Effects of neurotensin receptor antagonists on the firing rate of rat ventral pallidum neurons.

Using anaesthetized adult rats, we compared the effects of i.v. injections of neurotensin receptor antagonists (SR48692 and SR142948), haloperidol and clozapine on neuronal firing rate in the two ventral pallidal areas. SR48692 (0.5, 1 and 2 mg/kg, i.v.) induced a dose-dependent inhibition of firing rate in two thirds of neurons in the ventromedial part without any effect in the ventrolateral part. These effects are in keeping with the preferential neurotensin immunoreactivity distribution reported in the ventral pallidum. The classical antipsychotic drug haloperidol (0.5 mg/kg, i.v.) induced an inhibition of neuronal firing rate in both ventral pallidal areas whereas the atypical antipsychotic drug clozapine (20 mg/kg, i.v.), like the neurotensin receptor antagonist SR48692, inhibited cell firing only in the ventromedial part.

Electrophysiological evidence for putative subtypes of neurotensin receptors in guinea-pig mesencephalic dopaminergic neurons.

Electrophysiologically identified mesencephalic dopaminergic neurons were examined by means of extra- and intracellular microelectrodes in coronal slices of guinea-pig brain. Neurotensin and its C-terminal fragment (8-13) were equipotent in the enhancement of spontaneous neuronal firing rate (EC50 values 81.9 and 72.6nM, respectively). The duration of response was significantly longer and more variable for neurotensin compared to neurotensin fragment (8-13) (mean half-time of recovery 423+/-44 and 100+/-14 s, respectively, for peptides applied at 300 nM). The initial fast phase of excitatory responses to neurotensin receptor agonists was associated with membrane depolarization (when assessed in current-clamp mode) or with inward currents (when assessed in voltage-clamp mode), whereas prolonged excitation was associated with a slowly occurring and long-lasting change in the late afterhyperpolarization. Two kinetically distinct components were revealed in responses to neurotensin and neurotensin fragment (8-13) by the use of SR48692 and SR142948, two selective non-peptide neurotensin receptor antagonists. SR142948 (100 nM) potently antagonized responses to both agonists [response was reduced by 661 5% and 74+/-9% for neurotensin and neurotensin fragment (8-13), respectively] and caused a rightward shift in the concentration-response curve for neurotensin. On the other hand, SR48692 (100 nM) selectively inhibited the slow (late afterhyperpolarization-dependent) component, without altering the response amplitude; the half-time of recovery was reduced by 71+/-6% and 65+/-5% of control values for responses induced by neurotensin (300 nM) and neurotensin fragment (8-13) (300 nM), respectively. In addition, neurotensin, but not neurotensin fragment (8-13), provoked SR48692-sensitive and long-lasting attenuation of dopamine-induced inhibitory responses. It is suggested that two subtypes of neurotensin receptors are present in dopaminergic neurons, based on the differences in agonist and antagonist sensitivity, kinetic properties and the membrane mechanisms involved.

Effects of SR140333, a selective non-peptide NK1 receptor antagonist, on trigemino-thalamic nociceptive pathways in the rat.

Trigeminal stimulation of C-fibers increased c-fos expression within the trigeminal nucleus caudalis (NtV) and thalamic neuronal activity which both reflect the transmission of a nociceptive message. We examined the effects on both these phenomena of the selective NK1 and NK2 receptor antagonists, SR140333 and SR48968. SR140333 (0.3, 1 and 3 micrograms/kg intravenously [i.v.]) dose-dependently, reversibly and stereoselectively antagonized the increase of contralateral thalamic activity. This compound, when given i.v. (30 micrograms/kg) or orally (10 mg/kg), also reduced the number of Fos-like immunoreactive cells particularly at the medial and caudal level of the NtV. In contrast, SR48968 did not exert any antagonistic effect either on thalamic activity or on Fos-like immunoreactivity. The data strongly suggest a preferential involvement of NK1 vs NK2 receptors in nociceptive transmission following trigeminal ganglion stimulation. Taken together, our results indicate that SR140333 could provide a potent drug for the relief of pain occurring under excessive activity of sensory trigeminal fibers.

Tachykinin neurokinin-1 and neurokinin-3 receptor-mediated responses in guinea-pig substantia nigra: an in vitro electrophysiological study.

