A flexible technology platform to explore valuable drug targets.

The high-throughput screening platform implemented for drug discovery is driven by the therapeutic areas of interest. Therefore the speed and information derived is governed by these areas. Multiple technologies are needed to exploit this and it is also important to show reactivity to new advances in technology. In contrast with a drive over the last few years towards higher throughput and speed, higher information content will be instrumental in driving lead discovery in the future.

Clozapine-induced dopamine levels in the rat striatum and nucleus accumbens are not affected by muscarinic antagonism.

The effect of the muscarinic antagonist, scopolamine, was examined for a change in the increase in extracellular dopamine, dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), induced by haloperidol or clozapine in the striatum and nucleus accumbens of anaesthetised and awake rats, monitored using in vivo cerebral microdialysis. Rats received scopolamine (1 mg kg(-1); s.c.) or vehicle followed by haloperidol (1 mg kg(-1); s.c.) or clozapine (20 mg kg(-1); s.c.). Dopamine, DOPAC, HVA and 5-HIAA overflow into striatal or accumbens perfusates was determined using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Scopolamine failed to modify the clozapine- or haloperidol-induced efflux of dopamine or its metabolites in either the striatum or nucleus accumbens following systemic administration in anaesthetised or awake rats. Although pretreatment with scopolamine tended to produce a smaller increase in the clozapine-induced efflux of DOPAC in striatal perfusates than following clozapine treatment alone, this was not statistically significant. Furthermore, local infusion of scopolamine (100 microM) with clozapine (1 mM) via the microdialysis probe did not attenuate the elevated efflux of dopamine observed following clozapine alone, in either the striatum or nucleus accumbens, in anaesthetised rats. This treatment did prevent the clozapine-induced increase in DOPAC and HVA in the striatum but not the nucleus accumbens. Carbachol (50 microM) infused into the dorsolateral striatum or nucleus accumbens raised extracellular dopamine levels 200% and 150%, respectively above baseline. Our data suggest that the increased efflux of dopamine and its metabolites in the rat basal ganglia following clozapine administration is not significantly dependent upon the interaction of clozapine with muscarinic receptors.

Multiple sigma binding sites in guinea-pig and rat brain membranes: G-protein interactions.

1. Evidence is accumulating for multiple sigma (sigma) sites in the mammalian CNS. 2. We have addressed this problem and have examined sigma site - G-protein coupling in guinea-pig and rat brain membranes. 3. Ditolylorthoguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine (3PPP) and dextromethorphan displaced [3H]-DTG (3.4 nM) with low Hill slopes of 0.5, 0.6 and 0.6, respectively in guinea-pig brain membranes. 4. In the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p; 100 microM), the specific binding of [3H]-DTG was reduced by 36.7%, the Hill slope of 3PPP was increased to near unity, the ability of dextromethorphan to displace DTG was virtually abolished and the Hill slope for DTG remained low (0.7), indicating the presence of at least two binding sites. These data indicate that although Gpp(NH)p removes a dextromethorphan high affinity site, two DTG selective sites remain in the presence of Gpp(NH)p. 5. The present study suggests that DTG binds to at least three sites in guinea-pig brain membranes, at least one of which is G-protein linked. 6. In rat brain membranes, DTG displaced itself (3.4 nM) with a Hill slope near 1. 3PPP displacement of [3H]-DTG was comparable with the guinea-pig (Hill slope 0.5) and displaced from more than 1 site. Dextromethorphan did not displace [3H]-DTG at concentrations below 10 microM. 7. The heterogeneity of sigma sites appears to be less in rat than in guinea-pig brain membranes.

The sigma ligand 1,3-di-o-tolylguanidine depresses amino acid-induced excitation non-selectively in rat brain.

The sigma ligand 1,3-di-o-tolylguanidine (DTG) has been applied by microiontophoresis to neurones in the rat hippocampal slice and to neurones in the neocortex and hippocampus of rats anaesthetised with urethane. DTG depressed the excitatory responses of cells to both N-methyl-D-aspartate (NMDA) and quisqualate on a majority of the units tested, in no case causing an enhancement. Haloperidol had no consistent effect of its own and did not prevent the depressant effects of DTG. It is concluded that in the preparations used, DTG did not selectively modify neuronal sensitivity to NMDA.

