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The mode of action of the antipsychotic drugs clozapine, haloperidol and iloperidone was investigated in layer V of prefrontal cortex slices using extracellular field potential, intracellular sharp-electrode as well as whole-cell voltage clamp recording techniques. Intracellular investigations on a broad range of concentrations revealed that the typical neuroleptic haloperidol at higher concentrations significantly depressed the excitatory postsynaptic component induced by electrical stimulation of layer II. This was not seen with the atypical neuroleptics clozapine and iloperidone. None of the three compounds had any effect on the resting membrane potential, spike amplitude or input resistance at relevant concentrations. Synaptic plasticity was assessed by means of extracellular field potential recordings. Clozapine significantly facilitated the potentiation of synaptic transmission, whereas haloperidol and iloperidone showed no effects. In line with its facilitating effect on synaptic plasticity, it could be demonstrated by whole-cell voltage clamp recordings that clozapine increased N-methyl-D-aspartic acid receptor-mediated excitatory postsynaptic currents in the majority of prefrontal cortical neurones. These investigations were made with neuroleptic drugs applied to the bath in the micromolar concentration range in order to approach clinical brain concentrations that are reached after administration of therapeutic doses. The drug concentrations reached in the slices after the experiments were assessed by means of high-pressure liquid chromatography coupled with mass-spectrometric detection. Surprisingly, drug accumulation in the in vitro preparation was of similar degree as reported in vivo. In conclusion, the typical neuroleptic haloperidol significantly depressed excitatory synaptic transmission in layer V neurones of the prefrontal cortex. In contrast, the two atypical neuroleptics iloperidone and clozapine revealed no depressing effects. This feature of the atypical neuroleptics might be beneficial since a hypofunctionality of this brain area is thought to be linked with the pathophysiology of schizophrenia. Additionally, clozapine facilitated long-term potentiation, which might be linked with the clinically observed beneficial effects on certain cognitive parameters. The clozapine-induced increase of N-methyl-D-aspartic acid receptor-mediated currents suggests that clozapine facilitates the induction of long-term potentiation. Furthermore, the present study points to the importance of considering the significant accumulation of neuroleptic drugs in in vitro studies., (Copyright 2003 IBRO)

In vitro and in vivo electrophysiological studies were done to investigate the neuronal function of the hippocampus and prefrontal cortex in the amyloid precursor protein (APP) 23 transgenic mouse model for amyloidosis developed by Sturchler-Pierrat et al. [Proc Natl Acad Sci USA 94 (1997) 13287]. Brain slices were taken from 3, 6, 9, 12, 18 and 24 month old wildtype and hemizygous type APP23 mice. Extracellular field potentials were recorded from the CA1 region of the hippocampus while stimulating the Schaffer collaterals. In addition, extracellular field potentials were elicited from areas within layer V/VI of the prefrontal cortex by stimulating the same layer V/VI. Basic synaptic function in the hippocampus was reduced in hemizygous APP23 mice compared with their wildtype littermates at 12 and 18 months of age, whereas, it was unaltered within the prefrontal cortex. Long-term potentiation in the hippocampus and the prefrontal cortex of hemizygous APP23 mice was similar compared with their wildtype littermates. In vivo electrophysiological experiments were done in 3, 9, 18 and 24 month old wildtype and hemizygous APP23 mice. No differences were observed in the number of single spontaneously active units recorded within the prefrontal cortex of hemizygous APP23 mice compared with their wildtype littermates. Field potentials elicited during stimulation of cortico-cortical pathways to assess synaptic transmission and short-term synaptic plasticity were also unchanged in hemizygous APP23 mice. Furthermore, presumable antidromic field potentials recorded in the prefrontal cortex during stimulation of the striatum were similar between the hemizygous APP23 and wildtype mice at each age. The present study shows that amyloidosis impairs basic synaptic function but not long-term potentiation in the hippocampus, however, does not alter any of the neurophysiological functions measured within the prefrontal cortex. These findings suggest that amyloidosis may be involved in altering some neurophysiological functions within only certain brain structures. Although APP23 mice have impaired cognitive performance, long-term plasticity, a cellular model for memory, is not affected, raising the question on the relationship between these processes.

