Mario F. Pozza

Epilepsy, Hyperalgesia, Impaired Memory, and Loss of Pre- and Postsynaptic GABA B Responses in Mice Lacking GABA B(1)

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

CGP 35348 : a centrally active blocker of GABAB receptors

The biochemical, electrophysiological and pharmacological properties of the new GABAB receptor blocker CGP 35348 are described. In a variety of receptor binding assays CGP 35348 showed affinity for the GABAB receptor only. CGP 35348 had an IC50 of 34 microM at the GABAB receptor. The compound antagonized (100, 300, 1000 microM) the potentiating effect of L-baclofen on noradrenaline-induced stimulation of adenylate cyclase in rat cortex slices. In electrophysiological studies CGP 35348 (10, 100 microM) antagonized the effect of L-baclofen in the isolated rat spinal cord. In the hippocampal slice preparation CGP 35348 (10, 30, 100 microM) blocked the membrane hyperpolarization induced by D/L-baclofen (10 microM) and the late inhibitory postsynaptic potential. CGP 35348 appeared to be 10-30 times more potent than the GABAB receptor blocker phaclofen. Ionophoretic and behavioural experiments showed that GABAB receptors in the brain were blocked after i.p. administration of CGP 35348. This compound may be of considerable value in elucidating the roles of brain GABAB receptors.

[3H]CGP 39653: a new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain

CGP 39653 (D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) was initially discovered to inhibit the binding of [3H]L-glutamate and [3H]3-[+/-)2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid [( 3H]CPP) with Ki values of 230 and 5 nM, respectively. The radiolabeled compound [3H]CGP 39653 binds to rat frontal cortical membranes in a saturable and reversible manner. Analysis of saturation experiments revealed that the ligand labels one binding site with a Kd value of 6 nM. Competition experiments indicated that the order of potency of a number of competitive excitatory amino acid agonist and antagonist compounds was similar to that found previously for other N-methyl-D-aspartate (NMDA) receptor ligands. In contrast to these competitive inhibitors, which produced steep inhibition curves, glycine inhibited binding in a complex manner. When the functional activity of the unlabeled compound was explored, CGP 39653 blocked NMDA-evoked depolarizations in the rat cortical wedge in vitro and inhibited L-glutamate stimulated [3H]N(1-[2-thienyl]cyclohexyl)3,4-piperidine [( 3H]TCP) binding in cortical membranes. These results suggest that [3H]CGP 39653 selectively binds to the NMDA receptor as an antagonist with high affinity and is currently the ligand of choice for labeling the NMDA receptor.

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity.

1 The pharmacological properties of CGP 37849 (dl‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentenoic acid; 4‐methyl‐APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N‐methyl‐d‐aspartate (NMDA) receptor antagonist, 2‐amino‐5‐phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2 Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nm) and competitively inhibited NMDA‐sensitive l‐[3H]‐glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]‐(±)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonate (CPP), with a Ki of 35 nm, and was 4, 5 and 7 fold more potent than the antagonists ((±)‐cis‐4‐phosphonomethylpiperidine‐2‐carboxylic acid) (CGS 19755), CPP and d‐AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the d‐stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3 CGP 37849 and CGP 39551 were inactive as inhibitors of l‐[3H]‐glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4 In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA‐evoked increases in CA1 pyramidal cell firing rate. In slices bathed in medium containing low Mg2+ levels, concentrations of CGP 37849 up to 10 μm suppressed burst firing evoked in CA1 neurones by stimulation of Schaffer collateral‐commissural fibres without affecting the magnitude of the initial population spike; CGP 39551 exerted the same effect but was weaker. In vivo, oral administration to rats of either CGP 37849 or CGP 39551 selectively blocked firing in hippocampal neurones induced by ionophoretically‐applied NMDA, without affecting the responses to quisqualate or kainate. 5 CGP 37849 and CGP 39551 suppressed maximal electroshock‐induced seizures in mice with ED50s of 21 and 4 mg kg−1 p.o., respectively. 6 CGP 37849 and CGP 39551 are potent and competitive NMDA receptor antagonists which show significant central effects following oral administration to animals. As such, they may find value as tools to elucidate the roles of NMDA receptors in brain function, and potentially as therapeutic agents for the treatment of neurological disorders such as epilepsy and ischaemic brain damage in man.