Nicotinylalanine increases cerebral kynurenic acid content and has anticonvulsant activity.

1. Nicotinylalanine is an analogue of kynurenine which has been reported to inhibit the enzymes kynurenine hydroxylase and kynureninase. 2. In the present study rats were given a tryptophan load together with nicotinylalanine two hours before killing, and the brain, liver and kidneys analysed by HPLC for their kynurenic acid content. 3. Tryptophan alone produced a significant elevation of kynurenate but with the additional administration of nicotinylalanine, levels rose dramatically, including a 19-fold increase in brain. 4. In mice the same dose of nicotinylalanine reduced the incidence of seizures induced by leptazol or electroshock treatment. 5. Since kynurenic acid is an antagonist at excitatory amino acid receptors the results may herald a new approach to producing a pharmacological blockade of amino acid receptors in the brain.

Changes in neurotransmitter sensitivity in the mouse neocortical slice following propranolol and theophylline administration.

1. The mouse neocortical slice has been used to examine the sensitivity of neurones to isoprenaline, 5-hydroxytryptamine (5-HT) and adenosine acutely and following chronic treatment of animals with propranolol or theophylline. 2. While having little effect alone, all three agonists enhanced the d.c. depolarizing potential produced by N-methyl-D-aspartate (NMDA). The effect of (-)-isoprenaline (0.2 microM) was shared by (+)-isoprenaline at the much higher concentration of 10 microM. 3. Superfusion of slices with theophylline or 8-phenyltheophylline blocked responses to adenosine with evidence of selectivity. A single injection of theophylline 24 h before slice preparation did not alter agonist sensitivity, but when administered daily at 100 mg kg-1 for 14 days, the xanthine caused a loss of sensitivity to adenosine and (-)-isoprenaline but not 5-HT. The lower dose of theophylline, 10 mg kg-1 daily, also led to a loss of adenosine responses but no change of sensitivity to the amines. 4. Following the 14 day treatment with theophylline at 100 mg kg-1 daily in two groups of mice, responses to adenosine recovered to control levels after 20 days. 5. Propranolol superfusion blocked responses to both isomers of isoprenaline and 5-HT but did not affect sensitivity to adenosine. 6. Chronic treatment with propranolol at 25 mg kg-1 daily for 14 days induced a loss of sensitivity to (-)-isoprenaline and 5-HT but not adenosine. A lower dose of 5 mg kg-1 daily caused no change in responses to adenosine or 5-HT, but yielded an increased sensitivity to (-)-isoprenaline. 7. The results are discussed with respect to reports of receptor up-regulation in binding studies; caution is clearly required in extrapolating from such work to receptor activity in a functional system, especially in the case of theophylline and adenosine.

The lack of utility of the rat vas deferens as a functional bioassay for sigma ligands.

The present study examined the utility of the rat vas deferens preparation as a bioassay for sigma site ligands. sigma Ligands such as (+/-)-pentazocine, phencyclidine (PCP) and (+)-SK&F 10047 potentiated neurogenic twitch contractions. However, neither the order of potency nor the absolute potency of (+/-)-pentazocine and (+)-SK&F 10047 correlated with their affinity at central sigma sites. Furthermore, another potent sigma ligand, ditolyl-ortho guanidine (DTG) neither affected neurogenic twitch contractions nor inhibited twitch potentiation by PCP or (+)-SK&F 10047 at concentrations up to 30 mumol/l. These data indicate that the rat vas deferens is not a useful bioassay for the evaluation of sigma ligands. PCP, (+)-SK&F 10047 and (+/-)-pentazocine probably enhance neurogenic contractions in rat vas deferens primarily by inhibition of the neuronal uptake of noradrenaline.

Chronic benzodiazepine treatment and cortical responses to adenosine and GABA.

The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chloroadenosine or in the presence of 2-hydroxynitrobenzylthioguanosine. GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses.

Theophylline down-regulates adenosine receptor function.