The effects of tachykinin receptor agonists and antagonists were investigated using intra- and extracellular recordings on spontaneously firing nigral neurons in guinea-pig brain slices. In 70 of 76 electrophysiologically identified dopaminergic neurons, a concentration-dependent increase in firing rate was induced by the selective neurokinin-3 tachykinin agonist senktide and by the natural tachykinin agonists neurokinin B and substance P, with EC50 values of 14.7, 31.2 and 12200 nM respectively. These responses were inhibited in a concentration- and time-dependent manner by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 (1-100 nM; n=23), but neither by its S-enantiomer SR 142806 (100 nM; n=4) nor by selective antagonists of neurokinin-1 (SR 140333) or neurokinin-2 (SR 48968) receptors (both at 100 nM; n=3). The selective neurokinin-1 agonist [Sar9,Met(O2)11]substance P (30-100 nM; n=23) and the selective neurokinin-2 agonist [Nle10]neurokinin A(4-10)(30-100 nM; n=13) were without any effect on dopaminergic cells. In 13 of 21 electrophysiologically identified, presumably GABAergic neurons located in the pars compacta of the substantia nigra, excitatory responses were evoked concentration dependently by substance P and [Sar9,Met(O2)11]substance P, with EC50 values of 18.6 and 41.9 nM respectively. These responses were inhibited by SR 140333 (100 nM; n=3), but neither by its R-enantiomer SR 140603 nor by SR 142801 (both at 100 nM; n=3). Senktide and [Nle10]neurokinin A(4-10) (both at 30-100 nM; n=10) were without effect on these presumed GABAergic neurons. A small population (12%) of pars compacta neurons was insensitive to any of the three selective tachykinin agonists. In the nigral pars reticulata, 12 neurons were recorded which had an electrophysiological profile similar to that of presumed GABAergic neurons in the pars compacta. Of these 12 cells, seven did not respond to any of the selective tachykinin agonists tested, while five were excited by senktide in a concentration-dependent manner (EC50=98.5 nM). Although this value was significantly higher than that found for dopaminergic neurons in the pars compacta, senktide-evoked responses were inhibited by SR 142801 (100 nM; n=3). We conclude that, in the guinea-pig substantia nigra, tachykinins evoke excitatory responses in both dopaminergic and non-dopaminergic neurons; however, the sensitivity to tachykinin agonists (neurokinin-1 versus neurokinin-3) depends on both neuronal type and localization.

Electrophysiological, behavioural and biochemical evidence for activation of brain noradrenergic systems following neurokinin NK3 receptor stimulation.

The objective of the present in vitro and in vivo experiments was to examine the involvement of neurokinin NK3 receptors in the regulation of the noradrenergic function in gerbils and guinea-pigs. Application of senktide, a peptide NK3 receptor agonist, on guinea-pig locus coeruleus slices increased the firing rate of presumed noradrenergic neurons (EC50 = 26 nM) in a concentration-dependent manner. Given i.c.v., senktide (0.5-2 micrograms) and (MePhe7)neurokinin B (1-10 micrograms), another NK3 receptor agonist, reduced exploratory behaviour in gerbils in a dose-dependent manner (2 micrograms of senktide producing a 50% reduction of locomotor activity and rearing). In vivo microdialysis experiments in urethane-anaesthetized guinea-pigs showed that senktide (2-8 micrograms i.c.v.) induced a dose-dependent increase in norepinephrine release in the medial prefrontal cortex. The electrophysiological, behavioural and biochemical changes elicited by senktide were concentration- or dose-dependently reduce by SR 142801, the selective non-peptide NK3 receptor antagonist. In the locus coeruleus slice preparation, complete antagonism of senktide (30 nM) was observed with 50 nM of SR 142801, while injected i.p. (0.1-1 mg/kg) it abolished the senktide-induced norepinephrine release in guinea-pigs. In gerbils, SR 142801 (1-10 mg/kg i.p.) reversed the reduction of exploratory behaviour induced by senktide (1 microgram). By contrast, the 100-fold less active enantiomer, SR 142806, did not exert any antagonism in these models. Finally, the reduction of exploratory behaviour in gerbils was found to be reversed by prazosin (0.25-2.56 micrograms/kg i.p.) and to some extent by clonidine, drugs known to depress noradrenergic function. All these experiments strongly support the hypothesis that brain noradrenergic neurons can be activated by stimulation of neurokinin NK3 receptors.

Inhibition of long-term potentiation in rat hippocampal slices by anandamide and WIN55212-2: reversal by SR141716 A, a selective antagonist of CB1 cannabinoid receptors.