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

Electrophysiological experiments were performed in vitro and in vivo to characterize the inhibitory effects of 6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA 1021; licostinel) on rat brain glutamate receptors. In vitro, ACEA 1021 was tested on N-methyl-D-aspartate (NMDA)-induced depolarizations in the neocortical slice preparation and on epileptiform activity in Mg2+-free hippocampal slices, which is known to be NMDA receptor mediated. In both in vitro models, ACEA 1021 exhibited antagonistic effects on the NMDA receptor-mediated responses. Selectivity tests in the neocortical slice preparation, using NMDA, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) showed that 10 microM ACEA 1021 reduced NMDA and kainate responses to 27.9 and 79.9% of the control value, respectively, whereas responses to AMPA were increased by 2.4% above the control value, thus showing that at this concentration ACEA 1021 acts preferentially at NMDA receptors. However, at 30 microM, all the NMDA-, AMPA- and kainate-induced responses were reduced. In vivo, ACEA 1021 was tested on NMDA-induced excitation in the CA1 region. After systemic administration of ACEA 1021, central effects were observed at 10 mg/kg i.v. in the CA1 region. These results indicate that ACEA 1021 is centrally active and inhibits NMDA receptor-mediated responses. Interestingly, selectivity tests in the CA1 region did not show clear differences in the action of ACEA 1021 on NMDA- and AMPA-induced excitations. Furthermore, ACh-induced excitations were also reduced. Thus, at low concentrations, ACEA 1021 seems to be a selective antagonist at the strychnine-insensitive glycine site of the NMDA receptor. However, at 30 microM in vitro and at 10 mg/kg in vivo, non-NMDA receptor-mediated actions of ACEA 1021 are observed. Our results suggest that these additional effects of ACEA 1021 may contribute to its anticonvulsive properties in mice as well as to its neuroprotective properties in animal models of cerebral ischemia.

This study sought to investigate the influence of GABAB receptor activation on acoustically induced excitation within the rat inferior colliculus. To this end, the GABAB receptor antagonist, CGP 35348, was applied systemically and iontophoretically. Single and multibarrel electrodes were used for extracellular recordings within the central nucleus of the inferior colliculus. The experimental model, a paired-pulse stimulus paradigm, applied two identical acoustic stimuli, 200 msec apart, evoking corresponding responses characterized by the second being consistently weaker than the first. Abolishment of the acoustically evoked response, following iontophoretic application of the GABAB receptor agonist, L-baclofen, verified the existence of GABAB receptors in all inferior colliculus cells tested. Intravenous application of CGP 35348 (200 mg/kg) evoked a 24% overall increase in stimulus responses. Likewise, a 13% increase in total evoked excitation was observed, following iontophoretic application. There was no significant reduction of inhibition on the second evoked response in the paired-pulse model, following either systemic or iontophoretic application of CGP 35348. This result implies that the decreased magnitude of the second response, with an interpulse interval of 200 msec, is not influenced by GABAB receptor mediated inhibition. These findings do indicate, however, that GABAB receptors play a small, but significant role during the processing of acoustic information, within the inferior colliculus.

