Autoreceptor-mediated regulation of GABA release: role of uptake inhibition and effects of novel GABAB antagonists

Summary While the role of GABA_B autoreceptors in the regulation of GABA release in synaptosomes and brain slices is well established, little is known about their role in vivo. Doubts have arisen because there is an apparent discrepancy between the frequencies at which GABA neurons fire and the frequency range within which autoreceptor regulation is observed in vitro. To see whether this apparent mismatch could be due to the use of a GABA uptake inhibitor in the release experiments in slices, we have compared the frequency dependencies of GABA release in the presence and absence of uptake inhibition. Beforehand, the previously incomplete frequency curve in the presence of uptake inhibition was extended at the lower end. To achieve this, stimulation was performed by means of groups of 4 pseudo-one-pulses (POP's) at inter-POP intervals corresponding to frequencies of 0.015625-0.5 Hz. It could be shown that activation of the GABA_B autoreceptor by endogenously released GABA begins at a stimulation frequency as low as 0.0625 Hz. Experiments with the antagonist, CGP 35348, at inter-POP intervals of 1 min, at which the preceding POP has no longer an effect on GABA release during the next one, showed that basal release alone already substantially activated the autoreceptor. The frequency dependence in the absence as compared to the presence of uptake inhibition was shifted towards higher frequencies by a factor of 4. We do not consider this enough to remove our doubts about the in vivo operativity of GABA_B autoreceptors.Further experiments in the presence of uptake inhibition were made to see whether the expectation was met that autoreceptor-mediated regulation of GABA release could be blocked out by sufficiently high concentrations of potent antagonists. As judged from the frequency dependence in the presence of 1 mol/l of the potent compound CGP 55845 as well as from the similarity of the maximal increases of GABA release caused at 0.125 and 2 Hz by various other potent, novel GABA_B antagonists, this was not the case. Possible explanations are the occurrence of an agonist and an antagonist state of the autoreceptor prevailing at low GABA concentrations or, less likely, the occurrence of two autoreceptor subtypes with different relative sensitivities towards GABA and baclofen on the one hand and towards aminophosphonous acid antagonists on the other.Finally, it was shown that also in the absence of uptake inhibition, regulation of GABA release was mediated entirely by GABA_B autoreceptors. Both muscimol and bicuculline at 1 and 10 mol/1 were without effect. Correspondence to P. Waldmeier at the above address     

A novel type of GABA receptor in rat spinal cord?

Summary The depolarization-evoked release of -aminobutyric acid (GABA) and its possible modulation mediated by autoreceptors were studied in nerve endings isolated from rat spinal cord and prelabeled with the radioactive aminoacid. In the presence of the GABA uptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], used to minimize carrier-mediated homoexchange, exogenous GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA evoked by 15 mmol/l KCl. The GABA_A receptor agonist muscimol (10–100 µmol/l) did not affect the K⁺(15 mmol/l)-evoked³H-GABA release. Similarly ineffective was the GABA_B receptor agonist (–)-baclofen (3–100 µmol/l). The effect of GABA was not counteracted by the GABA_A receptor antagonists bicuculline,picrotoxinorSR95531 [2-(3-carbethoxy-2-propenyl)-3-amino-6-paramethoxy-phenyl-pyridazinium bromide]. In a set of experiments performed in parallel with spinal cord or cerebral cortex synaptosomes depolarized with 9 mmol/l KCI it was found that: (a) GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA in both brain regions and its effects were practically doubled when compared with those in synaptosomes depolarized with 15 mmol/l KCI; - (b) muscimol (1–100 µmol/l) was ineffective both in spinal cord and in cerebral cortex; - (c) (–)-baclofen (1-100 µmol/l) concentration-dependently inhibited³H-GABA release in cerebral cortex synaptosomes but was almost inactive in the spinal cord; the (+)-enantiomer of baclofen (100 µmol/l) did not affect significantly the release of³H-GABA; - (d) the GABA_B receptor antagonist phaclofen [3-amino-2-(4chlorophenyl)-propyl phosphonic acid] antagonized the effect of GABA in cerebral cortex but not in spinal cord synaptosomes. It is concluded that the release of GABA in rat spinal cord may be modulated by an autoreceptor which does not belong to the known GABA receptor subtypes. Send offprint requests to M. Raiteri at the above address     

