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The effects of compounds that open the GABAA receptor-chloride channel complex on the rapidly developed tolerance to the 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide(8-OH-DPAT)-induced hypothermia in rats were examined. The test compound was injected 15 min. before 1 mg/kg subcutaneous 8-OH-DPAT or saline and 24 hr later a challenge dose of 0.1 mg/kg subcutaneous 8-OH-DPAT was given. The rectal temperature was measured before the challenge dose and 30, 60, 90 and 120 min. thereafter. The hypothermic effect was calculated as the area under the curve. It was found that all the GABAergic compounds examined significantly counteracted the 8-OH-DPAT-induced tolerance to the hypothermic response: muscimol at 3 mg/kg subcutaneous, diazepam at 1 - 3 mg/kg subcutaneous, pentobarbitone sodium at 20 mg/kg subcutaneous, and chlormethiazole at 40 mg/kg subcutaneous. Combined treatment of the rats with the GABAA receptor antagonist, bicucculine, or the GABAA receptor-chloride channel blocker, picrotoxin and diazepam, pentoparbitone sodium or chlormethiazole significantly antagonised this counteraction of the 8-OH-DPAT-induced tolerance. Depletion of 5-HT by pretreatment of the rats with the tryptophan hydroxylase inhibitor p-chlorophenylalanine did not counteract the 8-OH-DPAT-induced tolerance to the hypothermic response. Pretreatment of the rats with dexamethazone did not change the development of the tolerance to 8-OH-DPAT-induced hypothermic effect which seems to exclude the involvement of the hypothalamo-pituitary-adrenocortical axis in the tolerance development. It is concluded that the results support the hypothesis that GABA neurones beyond the 5-HT neurones are involved in the mechanism causing tolerance to the 5-HT1A receptor-mediated hypothermia in rats.

The sensitivity of Sprague-Dawley rats from 4 different breeding colonies (ALAB, M&B, B&K, Charles River) and one breeding colony of Wistar rat (M&B) to the 5-hydroxytryptamine1A (5-HT1A) receptor stimulatory effect of 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) resulting in corticosterone secretion and hypothermia was compared. The dose-response curves of the increase in plasma corticosterone showed that ALAB and M&B rats were 3.5 times more sensitive to 8-OH-DPAT than B&K and Charles River rats, the Wistar rats being in between. The attenuation of the corticosterone response 24 hr after a single injection of 1 mg/kg 8-OH-DPAT was greater for the ALAB and M&B rats than for B&K, Charles River and Wistar rats. The comparison of the 8-OH-DPAT-induced hypothermia in the various rat colonies showed a similar pattern: the sensitivity of ALAB rats was about twice that of M&B, B&K and Wistar rats, Charles River rats being 9 times less sensitive. The attenuation of the response 24 hr after 1 mg/kg 8-OH-DPAT measured as the shift in dose-response showed similar shift factors (4.1 to 6.7) for all rat colonies except for the B&K rats (3.0). The hypothermic response at 0.1 mg/kg 8-OH-DPAT was significantly lower for the Charles River and B&K rats than for the ALAB rats. Similarly was the maximal attenuation of the hypothermic effect in these rats less than half of that of the ALAB rats. The possible cause of the observed differences in the response to 8-OH-DPAT between these rat colonies is discussed in terms of receptor reserves and the involvement of other transmitter systems in the responses.

The pharmacological properties of the 5-hydroxytryptamine (HT)(1A) receptor agonist (R)-3,4-dihydro-N-isopropyl-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide (NAE-086) were examined with in vitro and in vivo techniques. Receptor binding studies demonstrated that NAE-086 was a high-affinity and selective 5-HT(1A) receptor ligand with a K(i) value of 4.5 nM in membranes from rat hippocampus. Of 32 other receptors examined NAE-086 had a modest affinity only for the 5-HT(7) receptor (K(i) = 240 nM). NAE-086 inhibited VIP-stimulated adenylyl cyclase activity in GH(4)ZD10 cells with 79% of the efficacy of 5-HT. This inhibition was blocked by the 5-HT(1A) receptor (and beta-adrenoceptor) antagonist (-)alprenolol. A minor metabolite of NAE-086 in rats, (R)-3,4-dihydro-3-(N-isopropyl-N-propylamino)-2H-1-benzopyran-5-carboxamide had a similar receptor profile but had 17 times higher affinity for the 5-HT(1A) receptor (K(i) = 0.26 nM). In vivo, NAE-086 induced all the typical effects of a 5-HT(1A) receptor agonist in rats: it decreased 5-HT synthesis (5-HTP accumulation) and 5-HT turnover (measured as the ratio of 5-hydroxyindoleacetic acid/5-HT), increased corticosterone secretion, induced the 5-HT(1A) syndrome (flat body posture and forepaw treading), inhibited the cage-leaving response, and caused hypothermia. All the responses mediated by postsynaptic 5-HT(1A) receptors were attenuated after single or repeated treatment of the rats with NAE-086. Simultaneously with the development of the tolerance to 5-HT(1A) receptor-mediated responses, 5-HT(2A) receptor-mediated responses were enhanced, as judged from the increased number of spontaneous and/or agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-induced wet-dog shake responses. The significance of this behavioral effect in relation to clinical observations is discussed.