Chronic treatment of animals with caffeine or theophylline has been reported to increase the number of adenosine receptors in the CNS detected by ligand binding, but few functional studies have been performed. In the present study adenosine enhanced depolarising responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) in slices of mouse cerebral cortex. This effect was blocked acutely by theophylline but not by theophylline given 24 h previously. Twenty-four hours after 2 weeks of treatment with this antagonist, however (10 or 100 mg/kg/day) adenosine was no longer effective, sensitivity recovering subsequently. Theophylline treatment also reduced sensitivity to (-)-isoprenaline but not 5-hydroxytryptamine. It is concluded that changes of adenosine receptor number in the brain do not necessarily reflect the induced changes of receptor function.

NMDA-receptor-independent effects of low magnesium: involvement of adenosine.

Perfusion of hippocampal slices with magnesium-free media elicits epileptiform activity attributable partly to the activity of N-methyl-D-aspartate (NMDA) receptors, but recent reports have documented an NMDA-independent enhancement of orthodromic potentials by moderate reductions in magnesium concentration. The present experiments indicate that this enhancement is comparable with that produced by perfusion with an adenosine antagonist, 8-phenyltheophylline, and that superfusion with this compound or adenosine deaminase precludes any enhancement of potential size in low magnesium solutions. The low magnesium enhancement is probably attributable to the recently described magnesium dependency of presynaptic inhibition by adenosine.

Distribution of NADPH-diaphorase positive cells in the rat brain.

1. Recent work suggests that neurones in vivo and in culture which contain neuropeptide Y and somatostatin and which stain positively for the enzyme NADPH-diaphorase may be resistant to excitotoxins. 2. We have therefore examined the distribution of the enzyme throughout the rat brain. 3. Neurones were found intensely or moderately stained at all levels of the neuraxis, but with particularly dense clusters of cells in the periaqueductal grey area and dorsal raphe nucleus of the hindbrain, the pedunculopontine and interpeduncular nuclei, and the dorsal spinal trigeminal nucleus. 4. Intensely stained cells occurred with no clear pattern in neocortical and striatal areas, and in nucleus basalis. 5. The observed distribution of staining is consistent with previous studies in other species of limited regions of the CNS. 6. While no consistent functional or neurochemical correlate of the NADPH-diaphorase distribution could be proposed, the work provides a basis for more detailed investigations of neuronal sensitivity to excitotoxins.

Quinolinic acid neurotoxicity: protection by intracerebral phenylisopropyladenosine (PIA) and potentiation by hypotension.

In the present study we have used the purine agonist R-phenylisopropyladenosine (PIA) which suppresses excitatory amino acid release to assess its effect on quinolinate toxicity. Quinolinic acid was injected into the rat hippocampus alone or with PIA and the animals allowed to recover. After 4 days the brain was removed for histological examination. The extent of neuronal degeneration was assessed blind by an independent observer on a scale of 0 (no damage) to 10 (complete degeneration). Co-administration of PIA protected against the toxicity, and this protective action of PIA was blocked by a xanthine adenosine receptor antagonist. However, systemic injections of PIA or the non-purine ganglion blocking drug trimetaphan, both of which caused significant depression of blood pressure, potentiated quinolinate toxicity. The results may indicate an interaction between endogenous excitotoxins and episodes of hypotension which may be critical in determining cell death in the CNS.

Effect of changing extracellular levels of magnesium on spontaneous activity and glutamate release in the mouse neocortical slice.

1. The mouse neocortical slice preparation, maintained in a two compartment, grease gap bath, exhibits spontaneous depolarizing activity (with or without rhythmic after potentials) after perfusion with magnesium-free artificial cerebrospinal fluid. 2. If the magnesium concentration is decrementally lowered over an extended time period, then incrementally raised following a similar time course, the spontaneous depolarizing shift activity shows a hysteresis (with regard to both frequency and amplitude), the depolarizing shifts being more resistant to magnesium during the incremental period. 3. The amino acid content of the perfusing fluid was analysed by high performance liquid chromatography (h.p.l.c.). Although a basal efflux of 6 amino acids was quantifiable, only glutamate levels increased following superfusion of the preparation with magnesium-free, artificial cerebrospinal fluid. 4. Glutamate release increased to 266% of the resting release in the presence of magnesium within the first 12 min of the change into magnesium-free artificial cerebrospinal fluid. This increase in release preceded the onset of spontaneous depolarising activity. The release of glutamate remained elevated at 182% of control up to 60 min after perfusion with magnesium-free buffer, when depolarizing activity was well established. 5. A model is presented and discussed for the genesis and maintenance of the spontaneous depolarizing shifts. It is suggested that the maintenance of this spontaneous activity reflects a long term enhancement of neocortical neurone excitability which may be related to long term potentiation in the hippocampus.