Modulation of the GABA autoreceptor by benzodiazepine receptor ligands

The effects of various benzodiazepine receptor ligands on the GABA autoreceptor have been studied in slices of cerebral cortex of the rat. The GABAA receptor agonist muscimol inhibited the K+-stimulated release of [3H]GABA with a pIC25 of 7.65 +/- 0.11. This effect was antagonised by the GABAA receptor antagonist bicuculline, which had an IC50 of 0.36 +/- 0.03 microM. Small concentrations (less than 1 microM) of the benzodiazepine full agonist clonazepam did not significantly alter K+-evoked release of [3H]GABA but shifted the concentration-effect curve for muscimol to the left. This effect was blocked by the benzodiazepine antagonist flumazenil. By contrast, the benzodiazepine full inverse agonist methyl beta-carboline-3-carboxylate shifted the muscimol concentration-effect curve to the right and this too was blocked by flumazenil. The results suggest that the GABA autoreceptor in cortical slices from the rat is modulated by a benzodiazepine receptor.     

GABA release in rat cortical slices is unable to cope with demand if the autoreceptor is blocked

Electrically stimulated release of neurotransmitters in brain slices normally displays frequency dependence because of progressive activation of autoreceptors by endogenously released transmitter, which is abolished by blockade of autoreceptors. In consequence, the maximal increase caused by an autoreceptor antagonist in percent of the corresponding controls should be greater at higher than at lower frequencies. In the case of -aminobutyric acid (GABA), we have previously found a marked deviation from this expectation. Among several explanations envisaged, computer simulation suggested only one to be compatible with the experimental data: the release mechanism may not be able to cope with high demand.This hypothesis was tested by investigating the frequency dependence of the release of³H-GABA in the presence and absence of a high concentration of the potent GABA_B antagonist, CGP 55845, using extremely short stimulation periods. To this end, slices were stimulated with groups of 4 POPS (a POP — pseudo-one-pulse — consists of 4 pulses delivered at 100 Hz). The intervals between the POPs within a group were varied from 60-0.5 s, corresponding to frequencies within the POP group of 0.0167–2 Hz. Under such circumstances, the theoretically expected pattern was indeed observed: the GABA_B antagonist abolished the frequency dependence. In a second series of experiments, fractional release per POP was determined when 4–32 POPs were delivered at 2 Hz, with and without CGP 55845. The increase of GABA release elicited by the GABA_B antagonist gradually subsided with increasing number of POPS. It was about 50Q7o of that observed during the first 4 POPs in the 4 subsequent ones, and almost nil in the last 16 of a total of 32 POPs.The results of this study support the hypothesis generated with the help of computer simulation, that release may not be able to keep up with high demand. They further suggest that exhaustion of the releasable transmitter pool occurs surprisingly fast. It is not known whether this phenomenon is physiologically relevant or a consequence of metabolic stress to which the slices are subjected during preparation and superfusion, or of their exposure to transaminase and uptake inhibitors, but it provides an explanation for the anomalies observed in the studies of the effects of GABA_B antagonists on³H-GABA release. Correspondence to: P. C. Waldmeier at the above address     

Enhancement by GABA of the stimulation-evoked catecholamine release from cultured bovine adrenal chromaffin cells

Summary The possible involvement of GABAergic mechanisms in the catecholamine (CA) release from adrenal medulla was investigated in a primary culture of bovine adrenal chromaffin cells. GABA elicited CA release and enhanced acetylcholine (ACh)-, excess K⁺-and veratridine-evoked CA release. Muscimol, a selective GABA_A receptor agonist, mimicked the action of GABA on CA release. On the other hand, baclofen, a GABA_B receptor agonist, failed to affect basal or evoked CA release. Furthermore, bicuculline and picrotoxin blocked the enhancement by GABA of veratridine-evoked CA release without affecting basal CA release and CA release evoked by veratridine. In Ca²⁺-free medium, GABA failed to affect basal and caffeine-evoked CA release. ACh-evoked CA release was slightly reduced by bicuculline, whereas excess K⁺-evoked CA release was not, suggesting the involvement of endogenous GABA in CA release evoked by ACh. These results suggest a facilitatory modulation by GABA of basal and evoked release of CA from bovine adrenal medulla through GABA_A receptor-mediated mechanisms. Send offprint requests to A. Tsujimoto at the above address     

On the origin of extracellular GABA collected by brain microdialysis and assayed by a simplified on-line method

