Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.     

Detection of gamma-aminobutyric acid-induced glutamate release in acute mouse hippocampal slices with a patch sensor

gamma-Aminobutyric acid (GABA)-stimulated release of L-glutamate from various neuronal regions of acute mouse hippocampal slices was detected with a patch sensor that responds to L-glutamate at the sub-micromolar level. The response of the patch sensor to L-glutamate was evaluated in terms of an integrated current. The integrated current increased with the concentration of L-glutamate ranging from 0.50 to 5.0 microM. By using the patch sensor, GABA-induced L-glutamate release from acute mouse hippocampal slices was detected. The effect of antagonists for GABA(A) and GABA(B) receptors on the L-glutamate release was also investigated. The GABA (25 microM) stimulation induced the release of L-glutamate via GABA(A) receptor in the CA1 region, but GABA did not induce L-glutamate release in the CA3 region. However, in the presence of the GABA(B) receptor antagonist (3-aminopropyl)(diethoxymethyl)phosphinic acid (CGP-35348), release of L-glutamate in the CA3 region was evoked by GABA stimulation. The glutamate release was completely suppressed when both GABA(A) and GABA(B) receptor were inhibited. The current results show that the glutamate release in the CA3 region occurs via a GABA(A) pathway when GABA(B) receptors are inhibited.     

GABA(B)-receptor mediation of the inhibitory effect of gamma-hydroxybutyric acid on intestinal motility in mice

The effect of acutely administered gamma-hydroxybutyric acid (GHB) and GHB receptor antagonist, NCS-382, on the propulsive activity in the mouse small intestine was assessed by measuring the transit of an orally administered, non absorbable marker. Both GHB (0, 25, 50, 100, 200 and 300 mg/kg; i.p.) and NCS-382 (0, 25, 50 and 75 mg/kg; i.p.) induced a dose-dependent inhibition (up to 50-60%) of the marker transit. Pretreatment with the GABA(B) receptor antagonist, SCH 50911 (100 mg/kg; i.p.), resulted in the blockade of the inhibiting effect of both GHB and NCS-382. These results suggest that the constipating effect of GHB and NCS-382 are secondary to stimulation of the GABA(B) receptor.     

Presynaptic kainate receptors modulating glutamatergic transmission in the rat hippocampus are inhibited by arachidonic acid

Kainate receptors are ionotropic glutamate receptors located postsynaptically, mediating frequency-dependent transmission, and presynaptically, modulating transmitter release. In contrast to the excitatory postsynaptic kainate receptors, presynaptic kainate receptor can also be inhibitory and their effects may involve a metabotropic action. Arachidonic acid (AA) modulates most ionotropic receptors, in particular postsynaptic kainate receptor-mediated currents. To further explore differences between pre- and postsynaptic kainate receptors, we tested if presynaptic kainate receptors are affected by AA. Kainate (0.3-3 microM) and the kainate receptor agonist, domoate (60-300 nM), inhibited by 19-54% the field excitatory postsynaptic potential (fEPSP) slope in rat CA1 hippocampus, and increased by 12-32% paired-pulse facilitation (PPF). AA (10 microM) attenuated by 37-72% and by 62-66% the domoate (60-300 nM)-induced fEPSP inhibition and paired-pulse facilitation increase, respectively. This inhibition by AA was unaffected by cyclo- and lipo-oxygenase inhibitors, indomethacin (20 microM) and nordihydroguaiaretic acid (NDGA, 50 microM) or by the free radical scavenger, N-acetyl-L-cysteine (0.5 mM). The K+ (20 mM)-evoked release of [3H]glutamate from superfused hippocampal synaptosomes was inhibited by 18-39% by domoate (1-10 microM), an effect attenuated by 35-63% by AA (10 microM). Finally, the KD (40-55 nM) of the kainate receptor agonist [3H]-(2S,4R)-4-methylglutamate ([3H]MGA) (0.3-120 nM) binding to hippocampal synaptosomal membranes was increased by 151-329% by AA (1-10 microM). These results indicate that AA directly inhibits presynaptic kainate receptor controlling glutamate release in the CA1 area of the rat hippocampus.     