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.

1. The effect of various classes of compounds on the rapidly developed tolerance to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-induced corticosterone secretion was examined. 2. Compounds activating the gamma-aminobutyric acidA (GABAA) receptor-chloride complex, i.e. muscimol (3 mg kg-1), diazepam (5 mg kg-1), flunitrazepam (1 mg kg-1), sodium pentobarbitone (10-30 mg kg-1) and chlormethiazole ethane disulphonate (50 mg kg-1) counteracted the development of tolerance when injected before or simultaneously with, but not 15 min after 8-OH-DPAT. 3. At these doses the compounds produced an acute increase in serum corticosterone but had, with the exception of muscimol, no effect on the response to the challenge dose of 8-OH-DPAT 24 h later. Muscimol significantly decreased the response. 4. The GABAA chloride channel antagonist, picrotoxin (1 mg kg-1, s.c.), but not bicuculline (1 mg kg-1, i.p.) potentiated the development of tolerance to 8-OH-DPAT-induced corticosterone secretion. 5. A number of compounds with widely differing pharmacological actions were examined and found to have no effect on the development of tolerance to 8-OH-DPAT-induced corticosterone secretion.

The effects of 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) and some other 5-hydroxytryptamine1A (5-HT1A) receptor agonists (buspirone, ipsapirone and flesinoxan) on corticosterone secretion in rats were studied. The 5-HT1A receptors mediating the corticosterone secretion appear to be postsynaptic to the 5-HT neurons, since the response to 8-OH-DPAT was not decreased but potentiated by depletion of 5-HT with p-chlorophenylalanine pretreatment of the animals. Rapid attenuation of the response was developed after a single dose of a 5-HT1A receptor agonist. Thus, 1 mg/kg s.c. of 8-OH-DPAT attenuated the response of a challenge dose (0.1 mg/kg s.c.) of this compound within 4 h lasting between 7 and 14 d. The development of the subsensitivity was antagonized by pretreatment of the rats with the 5-HT1A receptor antagonist S-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((-)-UH 301). This compound also antagonized the acute effect of 8-OH-DPAT in increasing serum corticosterone. The subsensitivity development was specific for the 5-HT1A receptor-mediated corticosterone secretion, since the increase in serum corticosterone produced by stimulation of other receptor systems, e.g. alpha 2-adrenoreceptors (clonidine) or 5-HT2 receptors [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, (DOI)] was not affected.

The effect of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists dizocilpine and phenycyclidine, the competitive NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and the antagonist at the glycine modulatory site of the NMDA receptor, 3-amino-1-hydroxy-2-pyrrolidone (HA-966) on the long lasting attenuation of some post-synaptic 5-HT1A receptor-mediated responses in rats (increased corticosterone secretion and inhibition of the cage leaving response) produced by a single injection of the 5-hydroxytryptamine1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was studied. It was found that these antagonists counteracted the attenuation of these responses at dose levels known to block the NMDA receptor-ion channel complex in vivo. It is concluded that the long lasting attenuation of postsynaptic responses after a 5-HT1A receptor agonist is initiated through stimulation of glutamate NMDA receptors indicating a functional interaction between the 5-HT and glutamate systems in at least two different models.