Summary The present study describes a simplified on-line system for determination of GABA in brain dialysates. GABA was determined with an isocratic HPLC method after derivatization with o-phtaldialdehyde. One peristaltic pump was sufficient to transport both the perfusion fluid and the derivatizing reagent.The basal release of GABA was stimulated by infusion with either elevated K⁺ or the GABA uptake inhibitor (–)-nipecotic acid. Basal as well as stimulated GABA release were investigated for possible calcium-dependency by infusing submmolar amounts of the potent calcium antagonist cadmium. Infusion of cadmium did not modify the dialysate concentrations of GABA. In addition basal as well as nipecotic acid enhanced release of GABA dialysate concentrations were investigated for nerve-impulse dependency by infusing molar amounts of tetrodotoxin. No change in the GABA output was observed during infusion of TTX. From these results it is concluded that the basal as well as the nipecotic acid induced release of GABA did not fulfill the criteria for classic exocytotic release. Possible explanations for these unexpected findings are discussed. Send offprint requests to B. H. C. Westerink at the above address     

Pharmacological actions of thymol and an analogue at GABAB autoreceptors

GABA_B autoreceptors inhibit release of GABA from GABAergic nerve terminals. Agonists of these receptors (e.g. baclofen) inhibit, whereas antagonists (e.g. (+)‐(S)‐5,5‐dimethylmorpholinyl‐2‐acetic acid; Sch 50911) enhance release of the transmitter. The actions of thymol (2‐isopropyl‐5‐methylphenol) and the structurally related compound 2‐tert‐butyl‐4‐methylphenol, (4MP) on the release of [³H]‐GABA were examined in rat neocortical slices where the GABAergic nerves had been preloaded with [³H]‐GABA and subsequently stimulated electrically on two occasions (S₁ and S₂). Test agents, baclofen and Sch 50911 were added to the superfusion medium prior to the second period of stimulation (S₂). Stimulation‐induced overflow (SIO) of [³H]‐GABA as a consequence of these stimulations (SIO₁ and SIO₂) were calculated and the effects of agents determined by comparing the SIO₂/SIO₁ ratio in the presence of each agent with that in control tissue. Thymol potentiated the release of [³H]‐GABA (EC₅₀ 170 μmol/L), an action reversed by baclofen (2 μmol/L). Baclofen alone had little effect on GABA release. Release of [³H]‐GABA was inhibited by 4MP (IC₅₀ 3 μmol/L) and this effect was blocked by Sch 50911 (10 μmol/L). Alone, Sch 50911 markedly potentiated the release of GABA. These results imply that 4MP is an agonist of GABA_B autoreceptors; however, further studies are needed to confirm that thymol is indeed a GABA_B autoreceptor antagonist. Of interest are structural differences in these agents. Thymol has a propyl group in the ortho position relative to the phenolic hydroxyl, whereas in 4MP this is a butyl group and the methyl group moves from position 5 to 4. Whether one or both of these changes was responsible for the above actions is unknown.     

Lamina-specific differences in GABA(B) autoreceptor-mediated regulation of spontaneous GABA release in rat entorhinal cortex

Spontaneous synaptic inhibition plays an important role in regulating the excitability of cortical networks. Here we have investigated the role of GABA(B) autoreceptors in regulating spontaneous GABA release in the entorhinal cortex (EC), a region associated with temporal lobe epilepsies. We have previously shown that the level of spontaneous inhibition in superficial layers of the EC is much greater than that seen in deeper layers. In the present study, using intracellular and whole cell patch clamp recordings in rat EC slices, we have demonstrated that evoked GABA responses are controlled by feedback inhibition via GABA(B) autoreceptors. Furthermore, recordings of spontaneous, activity-independent inhibitory postsynaptic currents in layer II and layer V neurones showed that the GABA(B) receptor agonist, baclofen, reduced the frequency of GABA-mediated currents indicating the presence of presynaptic GABA(B) receptors in both layers. Application of the antagonist, CGP55845, blocked the effects of baclofen and also increased the frequency of GABA-mediated events above baseline, but the latter effect was restricted to layer V. This demonstrates that GABA(B) autoreceptors are tonically activated by synaptically released GABA in layer V, and this may partly explain the lower level of spontaneous GABA release in the deep layer.     