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

GABA(A)-mediated toxicity of hippocampal neurons in vitro

In the present study, we examined whether the elevation of GABA by gamma-vinyl-GABA protects cultured rat fetal hippocampal neurons against toxicity induced by a 20-min incubation with 100 microM L-glutamate. Neither a 24-h pretreatment nor posttreatment with gamma-vinyl-GABA (100 microM) had any neuroprotective effects, as determined by counting microtubule-associated protein-2 positive cells and lactate dehydrogenase assay 24 h after the glutamate treatment. Unexpectedly, gamma-vinyl-GABA alone induced a 20% loss of microtubule-associated protein-2-positive cells in a culture that was grown in medium containing 25 mM KCl. The toxic effect of gamma-vinyl-GABA was mimicked by a 24-h treatment with GABA (100 microM) and the GABA(A) receptor agonist, muscimol (10 microM), but not the GABA(B) receptor agonist, baclofen (10 microM). The GABA(A) receptor antagonist, bicuculline (10 microM), protected against gamma-vinyl-GABA and GABA-evoked toxicity. Neither gamma-vinyl-GABA nor GABA was toxic in culture medium containing 15 mM KCl. These data indicate that, under depolarizing conditions, an increased GABA level is toxic for a subpopulation of developing hippocampal neurons in vitro. The effect is GABA(A) receptor-mediated. These data provide a new view for understanding neurodegenerative processes, and raise a question of the safety of therapies aimed at increasing GABA concentration following brain insults, especially in immature brains.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT1A/5-HT2 receptor ligand adatanserin

Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.     

Modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit

We have previously shown that ionotropic glutamate receptors in the caudal portion of the nucleus tractus solitarii (NTS), especially in the commissural NTS, play a prominent role in the mediation of tracheobronchial cough and that substance P potentiates this reflex. This NTS region could be a site of action of some centrally acting antitussive agents and a component of a drug-sensitive gating mechanism of cough. To address these issues, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of centrally acting antitussive drugs into the caudal NTS of pentobarbitone-anesthetized, spontaneously breathing rabbits. [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and baclofen decreased baseline respiratory frequency because of increases in the inspiratory time only at the higher concentration employed (5 mM and 1 mM, respectively). DAMGO (0.5 mM) and baclofen (0.1 mM) significantly decreased cough number, peak abdominal activity, peak tracheal pressure, and increased cough-related total cycle duration. At the higher concentrations, these agents suppressed the cough reflex. The effects of these two drugs were counteracted by specific antagonists (10 mM naloxone and 25 mM CGP-35348, respectively). The neurokinin-1 (NK1) receptor antagonist CP-99,994 (10 mM) abolished cough responses, whereas the NK2 receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the caudal NTS is a site of action of some centrally acting drugs and a likely component of a neural system involved in cough regulation. A crucial role of substance P release in the mediation of reflex cough is also suggested.     

GABA-mediated neurotransmission in the ventrolateral NTS plays a role in respiratory regulation in the rat

Our purpose was to determine whether endogenously released GABA in the ventrolateral nucleus of the solitary tract (vlNTS) of the rat influences respiration. Experiments were carried out in anesthetized, vagotomized and spontaneously breathing rats, and diaphragm electromyogram activity was measured while drugs affecting GABAergic neurotransmission were microinjected into the vlNTS and medial NTS (mNTS). Bilateral microinjection of nipecotic acid, 5 or 25 nmol, into the vlNTS (but not in the mNTS) produced dose-dependent increases in inspiratory duration (Ti) frequently culminating in apneustic breathing. Neither unilateral microinjection of bicuculline nor CGP-35348 (GABA(B) receptor antagonist) reversed this response; however, a combination of both GABA receptor antagonists effectively reversed apneustic breathing. Bilateral microinjection of either muscimol or baclofen into the vlNTS mimicked the effect of nipecotic acid. Microinjection of the bicuculline produced apnea, whereas microinjection of CGP-35348 produced a decrease in Ti and an increase in expiratory duration. Immunohistochemical analysis of the vlNTS region revealed GABA(A) receptors densely localized to processes, whereas GABA(B) immunoreactivity was localized to cell bodies. Our data indicate that GABA activity in the vlNTS is important for respiratory function.     