The synaptic release of gamma-aminobutyric acid (GABA) is thought to be regulated by presynaptic GABA receptors of the B-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 microM) and D-2-amino-5-phosphonopentanoate (AP5 40 microM) 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 GABA-response is thought to result from the activation of presynaptic GABA receptors. The GABA-uptake inhibitor SKF 89976 (100 microM) increased the amplitude of the IPSP's 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 GABA-B 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 microM (maximal effect).(ABSTRACT TRUNCATED AT 250 WORDS)

The goal of this report is to present the results obtained with three new GABAB receptor antagonists. CGP 54062 has an IC50 in a GABAB binding test of 0.013 microM which is roughly 2500-fold lower than one of the most potent blockers known so far, CGP 35348 (IC50 = 34 microM). CGP 46381 and CGP 36742 have IC50s of 4.9 and 36 microM respectively. The latter two compounds are the first orally active GABAB receptor antagonists. All three compounds bind to the GABAB receptor selectively, and are inactive in a number of binding tests assessing the compounds' affinity to various other receptor sites. The effect of these blockers on GABAergic transmission was investigated in the CA1 area of hippocampal slices. The Schaffer collateral/commissural fibers were stimulated and the evoked postsynaptic potentials were recorded intracellularly in pyramidal neurons. The three antagonists blocked the late inhibitory postsynaptic potential with the following rank order of potency CGP 54062 > 46381 > 36742 approximately 35348. These findings support the hypothesis that these potentials are mediated by GABAB receptors. Orally administered CGP 36742 and CGP 46381 block the neuronal depression induced by iontophoretically applied baclofen in anaesthetised rats. Up to a dose of 10 mg/kg i.v. CGP 54062 was inactive and thus does not appear to cross the blood-brain barrier at this dose. In anaesthetised rats the effects of the three new GABAB antagonists and of CGP 35348 were investigated on the paired-pulse inhibition of the population spikes evoked in the CA1 area of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Understanding the mechanisms involved in long-term potentiation (LTP) should provide insights into the cellular and molecular basis of learning and memory in vertebrates. It has been established that in the CA1 region of the hippocampus the induction of LTP requires the transient activation of the N-methyl-D-aspartate (NMDA) receptor system. During low-frequency transmission, significant activation of this system is prevented by gamma-aminobutyric acid (GABA) mediated synaptic inhibition which hyperpolarizes neurons into a region where NMDA receptor-operated channels are substantially blocked by Mg2+ (refs. 5, 6). But during high-frequency transmission, mechanisms are evoked that provide sufficient depolarization of the postsynaptic membrane to reduce this block and thereby permit the induction of LTP. We now report that this critical depolarization is enabled because during high-frequency transmission GABA depresses its own release by an action on GABAB autoreceptors, which permits sufficient NMDA receptor activation for the induction of LTP. These findings demonstrate a role for GABAB receptors in synaptic plasticity.

We present a comparative study of the modulation of the N-methyl-D-aspartate (NMDA) receptor at the strychnine-insensitive glycine site in the spinal cord and in the cortex. The excitatory effect of NMDA was potentiated by D-serine (a glycine mimetic) in the hemisected rat spinal cord. The non-competitive NMDA antagonists 7-chlorokynurenic acid (7-Cl KYNA; 10 microM) and 3-amino-1-hydroxypyrrolid-2-one (HA-966; 100 or 200 microM) antagonized the effect of NMDA in the spinal cord and cortical wedge preparation. The antagonism was reversed by the addition of D-serine. This effect was strychnine-insensitive and hence not related to the inhibitory glycine receptor known to be present in the spinal cord. Our results suggest strongly that glycine positively modulates the NMDA system not only at a supraspinal level but also at the spinal level. As the positive modulation of NMDA responses by D-serine was also seen in the presence of tetrodotoxin, we conclude that the NMDA/glycine complex is (also) located on motoneurones in addition to the known glycine-mediated inhibitory system.

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.

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.

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 microM suppressed burst firing evoked in CAl 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 ionophoreticallyapplied NMDA, without affecting the responses to quisqualate or kainate. 5. CGP 37849 and CGP 39551 suppressed maximal electroshock-induced seizures in mice with ED50 s of 21 and 4 mg kg'- 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.