Functional evidence for different roles of GABAA and GABAB receptors in modulating mouse gastric tone

The aims of the present study were to investigate, using mouse whole stomach in vitro, the effects of γ-aminobutyric acid (GABA) and GABA receptor agonists on the spontaneous gastric tone, to examine the subtypes of GABA receptors involved in the responses and to determine the possible site(s) of action.GABA induced gastric relaxation, which was antagonized by the GABA_A-receptor antagonist, bicuculline, potentiated by phaclofen, GABA_B-receptor antagonist, but not affected by 1,2,5,6-Tetrahydropyridin-4-yl methylphosphinic acid hydrate (TPMPA), GABA_C-receptor antagonist. Muscimol, GABA_A-receptor agonist, mimicked GABA effects inducing relaxation, which was significantly reduced by bicuculline, Nω-nitro-l-arginine methyl ester (l-NAME), inhibitor of NO synthase or apamin, inhibitor of small conductance Ca²⁺-dependent K⁺ channels, which blocks the purinergic transmission in this preparation. It was abolished by tetrodotoxin (TTX) or l-NAME plus apamin. Baclofen, a specific GABA_B-receptor agonist, induced an increase in the gastric tone, which was antagonized by phaclofen and abolished by TTX or atropine. Bicuculline, but not phaclofen or TPMPA, per se induced an increase in gastric tone, which was prevented by l-NAME. In conclusion, our results suggest that GABA is involved in the regulation of mouse gastric tone, through modulation of intrinsic neurons. Activation of GABA_A-receptors mediates relaxation through neural release of NO and neurotransmitters, activating Ca²⁺-dependent K⁺ channels, likely purines, while activation of GABA_B-receptors leads to contraction through acetylcholine release.     

Weak effects of local and systemic administration of the GABA uptake inhibitor,SK&F 89976, on extracellular GABA in the rat striatum

Summary The effects of local and systemic administration of the potent GABA uptake inhibitor, SK&F 89976, on GABA overflow from the striatum of conscious rats were investigated in brain dialysis experiments. Administration of the compound via the dialysis probe at concentrations of 25 or 100 gmol/l significantly increased basal GABA overflow about 2-fold. Overflow evoked by 104 mmol/l K⁺ remained unaltered at the lower and was almost doubled at the higher concentration; this increase did, however, not reach statistical significance.Given systemically at 50 mg/kg i.p., a dose which is severalfold higher than those which exhibit anticonvulsant effects, SK&F 89976 caused a significant enhancement of K⁺-stimulated GABA overflow by about a factor of 2; the lower dose of 20 mg/kg i.p. was not effective. Basal GABA overflow was not significantly increased by either dose. These results suggest that the marked effects of nipecotic acid on basal GABA overflow reported by several authors seem to be related to GABA displacement rather than uptake inhibition, and that uptake inhibition does not improve the interpretability of measurements of GABA release by brain dialysis. They neither support the idea that the relative insensitivity of extracellular GABA to low Ca²⁺ and tetrodotoxin is indirectly due to very efficient removal of GABA by neuronal and/or glial uptake, leaving only residual amounts to be measured. Send offprint requests to P. Waldmeier at the above address     

International Union of Pharmacology. XXXIII. Mammalian gamma-aminobutyric acid(B) receptors: structure and function

The gamma-aminobutyric acid(B) (GABA(B)) receptor was first demonstrated on presynaptic terminals where it serves as an autoreceptor and also as a heteroreceptor to influence transmitter release by suppressing neuronal Ca(2+) conductance. Subsequent studies showed the presence of the receptor on postsynaptic neurones where activation produces an increase in membrane K(+) conductance and associated neuronal hyperpolarization. (-)-Baclofen is a highly selective agonist for GABA(B) receptors, whereas the established GABA(A) receptor antagonists, bicuculline and picrotoxin, do not block GABA(B) receptors. The receptor is G(i)/G(o) protein-coupled with mixed effects on adenylate cyclase activity. The receptor comprises a heterodimer with similar subunits currently designated 1 and 2. These subunits are coupled via coiled-coil domains at their C termini. The evidence for splice variants is critically reviewed. Thus far, no unique pharmacological or functional properties have been assigned to either subunit or the variants. The emergence of high-affinity antagonists for GABA(B) receptors has enabled a synaptic role to be established. However, the antagonists have generally failed to establish the existence of pharmacologically distinct receptor types within the GABA(B) receptor class. The advent of GABA(B1) knockout mice has also failed to provide support for multiple receptor types.     