CGP 55845A: a potent antagonist of GABAB receptors in the CA1 region of rat hippocampus.

The new GABAB receptor antagonist CGP 55845A was tested on pre- and post-synaptic GABAB receptors in the hippocampus. CGP 55845A (1 microM) blocked (-)-baclofen (5-10 microM)-induced postsynaptic hyperpolarization and depression of evoked IPSPs and EPSPs. It also blocked three physiological consequences of GABAB receptor activation: the late IPSP, paired-pulse depression of IPSCs, and heterosynaptic depression of EPSPs. Therefore, CGP 55845A is an antagonist at pre- and post-synaptic GABAB receptors in the hippocampus and is approximately three orders of magnitude more potent than previously described GABAB receptor antagonists.

Comparison of antagonist potencies at pre- and post-synaptic GABAB receptors at inhibitory synapses in the CA1 region of the rat hippocampus

Synaptic activation of γ‐aminobutyric acid (GABA)B receptors at GABA synapses causes (a) postsynaptic hyperpolarization mediating a slow inhibitory postsynaptic potential/current (IPSP/C) and (b) presynaptic inhibition of GABA release which depresses IPSPs and leads to paired‐pulse widening of excitatory postsynaptic potentials (EPSPs). To address whether these effects are mediated by pharmacologically identical receptors the effects of six GABAB receptor antagonists of widely ranging potencies were tested against each response. Monosynaptic IPSPBs were recorded in the presence of GABAA, AMPA/kainate and NMDA receptor antagonists. All GABAB receptor antagonists tested depressed the IPSPB with an IC50 based rank order of potency of CGP55679CGP56433=CGP55845A=CGP52432>CGP51176> CGP36742. Paired‐pulse EPSP widening was recorded as an index of paired‐pulse depression of GABA‐mediated IPSP/Cs. A similar rank order of potency of antagonism of paired‐pulse widening was observed to that for IPSPB inhibition. Comparison of the IC50 values for IPSPB inhibition and paired‐pulse EPSP widening revealed a close correlation between the two effects in that their IC50s lay within the 95% confidence limits of a correlation line that described IC50 values for inhibition of paired‐pulse EPSP widening that were 7.3 times higher than those for IPSPB inhibition. Using the compounds tested here it is not possible to assign different subtypes of GABAB receptor to pre‐ and post‐synaptic loci at GABAergic synapses. However, 5–10 fold higher concentrations of antagonist are required to block presynaptic as opposed to postsynaptic receptors when these are activated by synaptically released GABA.

GABAA and GABAB receptors in locus coeruleus: effects of blockers

Racemic baclofen, (-)-baclofen and muscimol depressed all spontaneously firing locus coeruleus neurons tested in a slice preparation. Racemic phaclofen (100 microM; 1 mM) moderately antagonized the effects of racemic baclofen without antagonizing those of muscimol. Bicuculline (10, 30, 100 microM) potently antagonized the action of muscimol without affecting the inhibition of baclofen. Phaclofen and bicuculline had no pronounced effect on the spontaneous discharge rate of cells. The results suggests that there are GABAA and GABAB receptors in the locus coeruleus.

Cholinomimetics induce θ rhythm and reduce hippocampal pyramidal cell excitability

Abstract The actions of cholinomimetics and of physostigmine were tested on two parameters reflecting hippocampal activity, namely θ activity and pyramidal cell excitability. In rats pretreated with methylscopolamine and anaesthetized with urethane i.v. administration of the cholinomimetics oxotremorine and arecoline and the cholinesterase blocker physostigmine evoked θ wave activity in the hippocampus, which was blocked by scopolamine. Spectral analysis demonstrated that the frequency of the θ waves induced was dose-related, ranging from about 3 Hz to between 5 and 6 Hz. θ Activity could not be induced by arecoline in animals with large septal lesions. Pyramidal cell excitability is known to be increased by endogenous acetylcholine released from cholinergic fibres. In the present study, however, i.v. injections of oxotremorine, arecoline and physostigmine in doses that induce θ activity diminished the excitability of CA1 pyramidal cells in a dose-dependent manner, as judged by the reduction in the amplitude of the population spike and the dendritic epsp. These depressant effects were attenuated by scopolamine but not by methylscopolamine. The depressant effect of arecoline was attenuated in rats with extensive lesions in the medial septal area. The present findings demonstrate that exogenously administered cholinomimetics only partly mimic the action of endogenous acetylcholine in the hippocampus. The central sites of action of exogenously administered cholinomimetics for mediation of θ activity and alteration of pyramidal cell excitability remain to be elucidated.