Increase in kynurenic acid in Huntington's disease motor cortex.

Huntington's disease is a neurological disorder characterised by a progressive chorea and dementia. Recent evidence has suggested that dysfunction involving endogenous excitatory amino acids may be important in the pathogenesis of this disease. Following the recent demonstration that kynurenic acid is present in the brain, we examined the levels in various areas of brain from patients who died with Huntington's disease and from age/sex-matched controls. Blocks (100-500 mg) of cortex (Brodmann's areas 4 and 10) and caudate nucleus and globus pallidus (lateral and medial parts) were obtained from the Cambridge Brain Bank. The tissue was then processed for the extraction and analysis of kynurenic acid. Whereas no differences in the content of kynurenic acid were observed in the caudate nucleus, lateral or medial globus pallidus, or prefrontal cortex (area 10) between controls' brains and those from patients who died with Huntington's disease, there was a 94% (p less than 0.01; n = 5) increase in the kynurenic acid content in the motor cortex (area 4) from Huntington's disease brains, relative to those of controls. Some time ago we suggested that a subtle change in the relative concentrations of quinolinic and kynurenic acids might be important in the pathogenesis of neurodegeneration. It is possible that the observation of raised kynurenic acid levels supports this supposition. Further work is now in progress to determine whether the change in kynurenic acid is a primary effect or a compensatory response to an increase in excitatory activity.

Excitatory amino acid antagonists and endogenous aspartate and glutamate release from rat hippocampal slices.

1. The effect of excitatory amino acid agonists and antagonists on the efflux of endogenous aspartate and glutamate from the rat hippocampus in vitro was studied. 2. None of the compounds tested had any effect on the basal efflux of endogenous aspartate and glutamate. 3. 2-Amino-5-phosphonovaleric acid (APV), 2-amino-7-phosphonoheptanoic acid (APH) and MK-801 all reduced the potassium-evoked efflux of aspartate and glutamate by between 14.9% and 34.3% (P less than 0.05). 4. The depression of efflux brought about by APV was still observed in the presence of tetrodotoxin. 5. Neither N-methyl-D,L-aspartate nor quinolinic acid had any effect on the potassium-evoked efflux of aspartate and glutamate. 6. These results imply the existence of presynaptic amino acid receptors that are capable of modulating the efflux of endogenous aspartate and glutamate.

Quinolinic acid effects on amino acid release from the rat cerebral cortex in vitro and in vivo.

1. The effect of quinolinic acid, N-methyl-D,L-aspartate (NMDLA) and kainate on the release of endogenous and exogenous amino acids from the rat cerebral cortex in vitro and in vivo was studied. 2. Neither quinolinic acid nor NMDLA had any effect on the basal or potassium-evoked release of [3H]-D-aspartate from slices of rat cerebral cortex either in the presence or absence of magnesium. Kainic acid failed to modify the basal efflux of [3H]-D-aspartate but significantly inhibited (by 34.4% +/- 0.04%, P less than 0.05) the potassium-evoked release. 3. Neither quinolinate nor NMDLA had any effect on the basal efflux of endogenous amino acids from rat cortical slices either in the presence or absence of magnesium ions at concentrations between 10 microM and 5 mM. 4. Both NMDLA (1 mM) and quinolinate (5 mM) produced an efflux of endogenous aspartate (371.4% +/- 11.6%; 389.3% +/- 12.1%) and glutamate (405.4% +/- 13.6%; 430.1 +/- 8.7%) respectively from the rat cerebral cortex in vivo (P less than 0.01). The quinolinic acid-evoked efflux was abolished by the NMDLA antagonist, 2-amino-5-phosphonovaleric acid (200 microM). 5. Kainic acid also caused an efflux of endogenous amino acids from the rat cerebral cortex in vivo. However, the profile of this release was different from that produced by quinolinate and NMDLA. 6. The results add further support to the suggestion that quinolinic acid acts at the NMDLA-preferring receptor and may also explain the requirement for intact afferent projections for the neurotoxic effects of quinolinate to be manifested.