Mice were premedicated with reserpine and alpha-methyl-p-tyrosine to deplete stores of dopamine (DA) (and other neurotransmitters) and to stop DA (and noradrenaline (NA] synthesis. In DA-depleted mice, the mixed alpha 1/alpha 2 agonist clonidine potentiated locomotor stimulation induced by a low dose of apomorphine as measured in automated activity cages. Clonidine and the slightly alpha 1-selective agonist ST587, but not ST91, an alpha-agonist which does not readily cross the blood brain barrier, produced marked stimulation when combined with the selective D2 agonist quinpirole. The D1 -selective agonist SKF38393 also produced marked excitation when combined with quinpirole. All the selective agonists, bar quinpirole which in some cases produced a significant locomotor stimulation, were relatively inactive when given alone. A "blind" observational analysis of the animals challenged with clonidine plus quinpirole indicated an increase in sniffing, rearing and shaking behaviour. In contrast, observation of the animals challenged with SKF38393 plus quinpirole indicated increased sniffing, rearing and biting and, in one case, increased grooming behaviour. Clonidine did not produce excitation (in automated cages) when combined with the selective D1 agonist SKF38393. The excitation produced by clonidine plus quinpirole was blocked by the selective D2 antagonist raclopride but not by the selective D1 antagonist SCH23390. The stimulation was also blocked by the alpha 1 antagonist prazosin but not by the alpha 2 antagonists idazoxan or yohimbine. Biochemical analysis in the striata of mice challenged with clonidine plus quinpirole did not provide any obvious biochemical basis for the behavioural interaction. It is concluded that alpha 1 receptor agonists in combination with D2 DA agonists can produce marked stimulation in DA depleted mice.

The uptake and efflux of 3H-bretylium in slices of the rat vas deferens were examined. The uptake of bretylium was temperature, Na+ and Cl- dependent, sensitive to 6-hydroxydopamine but insensitive to reserpine. The uptake was inhibited by desipramine, (+)-amphetamine, imipramine methiodide and cocaine, whereas the local anaesthetics millicaine and lidocaine were poor inhibitors. The spontaneous efflux was very slow at 0 degrees C and slow at 37 degrees. Desipramine accelerated the efflux probably by inhibiting the re-uptake of bretylium. (+)-Amphetamine, low external Na+, high external K+ and ouabain caused a pronounced increase in the efflux at 37 degrees C but was almost without effect at 0 degrees C. The efflux evoked by (+)-amphetamine, low Na+ and ouabain was inhibited by desipramine, imipramine methiodide and cocaine, whereas millicaine and lidocaine had very poor effects. The increase of bretylium efflux by hypertonic K+ was only slightly inhibited by desipramine and partially antagonized by omission of Ca2+ in the medium. The results obtained indicate that bretylium was taken up in noradrenergic neurones by the noradrenaline transport mechanism and that the efflux of bretylium produced by (+)-amphetamine, low external Na+, low external Cl- and by ouabain occurred by the same transport mechanism as the uptake but now working in the outward direction whereas the efflux induced by high K+ seemed to occur mainly with another mechanism.

The effect of repeated treatment of rats for 21 days with the monoamine reuptake inhibitors imipramine, zimeldine, alaproclate (in each case 10 mumol/kg b.i.d.) and the reversible monoamine oxidase-A inhibitor amiflamine (3 mumol/kg b.i.d.) on brain noradrenergic mechanisms measured at different times of the day and night was investigated. Imipramine treatment produced a down-regulation of the Bmax for 3H-dihydroalprenolol binding to cortical beta-adrenoceptors that was not dependent upon the time of day the animals were killed. Zimeldine, on the other hand, reduced both Bmax and Kd of binding for day-time, but not night-time samples. Alaproclate and amiflamine were without effect on the binding. Twenty-four hour mean values for 1 nM 3H-p-aminoclonidine binding to alpha 2-adrenoceptors were lower for the zimeldine-treated rats than for the saline-treated rats. Pineal melatonin concentrations, which are regulated by beta-adrenoceptors, showed a pronounced diurnal rhythm, with the highest concentrations being found at 02:00. At this time point, a lower pineal melatonin content was found after amiflamine treatment, whereas imipramine, zimeldine and alaproclate were without significant effect. The importance of the use of more than one time point and the use of more than one biochemical test for the determination of the effects of repeated antidepressant treatment on central noradrenergic systems measured ex vivo is discussed.