It has been reported previously that delta 9-tetrahydrocannabinol and the synthetic cannabinoid agonist HU-210 [(--)-11-OH-delta 8-dimethylheptyl tetrahydrocannabinol] prevent long-term potentiation (LTP) induction in rat hippocampal slices. In this study we confirm that both WIN55212-2 ¿R-(+)-(2,3-dihydro-5-methyl-3-[¿4-morpholinyl¿ methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl) (1-naphtalenyl) methanone monomethanesulphonate¿ (3 and 10 microM), another synthetic cannabinoid agonist, and anandamide (10 microM), considered to be the endogenous ligand of cannabinoid receptors, inhibit LTP formation in the Schaffer collateral-CA1 field complex. In addition, we show that SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamide hydrochloride] at 0.1-10 microM, a potent and selective antagonist of CB1 cannabinoid receptors, concentration-dependently reversed the inhibition of LTP induced by both WIN55212-2 and anandamide. These data indicate that cannabinoid receptor agonists inhibit hippocampal LTP formation through CB1 receptor activation and that anandamide could be a candidate for an endogenous neuromessenger involved in memory processes.

SR 57227A: a potent and selective agonist at central and peripheral 5-HT3 receptors in vitro and in vivo.

SR 57227A (4-amino-(6-chloro-2-pyridyl)-1 piperidine hydrochloride) is a novel compound with high affinity and selectivity for the 5-HT3 receptor. The compound had affinities (IC50) varying between 2.8 and 250 nM for 5-HT3 receptor binding sites in rat cortical membranes and on whole NG 108-15 cells or their membranes in vitro, assayed under various conditions with [3H]S-zacopride or [3H]granisetron as radioligand. Like reference 5-HT3 receptor agonists, SR 57227A stimulated the uptake of [14C]guanidinium into NG 108-15 cells in the presence of substance P (EC50 = 208 +/- 16 nM) and contracted the isolated guinea-pig ileum (EC50 = 11.2 +/- 1.1 microM), effects that were antagonised by the 5-HT3 receptor antagonist tropisetron. The agonist effect of SR 57227A was also observed in vivo, as the compound elicited the Bezold-Jarisch reflex in anesthetised rats (ED50 = 8.3 micrograms/kg i.v.), an effect that was blocked by tropisetron and R,S-zacopride, but not by methysergide. When injected unilaterally into the mouse striatum, SR 57227A, like 2-methyl-5-HT, elicited contralateral turning behaviour which was antagonised by ondansetron. Furthermore, microiontophoretic application of SR 57227A markedly inhibited the firing rate of rat cortical neurones, an effect antagonised by tropisetron. Finally, in contrast to reference 5-HT3 agonists, SR 57227A bound to 5-HT3 receptors on mouse cortical membranes after systemic administration (ED50 = 0.39 mg/kg i.p. and 0.85 mg/kg p.o.). These results suggest that SR 57227A is a potent agonist at peripheral and central 5-HT3 receptors, both in vitro and in vivo. In view of the dearth of 5-HT3 receptor agonists which are capable of crossing the blood-brain barrier, SR 57227A may be useful in the characterisation of the neuropharmacological effects produced by the stimulation of these receptors.

SR 46559A: a novel and potent muscarinic compound with no cholinergic syndrome.

The cholinergic activities of SR 46559A, 3-[N-(2 diethyl-amino-2-methylpropyl)-6-phenyl-5-propyl] pyridazinamine sesquifumarate, have been investigated in vitro and in vivo, in rodents. Using rat brain cortical membranes, SR 46559A was a competitive ligand (Ki = 112 nM) at muscarinic M1 receptors, its affinity for muscarinic M2 (cardiac) and M3 (glandular) receptors being 6-7 times lower. SR 46559A did not interact with brain nicotinic receptors and high affinity choline uptake sites nor did it inhibit brain acetylcholinesterase activity. In contrast to reference muscarinic agonists, SR 46559A (1 mM) did not inhibit the forskolin-induced activation of cAMP synthesis nor did it stimulate phosphoinositides breakdown in various brain preparations. However, this compound enhanced (+67% at 1 mM) diacylglycerol formation in rat striatal miniprisms, an effect fully reversed by atropine. As shown with reference agonists, SR 46559A inhibited (IC50 = 10 microM) the K(+)-evoked release of [3H]GABA from rat striatal slices and reduced at 0.5 and 1 microM, the population spike amplitude of the CA1 pyramidal cells induced by stimulation of the Schaffer's collateral commissural pathway in rat hippocampal slices. In mice, SR 46559A at a near lethal dose (200 mg/kg PO) did not induce the typical cholinergic syndrome nor did it modify at 30 mg/kg PO the oxotremorine-induced hypothermia. Like muscarinic agonists, SR 46559A (1 mg/kg PO) potentiated haloperidol-induced catalepsy in rats and inhibited (ED50 = 0.12 mg/kg PO) rotations induced in mice by intrastriatal injection of pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of the aminopyridazine SR 95639A, a selective M1 muscarinic agonist.