Characterization of the interaction between fenamates and hippocampal neuron GABA(A) receptors

Fenamate NSAIDs have several central effects, including anti-epileptic and neuroprotective actions. The underlying mechanism(s) of these actions are not presently understood. In this study, the effects of five members of the fenamate NSAID group were investigated on native ligand-gated ion channels expressed in cultured rat hippocampal neurons. All fenamates tested (1-100 microM) dose-dependently potentiated GABA-evoked currents; mefenamic acid (MFA) was the most potent and efficacious and was found to shift the GABA dose-response curve to the left without effect on the maximum amplitude or the GABA Hill Slope. The modulation of GABA receptors by MFA was not reduced in the presence of the benzodiazepine antagonist, flumazenil (10 microM) and was moderately voltage-dependent. MFA at concentrations >or=10 microM evoked dose-dependent currents in the absence of GABA. These currents were potentiated by diazepam (1 microM) and blocked by bicuculline (10 microM). The MFA (50 microM) current-voltage relationship and reversal potential were similar to that evoked by GABA. MFA (1-100 microM) had no effects on sub-maximal glycine, glutamate or NMDA evoked currents. These data show that fenamate NSAIDs are a highly effective class of GABA(A) receptor modulator and activators.     

Contribution of nicotinic receptors to the function of synapses in the central nervous system: The action of choline as a selective agonist of α7 receptors

The α7-nicotinic receptor (nAChR)-selective agonist choline and nAChR-subtype-selective antagonists led to the discovery that activation of both α7 and α4β2 nAChRs located in CA1 interneurons in slices taken from the rat hippocampus facilitates the tetrodotoxin (TTX)-sensitive release of γ-aminobutyric acid (GABA). Experiments carried out in cultured hippocampal neurons not only confirmed that preterminal α7 and α4β2 nAChRs modulate the TTX-sensitive release of GABA, but also demonstrated that evoked release of GABA is reduced by rapid exposure of the neurons to acetylcholine (ACh, 10 μM-1 mM) in the presence of the muscarinic receptor antagonist atropine (1 μM). This effect of ACh, which is fully reversible and concentration-dependent, is partially blocked by superfusion of the cultured neurons with external solution containing either the α7-nAChR-selective antagonist methyllycaconitine (MLA, 1 nM) or the α4β2-nAChR-selective antagonist dihydro-β-erythroidine (DHβE, 100 nM). A complete blockade of ACh-induced reduction of evoked release of GABA was achieved only when the neurons were perfused with external solution containing both MLA and DHβE, suggesting that activation of both α7 and α4β2 nAChRs modulates the evoked release of GABA from hippocampal neurons. Such mechanisms may account for the apparent involvement of nAChRs in the psychological effects of tobacco smoking, in brain disorders (e.g., schizophrenia and epilepsy), and in physiological processes, including cognition and nociception.     

In vivo co-ordinated interactions between inhibitory systems to control glutamate-mediated hippocampal excitability

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA(A) receptor labelling in the hippocampal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extracellular levels were studied in control and lesioned rats. In vivo effects of 100 mm KCl perfusion and adenosine A(1) receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA(A) receptors and decreased glutamate neurotransmission. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.     

GABA(B) receptors on vagal afferent pathways: peripheral and central inhibition

To investigate GABA(B) receptors along vagal afferent pathways, we recorded from vagal afferents, medullary neurons, and vagal efferents in ferrets. Baclofen (7-14 micromol/kg i.v.) reduced gastric tension receptor and nucleus tractus solitarii neuronal responses to gastric distension but not gastroduodenal mucosal receptor responses to cholecystokinin (CCK). GABA(B) antagonists CGP-35348 or CGP-62349 reversed effects of baclofen. Vagal efferents showed excitatory and inhibitory responses to distension and CCK. Baclofen (3 nmol i.c.v. or 7-14 micromol/kg i.v.) reduced both distension response types but reduced only inhibitory responses to CCK. CGP-35348 (100 nmol i.c.v. or 100 micromol/kg i.v.) reversed baclofen's effect on distension responses, but inhibitory responses to CCK remained attenuated. They were, however, reversed by CGP-62349 (0.4 nmol i.c.v.). In conclusion, GABA(B) receptors inhibit mechanosensitivity, not chemosensitivity, of vagal afferents peripherally. Mechanosensory input to brain stem neurons is also reduced centrally by GABA(B) receptors, but excitatory chemosensory input is unaffected. Inhibitory mechano- and chemosensory inputs to brain stem neurons (via inhibitory interneurons) are both reduced, but the pathway taken by chemosensory input involves GABA(B) receptors that are insensitive to CGP-35348.     

