABA release to be linearly correlated with the frequency of electrical stimulation (5, 10, 20 Hz). Electrically evoked GABA release (10 Hz) was tetrodotoxin-sensitive and Ca²⁺-dependent and was under GABA_B autoreceptor control. Under these experimental conditions, acetylcholine (0.1–10 μM) and physostigmine (30 μM) decreased the electrically evoked GABA release while the M₂ receptor antagonist AFDX-116 (0.01–0.1 μM) counteracted these effects. Similar results were also observed in a cortical synaptosomal preparation stimulated with K⁺ (10 mM). These findings demonstrate an inhibitory cholinergic regulation of electrically evoked GABA release via M₂ receptors located on cortical GABAergic terminals.     

Separation of carrier mediated and vesicular release of GABA from rat brain slices.

In this study the temperature dependence of [3H]GABA release from brain slices evoked by electrical field stimulation and the Na+/K+ ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S2/S1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 microM) inhibited stimulation-induced [3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 microM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

Mechanism by which GABA, through its GABA(A) receptor, modulates glutamate release from rat cortical neurons in culture

In cortical neurons, the GABA(A) agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99 +/- 7 microM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABA(A) antagonist. When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCl concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30-100 microM of KCl), it produced a hyperpolarization in these cells. The mechanism by which the GABA-Cl(-)-channel permits Cl- fluxes, inward or outward, depending on the membrane potential.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

Effect of pyrithiamine treatment of potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enchanced Na⁺/K⁺ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K⁺ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K⁺ was monitored directly with a K⁺-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K⁺ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K⁺ upon tetrodotoxin addition was inversely related to the extracellular Ca²⁺ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.     

Oxalate accumulation in rat renal cortical slices

Tubular transport of oxalate is thought to be an energy-mediated process which may contribute to the renal deposition of calcium oxalate in a variety of pathologic states. In order to examine this possibility, the renal handling of oxalate was investigated in rat renal cortical slices in vitro. Slices incubated in vitro with 1 microM [14C]oxalate in Krebs-Ringer bicarbonate buffer at 25 degrees C for 180 min achieved a mean slice to medium ratio of 2.8 +/- 0.08 (SEM) and a mean tissue concentration of 7.7 +/- 0.2 mumol/kg dry wt (N = 64). Section freeze-dry autoradiographs demonstrated maximum uptake within proximal tubule cells but no crystals were evident. Substituting N2 for O2, adding KCN, or removing Ca2+ increased uptake of 14C-oxalate. Dinitrophenol (DNP) and iodoacetamide (IoAc), however, significantly decreased, and O degrees C eliminated slice uptake. Slices incubated with 100 microM [14C]oxalate showed a further increase in tissue accumulation and the appearance of [14C]oxalate crystals. Crystals formed in vitro were deposited throughout the tissue. Oxalic acid did not appear to share the organic acid by renal cortical slices in vitro is largely independent of energy-mediated mechanisms.     

Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with 3H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 microM. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro.     

Effect of pyrithiamine treatment on potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enhanced Na+/K+ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K+ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K+ was monitored directly with a K+-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K+ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K+ upon tetrodotoxin addition was inversely related to the extracellular Ca2+ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.     

Possible reasons for the failure of glutamine to influence GABA release in rat hippocampal slices; Effect of nipecotic acid and methionine sulfoximine

Although labelled glutamine is readily incorporated into labelled releasable GABA, it has been shown recently that high concentrations (0.1–0.5 mM) glutamine do not increase the release of GABA from brain slices, while greatly enhancing that of glutamate. Two possible reasons for this discrepancy were investigated: (a) That released GABA, in contrast to glutamate is not freshly synthesized but derives from GABA taken up by terminals. The possibility was made unlikely by the present finding which showed that even in the presence of the uptake inhibitor nipecotic acid, glutamine failed to enhance GABA release. (b) That glutamine is transported into GABA-ergic terminals by a high-affinity transport system which is saturated even at low glutamine concentrations obtained without adding glutamine to the superfusion fluid. However, when glutamine efflux was further reduced by prolonging depolarization with 50 mM K⁺ and by pretreatment with the glutamine synthetase inhibitor methionine sulfoximine, GABA release was depressed only very little and this decrease was related to the duration of depolarization and not to extracellular glutamine levels. These results can be reconciled with the ready incorporation of labelled glutamine into releasable GABA by assuming that GABA originates from a glutamate pool to which both glutamine and glucose contribute. The formation of releasable GABA however, is not governed by the supply of glutamate in this pool but by the activity of the rate-limiting enzyme glutamate decarboxylase.     