In order to design a selective M1 muscarinic agonist, we synthesized SR 95639A (morpholinoethylamino-3-benzocyclohepta-(5,6-c)-pyridazine, dihydrochloride), a semi-rigid analogue of the aminopyridazine antidepressant drug minaprine. SR 95639A displaced [3H]pirenzepine from its binding sites in rat hippocampal membranes with an IC50 value of 0.27 microM. It only weakly displaced [3H]N-methylscopolamine from cerebellar, cardiac and ileal membranes (10-48 microM), and, up to 100 microM, did not interact with the main other receptors of the rat brain. In rat isolated sympathetic ganglia, SR 95639A induced dose-dependent depolarizations which were antagonized by pirenzepine, and dose dependently suppressed the M current. These latter effects were also pirenzepine-sensitive. After i.p. or oral treatment in mice, SR 95639A never induced the classical cholinergic syndrome, up to lethal doses. Finally, SR 95639A (i.p. and p.o.) antagonized contralateral rotations induced by intrastriatal injection of pirenzepine, in mice. These results suggest that SR 95639A is a selective agonist at central muscarinic M1 receptors and may represent a useful tool for further characterization of the nature and function of muscarinic receptor subtypes.

Interactions between three pyridazinyl-GABA derivatives and central GABA and glycine receptors in the rat, an in vivo microiontophoretic study.

Three pyridazinyl-gamma-aminobutyric acid (GABA) derivatives, SR 95103, SR 42641 and SR 95531, have previously been shown to be specific, competitive and reversible GABAA antagonists. For all three compounds selectivity was claimed mainly on the basis of biochemical results. However the absence of an interaction with the binding site for strychnine does not preclude an interaction with the glycine receptor. Therefore the interaction between these compounds and GABA- and glycine-elicited responses in the rat cuneate nucleus was examined in vivo by microiontophoretic techniques using extracellular recordings. Preliminary experiments in the somesthetic cortex of the rat (18 cells) confirmed that SR 95103 (100 mM; 0-100 nA) blocked GABA-evoked responses. In the cuneate nucleus SR 95103 (100 mM), SR 42641 (5 mM), SR 95531 (5 mM) and bicuculline methochloride (BMC; 5 mM) blocked GABA-elicited responses in a dose-dependent, competitive and reversible fashion for ejection currents up to 100 nA. The compound SR 95103 appeared to be less potent than bicuculline and also antagonized glycine-induced responses. Both SR 42641 and SR 95531 appeared to be equipotent to bicuculline and selective for the GABA receptor. Based on these results, it is postulated that SR 42641 and SR 95531 are potent and reversible GABAA antagonists and could be useful tools to further characterize the GABA receptor.

EEG effects of i.v. infusion of pentylenetetrazol in rats: a model for screening and classifying antiepileptic compounds.

In order to validate a new animal model predictive of the profile of antiepileptic drugs, we studied the antagonism by standard antiepileptics of the EEG modifications induced by low-speed IV infusion of pentylenetetrazol (PTZ) in rats. The activity of the drugs was measured by their effects on temporal characteristics of the PTZ-induced EEG paroxysms. Most compounds had moderate to potent anti-PTZ effects, as shown by the changes in the EEG temporal parameters. However, these effects depended on the drugs and doses. Cluster analysis showed that drugs and doses which evoked similar changes were closely related and were included in separate clusters with respect to one another. In particular, the present results showed that benzodiazepines and antiepileptics cluster differently in their effects. Thus, this model could be a useful tool for assessing new antiepileptic drugs.

Mild insomnia induced by environmental perturbations in the rat: a study of this new model and of its possible applications in pharmacological research.

This paper describes an experimental model for testing new products in a situation where sleep is impaired by a nonpharmacological procedure. This model consists in studying the effect of hypnogenic substances on sleep perturbations which take place when chronically implanted rats are moved to a novel individual cage, connected for the first time to their recording cable and dosed intragastrically with test products, vehicle or placebo. Continuous polygraphic recordings were taken throughout the daily treatment period (15 days) and scored for the presence of slow wave sleep (SWS), and rapid eye movement sleep (REM). The hourly vigilance stages were also computed to determine the parameters of their nycthemeral rhythms. In vehicle-treated rats, a decrease in SWS and REM values and a delay of SWS and REM phases of nycthemeral rhythms occurred on the first day as compared with the end of the recording period. Phenobarbital (5 mg/kg p.o.), mecloqualone (5 mg/kg p.o.) and a new benzodiazepine CM 7302 (7 mg/kg p.o.) led to an immediate or fast recovery, whereas no improvement was observed with placebo and with clorazepate dipotassium (9 mg/kg p.o.). This model can be considered as an attractive model for studying mild insomnia because it allows "true" hypnogenic drugs to be selected in a minimum amount of time.