1. The inhibition of monoamine oxidase (MAO) by 4, alpha-dimethyl-m-tyramine (H77/77) and 4-methyl-alpha-ethyl-m-tyramine (H75/12), two amine releasing compounds, within monoaminergic neurons in the rat hypothalamus and striatum in vivo was determined. This was performed by measuring the protection of MAO by the test compound against the irreversible inhibition produced by phenelzine. The MAO activity inside and outside monoaminergic synaptosomes in homogenates of brain tissue was measured in the absence and presence of selective uptake inhibitors at low concentrations of 14C-labelled 5-hydroxytryptamine, noradrenaline or dopamine. 2. It was found that H77/77 and H75/12 produced a pronounced protection against phenelzine within the serotonergic and noradrenergic neurons, whereas much less effect was observed outside these neurons. 3. It was shown that the protection by H75/12 within the serotonergic neurons was somewhat reduced in 5-HT depleted reserpinized rats and that the protection outside these neurons was abolished. Some of the protection of MAO might therefore have been brought about by 5-HT molecules released by H75/12. 4. The marked inhibition of MAO within serotonergic and noradrenergic neurons was counteracted by amine uptake inhibitors and is accordingly brought about by the high concentrations of the accumulated compounds. 5. In contrast to other neuron selective MAO inhibitors, H75/12 decreased the 5-HT concentration in the hypothalamus showing that the releasing effect dominated over the MAO inhibitory effect.

Twenty-four hours after [3H]dopamine ([3H]DA) injections into different parts of the ventral tegmentum the radioactivity recovered in the forebrain showed a highly regional distribution which corresponded to well-known DA projections. Selective degeneration of DA-neurons with 6-hydroxydopamine (6-OHDA) markedly reduced the axoplasmic transport after [3H]DA injections. The ventral tegmental area (VTA) was found to project primarily to the nucleus accumbens, olfactory tubercle and amygdala. The DA-innervation of the entorhinal cortex and the amygdala derive from cells widely distributed in the VTA and SN.

The inhibitory effect on monoamine oxidase (MAO) of the reversible MAO-A inhibitor (+)-4-dimethylamino-2,alpha-dimethylphenethylamine [amiflamine, FLA 336(+)] was evaluated in the rat after acute and repeated (twice daily for two weeks) oral treatment. MAO activity was measured ex vivo in slices from the hypothalamus and the duodenum for both MAO-A and MAO-B. Amiflamine selectively inhibited the A form of MAO after repeated as well as after acute treatment (ED50 approximately 7 mumoles/kg both acute and repeated). In the brain slices this inhibition corresponded to a decrease in the concentration of 5-hydroxyindoleacetic acid (5-HIAA) and to an increase in the concentration of 5-HT in the hypothalamus, the hippocampus and the striatum. The concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) was decreased in the striatum to the same extent as the decrease in the 5-HIAA concentrations. The effect on the homovanillic acid (HVA) concentration was somewhat weaker as was the increase in the concentration of dopamine. No essential difference was found after acute and chronic treatment on the amine and metabolite levels. The MAO activity returned to normal 24 hours after final dosing. A large difference between the neuronal and the extraneuronal protection against the phenelzine-induced irreversible MAO inhibition in the hypothalamus was found after both acute and repeated treatment. The ED50 of the protection within the serotonergic neurons was 1.3 mumoles/kg p.o. (acute) and 0.75 mumoles/kg p.o. (repeated). Amiflamine was 3 times less potent within noradrenergic neurons than within serotonergic neurons. A brain to plasma ratio of about 20:1 was found for amiflamine and its metabolites. The plasma and the brain concentrations of the N-demethylated metabolite [FLA 788(+)] exceeded that of amiflamine after a single dose, whereas the N,N-demethylated [FLA 668(+)] was found in low concentrations. The effect on MAO-A correlated significantly with the plasma and the brain concentration of FLA 788(+).

The inhibitory potencies of 27 compounds on the accumulation of 3H-dopamine (DA) in synaptosome-rich striatal homogenates of normal and reserpinized rats were determined. It was found that some compounds, e.g. amphetamine derivatives, phenmetrazine, phenethylamine derivatives and tryptamine derivatives were considerably more potent in the reserpinized preparation than in the normal one. Other compounds, e.g. amfonelic acid, mazindol, EXP 561, benztropine, pipradrol, nomifensine, methylphenidate and cocaine had similar potencies in the two preparations. It is suggested that the compounds enhanced by reserpine are more potent as DA releasing agents than as inhibitors of the DA uptake, whereas the compounds in the other group are most potent as uptake inhibitors. Interestingly, these two groups completely agree with the two groups of central stimulatory agents, viz. the amphetamine-like and the methylphenidate-like drugs.