Extracellular GABA in the Ventrolateral Thalamus of Rats Exhibiting Spontaneous Absence Epilepsy: A Microdialysis Study

Abstract: There is compelling evidence that excessive GABA-mediated inhibition may underlie the abnormal electrical activity, initiated in the thalamus, associated with epileptic absence seizures. In particular, the GABA_B receptor subtype seems to play a critical role, because its antagonists are potent inhibitors of absence seizures, whereas its agonists exacerbate seizure activity. Using a validated rat model of absence epilepsy, we have previously found no evidence of abnormal GABA_B receptor density or affinity in thalamic tissue. In the present study, we have used in vivo microdialysis to monitor changes in levels of extracellular GABA and other amino acids in this brain region. We have shown that basal extracellular levels of GABA and, to a lesser extent, taurine are increased when compared with values in nonepileptic controls. However, modifying GABAergic transmission with the GABA_B agonist (−)-baclofen (2 mg/kg i.p.), the GABA_B antagonist CGP-35348 (200 mg/kg i.p.), or the GABA uptake inhibitor tiagabine (100 µ M ) did not produce any further alteration in extracellular GABA levels, despite the ability of these compounds to increase (baclofen and tiagabine) or decrease (CGP-35348) seizure activity. These findings suggest that the increased basal GABA levels observed in this animal model are not simply a consequence of seizure activity but may contribute to the initiation of absence seizures.     

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.     

Both dopamine and the putative dopamine D3 receptor antagonist PNU-99194A induce a biphasic inhibition of phorbol ester-stimulated arachidonic acid release from CHO cells transfected with the dopamine D3 receptor

In Chinese hamster ovary (CHO) cells transfected with the cDNA for the dopamine D3 receptor, low concentrations of dopamine (IC50: 0.5 nM) counteracted the release of arachidonic acid (AA) induced by the protein kinase C activator TPA (maximal inhibition: 15% at 10 - 30 nM). The effect of dopamine -- which was antagonized by pretreatment with pertussis toxin (PTX) or by the dopamine receptor antagonist haloperidol -- was biphasic; thus, at increasing concentrations of dopamine (100 nM - 1 microM), AA levels approached baseline. The preferential dopamine D3 receptor ligand PNU-99194A displayed an effect similar to that of dopamine; thus, whereas low concentrations of PNU-99194A (IC50: 1.9 nM) reduced TPA-induced AA release (maximal inhibition: 15% at 30 - 100 nM), higher concentrations (> or =1 microM) were ineffective. When dopamine and PNU-99194A were administered together at concentrations yielding maximal inhibition of AA release, no additive effect was observed; moreover, a high concentration of dopamine counteracted the AA-reducing effect of a low concentration of PNU-99194A and vice versa. It is suggested that D3 receptors in transfected CHO cells may exert mainly an inhibitory, but also a stimulatory influence on TPA-induced AA release, and that PNU-99194A acts as an agonist in this system.     

GABA in Paraventricular Nucleus Regulates Adipose Afferent Reflex in Rats

Background

Chemical stimulation of white adipose tissue (WAT) induces adipose afferent reflex (AAR), and thereby causes a general sympathetic activation. Paraventricular nucleus (PVN) is important in control of sympathetic outflow. This study was designed to investigate the role of γ-aminobutyric acid (GABA) in PVN in regulating the AAR.

Methodology/Principal Findings

Experiments were carried out in anesthetized rats. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. AAR was evaluated by the RSNA and MAP responses to electrical stimulation of the right epididymal WAT (eWAT) afferent nerve. Electrical stimulation of eWAT afferent nerve increase RSNA. Bilateral microinjection of the GABA_A receptor agonist isoguvacine or the GABA_B receptor agonist baclofen attenuated the AAR. The effect of isoguvacine on the AAR was greater than that of baclofen. The GABA_A receptor antagonist gabazine enhanced the AAR, while the GABA_B receptor antagonist CGP-35348 had no significant effect on the AAR. Bilateral PVN microinjection of vigabatrin, a selective GABA-transaminase inhibitor, to increase endogenous GABA levels in the PVN abolished the AAR. The inhibitory effect of vigabatrin on the AAR was attenuated by the pretreatment with gabazine or CGP-35348. Pretreatment with combined gabazine and CGP-35348 abolished the effects of vigabatrin.

Conclusions

Activation of GABA_A or GABA_B receptors in the PVN inhibits the AAR. Blockade of GABA_A receptors in the PVN enhances the AAR. Endogenous GABA in the PVN plays an important role in regulating the AAR.