Effect of experimental diabetes on GABA-mediated inhibition of neurally induced contractions in rat isolated trachea

1. In the present study, we investigated the effect of GABA and selective GABA agonists and antagonists on neurally induced tracheal contractions in streptozotocin (STZ) diabetic rats. 2. Contractile responses to electrical field stimulation (EFS) in rat tracheal rings were completely abolished by atropine and tetrodotoxin, but were unaffected by the ganglion blocker hexamethonium, indicating that they were mediated via neuronal release of acetylcholine (ACh). 3. Contractions induced by EFS, but not by exogenous ACh, were inhibited by GABA and the selective GABA(B) receptor agonist baclofen, but not by the selective GABA(A) receptor agonist 3-aminopropane sulphonic acid. The inhibitory effects of GABA or baclofen were not affected by the GABA(A) antagonist bicuculline, but were significantly reversed by the GABA(B) antagonist phaclofen. 4. The inhibitory effects of both GABA and baclofen were found to be significantly greater in trachea from control rats compared with tissues from diabetic rats. 5. Non-adrenergic, non-cholinergic relaxation responses elicited by EFS in precontracted tracheal rings from diabetic and control rats were similar in magnitude and were unaffected by GABA or GABA analogues. 6. These results suggest that GABA decreases the response to EFS by directly inhibiting the evoked release of ACh through GABA(B) receptors in rat trachea and that STZ-induced diabetes causes an impairment in the inhibitory effect of GABA on neurally induced contractions in this tissue.     

Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons

γ-Aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of μ-opioid receptors. Bath application of μ-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby μ-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that μ-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective μ-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca²⁺ ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K⁺ channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which μ-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K⁺ channels are implicated in the inhibition of the efficacy of the secretory process by μ-opioid receptors.     

Frequency-dependence of serotonin autoreceptor but not α2-adrenoceptor inhibition of [3H]-serotonin release in rat hypothalamic slices

Summary The overflow of tritium from stimulated rat hypothalamic slices preincubated with [³H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT with IC₅₀ values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [³H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC₅₀ values for inhibition of [³H]-5-HT release by the selective ₂-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by ₂-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization. Send offprint requests to S. Z. Langer at the above address     

Systemic administration of baclofen and the GABAB antagonist,CGP 35348, does not affect GABA,glutamate or aspartate in microdialysates of the striatum of conscious rats

Summary Previous in vitro experiments have shown that the GABA_B agonist, baclofen, and the antagonist, CGP 35348, respectively, decrease and increase the autoreceptor-mediated release of GABA in brain slices and synaptosomes. Since it is not clear whether these autoreceptors are operative in vivo, an attempt was made to reproduce these results in brain dialysis experiments, knowing that only positive results would permit a conclusion in view of the doubts expressed in the literature with respect to the origin of extracellular GABA. Because of older reports of an inhibitory action of baclofen on the in vitro release of glutamate, which might be ascribed to the action of presynaptic GABA_B heteroreceptors, extracellular glutamate and aspartate were also measured.Neither (–)-baclofen, administered systemically at a dose of 20 mg/kg i.p., nor the GABA_B antagonist, CGP 35348 (300 mg/kg i.p.) had significant effects on basal overflow of GABA, glutamate, or aspartate nor on that evoked by 100 mmol/l K⁺ in the striatum of the conscious, freely moving rat. To ascertain this result, (–)-baclofen was also administered between two K⁺ stimulations, so that the first stimulation could serve as an intraindividual control of the second. The compound did not significantly affect K⁺ evoked overflow of any of the three transmitter amino acids under these conditions.It must be emphasized that these data do not exclude the operativity of presynaptic GABA_B auto- and hetero-receptors in vivo. They only suggest that this question must, in all probability, be addressed by other techniques than brain dialysis. Send offprint requests to P. Waldmeier at the above address     