Contribution of presynaptic GABA-B receptors to paired-pulse depression of GABA responses in the hippocampus

The synaptic release of γ-aminobutyric acid (GABA) is thought to be regulated by presynaptic GABA receptors of the 13-type. It was the goal of this study to validate this concept electrophysiologically using four selective antagonists of GABA-B receptors. Experiments were performed in hippocampal slices exposed to 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX 30 μM) and d-2-amino-5-phosphonopentanoate (AP5 40 μM) in order to block excitatory transmission. Consequently, electrical stimulation of the Schaffer collateral/commissural fibers evoked monosynaptic inhibitory potentials (IPSP) recorded intracellularly from CA 1 pyramidal neurons. In a test called paired-pulse paradigm two identical stimuli were applied at intervals ranging from 350 to 4000 ms. The IPSP evoked by the second stimulation was smaller in its amplitude over the entire interval range. This reduction of the second GABAresponse is thought to result from the activation of presynaptic GABA receptors. The GABA-uptake inhibitor SKF 89976 (100 μM) increased the amplitude of the IPSPs and increased the ratio of the first to the second IPSP amplitude. These findings indicate that the drug increases the GABA content in the synaptic cleft leading to a facilitation of paired-pulse depression.The actions of four bath-applied GABAB receptor antagonists were examined in the paired-pulse paradigm. None of these compounds abolished paired-pulse inhibition completely even at concentrations higher than those required to block postsynaptic GABA-B responses. The potent GABA-B antagonists CGP 55845 and CGP 52432 reduced paired-pulse depression by 80% at 10 μM (maximal effect). The other two compounds, CGP 46381 and CGP 36742 had no significant or only a subtle effect respectively.The adenosine receptor antagonist, caffeine (100 μM) and the metabotropic excitatory amino acid receptor antagonist (1SS, 3 R)-1-aminocyclopentane-t,3-dicarboxylic acid (MCPG, 1 mM) had no effect on paired-pulse depression in the presence of CGP 55845 (10 μM)In conclusion since all CGP compounds are GABA-B antagonists at postsynaptic sites these findings suggest that there may be differences between the pre- and postsynaptic GABA-B receptors. Apart from presynaptic GABA-B receptors there appear to exist additional mechanisms involved in paired-pulse depression that remain to be elucidated.

Electrophysiological characterization of a novel potent and orally active NMDA receptor antagonist: CGP 37849 and its ethylester CGP 39551

The selectivity and potency of the novel competitive N-methyl-D-aspartate (NMDA) receptor antagonists, CGP 37849 and CGP 39551, were investigated in vitro and in vivo using electrophysiological approaches. Like the reference blocker DL-AP5, both compounds acted in vitro (hippocampus, substantia nigra, spinal cord) to antagonize the excitatory actions of exogenously administered NMDA as well as the synaptically elicited, physiological NMDA receptor responses in hippocampus and spinal cord. In all isolated preparations CGP 37849 was more potent than CGP 39551, and 5- to 10-fold more potent than DL-AP5. Neither compound showed any marked effect on responses evoked by quisqualate and kainate. NMDA excited dopaminergic cells in the pars compacta region of the substantia nigra in a concentration-dependent manner. This effect also could be selectively antagonized by CGP 37849 and CGP 39551. In the anaesthetized rat, excitatory responses of hippocampal pyramidal cells evoked by iontophoretic application of NMDA were antagonized by CGP 37849 and CGP 39551 following their oral administration without reducing quisqualate or kainate responses. In contrast to the in vitro situation, CGP 39551 was more potent than CGP 37849 in vivo. Effective doses were 30 mg/kg p.o. for CGP 39551 and 100 mg/kg p.o. for CGP 37849. In conclusion, it is demonstrated that CGP 37849 and CGP 39551 selectively antagonize NMDA evoked neuronal responses in vivo and in vitro and that the drugs are centrally active following their oral administration.