The electrophysiological effects of neurotensin (NT), its analog xenopsin, and the fragment NT-(1-8) on the activity of dopaminergic neurons in rat substantia nigra slices were compared. Xenopsin and NT produced a concentration-dependent increase in the firing rate. The mean firing rate was increased by 58.5% with xenopsin (0.1 microM) and 49.1% with NT (0.1 microM). NT-(1-8) was inactive at this concentration. Dopamine (100 microM) inhibited firing in these neurons by 54.7%. These data provide evidence that xenopsin mimics the physiological effects of NT.

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.

In anesthetized rats, injections of a 0.5 mM glutamate solution into the locus coeruleus (LC) reversibly increased the amplitude of the population spike evoked in CA1 by stimulation of the Schaffer-commissural fiber tract. This effect was absent in N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)-treated, noradrenaline (NA)-depleted animals. The excitatory postsynaptic potential recorded in the stratum radiatum was unaffected following the glutamate injections. Systemic administration of the NA-uptake inhibitor desipramine also produced an increase in population spike amplitude. The findings demonstrate that activation of LC neurons increases pyramidal cell excitability in vivo.

It has previously been claimed that the anticonvulsant valproate acts by augmenting GABA-ergic transmission, however, the data supporting this claim is controversial. Here we demonstrate that valproate strongly and reversibly potentiates the depressant effects of the GABA-A receptor agonist muscimol on locus coeruleus neurones recorded extracellularly from a midpontine slice preparation of the rat. The depressant effect of muscimol (2 microM) is augmented by bath applied valproate at concentrations of 50 microM, 100 microM and 1 mM. The effect of GABA is also potentiated by valproate. The potentiating effect is selective since the cell inhibition elicited by the GABA-B receptor agonist baclofen is not affected. Valproate on its own had no effect on the firing frequency.

The goal of this study was to investigate whether locus coeruleus neurons of the rat are sensitive to agonists of the different excitatory amino acid receptors. All experiments were performed on a midpontine rat slice preparation. Bath-applied L-glutamate, kainate, N-methyl-D-aspartate (NMDA) and quisqualate induced concentration-dependent activations of all neurons which were reflected in an increase of the neurons' mean discharge rate. The rank order of cell activation was kainate approximately quisqualate greater than NMDA greater than L-glutamate. None of the agonists induced a bursting-type of discharge. The NMDA-receptor blocker DL-2-amino-5-phosphonovaleric acid (APV, 30 microM) selectively antagonized the NMDA-induced increase in cell firing. Kynurenic acid (100 microM) non-selectively attenuated the response to NMDA, kainate and quisqualate. Neither APV nor kynurenic acid per se had any effect on the spontaneous firing rate. If the Mg2+ concentration in the superfusion medium was lowered from 2 mM to nominally zero the response to NMDA was selectively increased. In conclusion, locus coeruleus neurons share with other neurons their sensitivity to agonists of all three types of excitatory amino acid receptors. However, in contrast to other neurons, they do not respond with a bursting type of discharge.

The actions of cholinomimetics and of physostigmine were tested on two parameters reflecting hippocampal activity, namely theta 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 theta wave activity in the hippocampus, which was blocked by scopolamine. Spectral analysis demonstrated that the frequency of the theta waves induced was dose-related, ranging from about 3 Hz to between 5 and 6 Hz. theta 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 theta 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 theta activity and alteration of pyramidal cell excitability remain to be elucidated.

A considerable number of neuropeptides have been localized immunohistochemically in the area of the locus coeruleus of the rat. The objective of this study was to assess the actions of some of these transmitter candidates on spontaneously active locus coeruleus neurons in vitro. The effects of bath-applied peptides on the discharge rate of individual locus coeruleus neurons were investigated. A midpontine slice preparation of the gerbil brain was used. Excitatory dose-dependent effects were found with four peptides with the following rank order of potency: Substance P, (Arg8)-vasopressin, neurotensin, ACTH1-24. Somatostatin hyperpolarized all neurons tested. Given the pronounced effects seen with substance P, somatostatin and vasopressin in the nanomolar range, it is conceivable that these peptides may have a role in regulating neuronal activity in locus coeruleus.