My close encounter with GABA(B) receptors

In this review, I summarize the sequence of events involved in characterizing the functional role of GABA(B) receptors in the CNS and their involvement in synaptic transmission. The story was launched with the realization that baclofen was a selective agonist of GABA(B) receptors. This lead to the discovery in the CNS that GABA(B) receptor activation could result in a presynaptic inhibition of transmitter release as well as a postsynaptic increase in potassium conductance. Based on this information, it was found that GABA also activated a potassium conductance. A role for GABA(B) receptors in synaptic transmission was suggested by the fact that activation of GABAergic interneurons could generate a slow IPSP mediated by an increase in potassium conductance. To link this slow IPSP to GABA(B) receptors required a selective GABA(B) antagonist. Phaclofen was the first antagonist developed and was found to antagonize the action of baclofen and the GABA(A) independent action of GABA. Most importantly, it blocked the slow IPSP. The properties of GABA(A) and GABA(B) IPSPs are remarkably different. GABA(A) IPSPs powerfully inhibit neurons and rapidly curtail excitatory inputs. This greatly enhances the precision of excitatory synaptic transmission. GABA(B) IPSPs are recruited with repetitive and synchronous activity and are postulated to modulate the rhythmic network activity of cortical tissue.     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9-15days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100microM), a selective agonist at GABA(A) receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA(A) receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100microM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100microM cadmium added to the bath solution. Choline 10mM, an agonist at alpha7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10microM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1microM of dihydro-beta-erythroidine (DHbetaE), an antagonist of non-alpha7 nAChRs but was insensitive to alpha7 nAChRs antagonists (strychnine, alpha-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10microM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non alpha7 nAChRs.     

Differences in kinetics of structurally related competitive GABA(A) receptor antagonists

Previously, 4-alkyl and 4-aryl substituted analogues of the low-efficacy partial GABA(A) receptor agonist 5-(4-piperidyl)-3-isothiazole (4-PIOL) have been identified as competitive GABA(A) receptor antagonists. These structurally related competitive antagonists show marked differences in their kinetic properties. The kinetics of 20 4-alkyl and 4-aryl substituted analogues of 4-PIOL, two 4-arylalkyl substituted 3-isothiazolol analogues and the classical GABA(A) receptor antagonist SR95531 was studied in cultured cerebral cortical neurons using whole-cell patch-clamp techniques. The kinetics of the antagonists was studied indirectly by measuring the changes in the response of the full GABA(A) receptor agonist isoguvacine (IGU) induced by concurrent application of an antagonist. When added, the majority of the antagonists did not affect the rate of deactivation of the IGU-induced responses. When removed, however, the majority of the antagonists slowed the reactivation phase of IGU implying that the dissociation of the antagonist from the GABA(A) receptor is the rate-limiting step. Surprisingly, the functional off-rates of the antagonists seemed to correlate better with the lipophilicity of the compounds than with the affinity and potency. This suggests that the dissociation of the tested antagonists from the GABA(A) receptor is restricted by lipophilic interactions, perhaps with the aromatic amino acids surrounding the GABA binding site.     

Effects of subtype-selective group I mGluR antagonists on synchronous activity induced by 4-aminopyridine/CGP 55845 in adult guinea pig hippocampal slices

Co-application of the convulsant 4-aminopyridine (4-AP) and the GABA(B) receptor antagonist CGP 55845 to adult guinea pig hippocampal slices elicits giant GABA-mediated postsynaptic potentials (GPSPs) and epileptiform discharges. Here we tested the effects of the group I metabotropic glutamate receptor (mGluR) subtype-selective antagonists LY 367385 (mGlu1, 100 microM), MPEP (mGlu5, 10 microM), and MTEP (mGlu5, 500 nM) on this synchronous activity. Electrophysiological field recordings were performed in the CA3 region of hippocampal slices from adult guinea pigs. The mGlu5 receptor antagonists increased GPSP rate, but the mGlu1 receptor antagonist did not. This ability of mGlu5 receptor antagonists to increase the rate of GPSPs indicates that enough endogenous glutamate is released under these conditions to activate group I mGluR; nevertheless, co-application of a mGlu1 receptor antagonist (LY 367385 or JNJ 16259685) and MPEP did not decrease pre-existing epileptiform activity. Furthermore, co-application of LY 367,385 and MPEP did not prevent the emergence of epileptiform activity. When ionotropic glutamate receptor (iGluR) antagonists were present, neither MPEP nor the group I mGluR agonist DHPG changed GPSP rate, suggesting that pyramidal cell-to-interneuron iGluR-mediated synaptic connections are involved in the rate change mechanism. In contrast to the lack of effect of group I mGluR antagonists on epileptiform activity in the 4-AP/CGP 55845 model, group I mGluR antagonists blocked the emergence of longer epileptiform events and decreased the overall amount of synchronous activity in the GABA(A) antagonist/4-AP model. In conclusion, in the 4-AP/CGP 55845 model, enough glutamate was released to activate group I mGluRs and affect GPSP rate via mGlu5 receptors; however, this group I mGluR activation was not required for the generation of the epileptiform activity.