The efflux of 3H-bretylium in rat vas deferens in vitro evoked by a number of sympathomimetic amines was examined. The tissue was preloaded with 3H-bretylium (2 x 10(-7) M) and the efflux of bretylium to the incubation medium in the presence of the amines during 20 min, was determined. The efflux evoked by (+)-amphetamine was almost abolished at 0 degrees and by desipramine. The dose response curves showed that some of the amines at high concentrations markedly antagonized their own effect. It could be demonstrated that (+)-amphetamine and N-methylamphetamine at a high concentration also antagonized the efflux evoked by low external Na+ concentrations and it is suggested that this effect is produced by inhibition of the outward transport of bretylium through the neurone membrane. The structure activity relationship obtained shows that the non-hydroxylated amines are most potent in evoking bretylium efflux and that the potency diminished with the number of hydroxyl groups. The tertiary amine analogue of amphetamine is less active and the quaternary derivative much less active than amphetamine and N-methylamphetamine. A hydroxyl group at 3-position gives a somewhat higher potency than that at 4-position. This structure activity relationship is similar to that previously reported for the sympathomimetic amines in reversing the adrenergic neurone blockade for bretylium and it is proposed that this reversal is induced by the reduction of the intraneuronal concentration of bretylium as a result of the evoked efflux.

The effect of the depolarizing agent veratridine on the accumulation and release of of 3H-noradrenaline and 3H-bretylium in in the rat vas deferens in vitro was examined. Veratridine produced inhibition of the accumulation and induced marked release of the amines. In vas deferens from non-reserpinized rats the release of noradrenaline evoked by veratridine was partially antagonized by omission of Ca2+ in the incubation medium and partially inhibited by low concentrations of desipramine. In reserpinized vas deferens the release of noradrenaline like that of bretylium in normal vas was not affected by omission of Ca2+ but inhibited by low concentrations (3-5 X 10(-7) M) of desipramine. Tetrodotoxin and the local anesthetics millicaine and lidocaine antagonized the effect of veratridine on the accumulation and release of the amines, probably due to prevention of the depolarization. High concentration (3 X 10(-5) M) of desipramine had a similar effect on the release of noradrenaline in normal tissue in presence of external Ca2+. It is concluded that noradrenaline is released by veratridine from normal vas deferens by two mechanisms: 1) an exocytosis release, 2) a carrier mediated desipramine sensitive outward transport; In reserpinized tissue the second mechanism is solely responsible for the release of noradrenaline. Bretylium is only released by the second mechanism. It is suggested that the inhibiton of the amine accumulation by veratridine is due to an equilibrium of the influx at a low tissue to medium ratio.

The efflux of 3H-5-HT from a crude synaptosome preparation of the cerebral cortex of reserpinized rats was examined. The synaptosomes were loaded with 3H-5-HT by pre-incubation of the homogenate in presence of pargyline and desipramine in order to inhibit deamination of 5-HT and uptake into noradrenergic neurons. The synaptosomes were collected by centrifugation, washed and resuspended in 0.25 M sucrose. No spontaneous efflux of 5-HT was detectable at 0 degrees C but marked efflux was observed at 27 degrees C and 37 degrees C. 4-Chloroamphetamine, low external Na+ concentration, ouabain and the depolarizing agent veratridine markedly accelerated the initial (5 min) efflux. Inhibitors of the neuronal 5-HT uptake, e.g. chlorimipramine, H 102/09 and A 23189, antagonized the 5-HT efflux evoked by these means, whereas desipramine, which is a poor inhibitor of the 5-HT uptake, had only slight effect on the 5-HT efflux. It is suggested that 5-HT can be actively transported out from the synaptosomes by the reversed 5-HT uptake mechanism.

The release of 3H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 X 10(-6) M) or low external Na+ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca2+ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca2+ but almost completely in absence of Ca2+. The release by high K+ (117 mM)+low Na+ (26 mM) in normal tissue was dependent on the presence of Ca2+ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca2+ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.