GABA transaminase inhibitors enhance the release of endogenous GABA but decrease the release of beta-alanine evoked by electrical stimulation of slices of the rat medulla oblongata

Slices of the rat medulla oblongata were superfused and electrically stimulated. The amount of endogenous GABA, beta-alanine and glutamate release from the slices was determined by high performance liquid chromatography with fluorometric detection. Inhibitors of GABA-transaminase (GABA-T), aminooxyacetic acid (10(-5) M), gamma-acetylenic GABA (10(-4) and 10(-3) M) and gabaculine (10(-5) M), enhanced the stimulus-evoked release of GABA and reduced that of beta-alanine, while no change was observed in the release of glutamate. These changes in amino acid release from the slices were accompanied by an increase in the content of GABA and a decrease in that of beta-alanine. The stimulus-evoked release of these amino acids was abolished by Ca2+-deprivation, in either the presence or absence of GABA-T inhibitors. These results suggest a modulatory role of GABA-T for synaptically releasable GABA and involvement of this enzyme in the synthesis of releasable beta-alanine.     

Uptake and release ofmeta-tyramine,para-tyramine,and dopamine in rat striatal slices

The uptakes of high-affinity concentrations (10^–8 M) ofmeta-tyramine (m-TA),para-tyramine (p-TA), and dopamine (DA) into rat striatal slices have been shown to be inhibited by DNP and ouabain. We now demonstrate that cocaine (5×10^–6 M) and low concentrations of sodium ion (26×10^–3 M) also reduced these uptakes. The spontaneous efflux and the release [induced by an elevated concentration of potassium ion (5×10^–2 M)] of each of the previously accumulated amines were studied in the presence and absence of added calcium ions. The spontaneous efflux of each amine (especially the tyramines) was enhanced by the absence of calcium ions. Part of this enhancement seemed to be due to an inhibition of a calcium-dependent reuptake. The elevated concentration of potassium ion proved to be an effective releaser of each amine; and for DA, such release was decreased by the removal of calcium. Form- andp-TA, however, the removal of calcium either did not reduce or completely abolished the releases depending upon the duration of the calcium removal. The significance of these findings is discussed.     

Characteristics of GABA release modified by glutamate receptors in mouse hippocampal slices

The major part of hippocampal innervation is glutamatergic, regulated by inhibitory GABA-releasing interneurons. The modulation of [(3)H]GABA release by ionotropic and metabotropic glutamate receptors and by nitric oxide was here characterized in superfused mouse hippocampal slices. The ionotropic glutamate receptor agonists kainate, N-methyl-D-aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate potentiated the basal GABA release. These effects were blocked by their respective antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX), dizocilpine and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX), indicating receptor-mediated mechanisms. The NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), sodiumnitroprusside and hydroxylamine enhanced the basal GABA release. Particularly the sodiumnitroprusside-evoked release was attenuated by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) and the inhibitor of soluble guanylyl cyclase 1H-(1,2,4)oxadiazolo(4,3a)quinoxalin-1-one (ODQ), indicating the involvement of the NO/cGMP pathway. This inference is corroborated by the enhancing effect of zaprinast, a phosphodiesterase inhibitor, which is known to increase cGMP levels. The K(+)-stimulated hippocampal GABA release was reduced by the groups I and III agonists of metabotropic glutamate receptors (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4), which effects were abolished by their respective antagonists (RS)-1-aminoindan-1,5-dicarboxylate (AIDA) and (RS)-2-cyclopropyl-4-phosphonophenylglycine (CPPG), again indicating modification by receptor-mediated mechanisms.     

Effect of endothelin-1 on the excitability of rat cortical and hippocampal slices in vitro

Endothelin-1 (ET-1) is a neuroactive protein produced in most brain cell types and participates in regulation of cerebral blood flow and blood pressure. In addition to its vascular effects, ET-1 affects synaptic and nonsynaptic neuronal and glial functions. Direct application of ET-1 to the hippocampus of immature rats results in cerebral ischemia, acute seizures, and epileptogenesis. Here, we investigated whether ET-1 itself modifies the excitability of hippocampal and cortical circuitry and whether acute seizures observed in vivo are due to nonvascular actions of ET-1. We used acute hippocampal and cortical slices that were preincubated with ET-1 (20 μM) for electrophysiological recordings. None of the slices preincubated with ET-1 exhibited spontaneous epileptic activity. The slope of the stimulus intensity-evoked response (input-output) curve and shape of the evoked response did not differ between ET-1-pretreated and control groups, suggesting no changes in excitability after ET-1 treatment. The threshold for eliciting an evoked response was not significantly increased in either hippocampal or cortical regions when pretreated with ET-1. Our data suggest that acute seizures after intrahippocampal application of ET-1 in rats are likely caused by ischemia rather than by a direct action of ET-1 on brain tissue.