Zimelidine decreases seizure susceptibility in stressed mice.

To further evaluate whether selective serotonin reuptake inhibitors (SSRIs) have pro- or anticonvulsant properties and whether these properties will be modified by stress, we studied the effect of zimelidine on the convulsions produced by picrotoxin, a GABA(A) receptor antagonist, in unstressed and swim stressed mice. Zimelidine potentiated the ability of swim stress to enhance the threshold doses of intravenously administered picrotoxin producing convulsant signs and death, without having an effect in unstressed mice. The anticonvulsant effect of zimelidine was counteracted with mianserin, the antagonist of 5-HT(2A/2C), and diminished with WAY-100635, a selective antagonist of 5-HT(1A) receptors. In stressed mice, WAY-100635 prevented the anticonvulsant effect of 8-OH-DPAT, a 5-HT(1A) receptor agonist. SB-269970 and ketanserin, the antagonists of 5-HT(7) and 5-HT(2A) receptors, respectively, failed to reduce the effect of zimelidine. The results suggest the involvement of 5-HT(2C) and 5-HT(1A) receptors in the anticonvulsant effects of zimelidine and possibly other SSRIs in stress.

The involvement of alpha2-adrenoceptors in the anticonvulsive effect of swim stress in mice.

RATIONALE: Various studies have shown that stressful manipulations in rats and mice lower the convulsant potency of GABA-related, but also some GABA-unrelated convulsants. The mechanism of this anticonvulsive effect of stress is still unknown. OBJECTIVES: We tested the possible involvement of alpha2-adrenoceptors in the previously observed anticonvulsive effect of swim stress. METHODS: The mice were, prior to exposure to swim stress and the IV infusion of picrotoxin, pre-treated with clonidine (an alpha2-adrenoceptor agonist), yohimbine (a non-selective alpha2-adrenoceptor antagonist), idazoxan (a selective alpha2-adrenoceptor antagonist), or niguldipine (an alpha1-adrenoceptor antagonist), and the latency to the onset of two convulsant signs was registered. RESULTS: In control unstressed animals clonidine (0.1 and 1 mg/kg IP), yohimbine (2 mg/kg IP) and idazoxan (1 mg/kg IP) failed to affect the doses of picrotoxin needed to produce convulsant signs, while niguldipine (5 mg/kg IP) prolonged the latency, i.e. it enhanced the doses of picrotoxin producing running/bouncing clonus and tonic hindlimb extension. In swim stressed mice clonidine enhanced, while idazoxan decreased doses of picrotoxin needed to produce two convulsive signs. Yohimbine decreased the dose of convulsant needed to produce tonic hindlimb extension, while niguldipine enhanced doses of picrotoxin needed to produce both symptoms. CONCLUSIONS: The results demonstrate the alpha2-adrenoceptor agonist-induced potentiation and alpha2-adrenoceptor antagonist-induced diminution of the anticonvulsive effect of stress. Additionally, they show the anticonvulsive effect of niguldipine in unstressed and stressed animals. Hence, the results suggest that alpha2-adrenoceptors are involved in the anticonvulsive effect of swim stress in mice.

[3H]Flunitrazepam binding to recombinant alpha1beta2gamma2S GABAA receptors stably expressed in HEK 293 cells.

The interaction of selected compounds with the binding of the benzodiazepine [3H]flunitrazepam to membranes isolated from human embryonic kidney (HEK) 293 cells, stably transfected with the aI( 2 2S subtype of GABAA receptors, was studied. This subtype of GABAA receptors is the most common type of GABAA receptor found in the brain, and benzodiazepines are drugs known to enhance the effects of the inhibitory neurotransmitter gamma-amino butyric acid (GABA) by binding to the benzodiazepine binding sites which are part of the GABAA receptor complex. Scatchard analysis of binding data revealed the existence of a single type of binding site for [3H]flunitrazepam. GABA and thiopental enhanced, while the antagonist of central benzodiazepine binding sites--flumazenil, benzodiazepines such as clonazepam, flunitrazepam and diazepam, and the triazolopyridazine CI 218,872--displaced with nanomolar potency the binding of [3H]flunitrazepam. A partial displacement was obtained with the antagonist of the peripheral benzodiazepine binding sites--PK 11195--and with the neurosteroid dehydroepiandrosterone sulfate. The potency of drugs to enhance or inhibit [3H]flunitrazepam binding mainly corresponded to that observed for the modulation of the binding of [3H]flunitrazepam to the native type 1 benzodiazepine binding sites. This, as well as a high density of expressed binding sites, makes the cell line under study a very reliable and economical model for the testing of effects of different compounds at the GABAA receptor.

Swim stress alters the behavioural response of mice to GABA-related and some GABA-unrelated convulsants.

To elucidate the relationship between stress and seizures, the effect of a single swim stress on the convulsive signs and death produced by several GABA-related and GABA-unrelated convulsants, and the effect of repeated swim stress on picrotoxin-induced convulsions was studied. Mice were subjected to swim stress (10 min swimming at 18-19 degrees C), and the i.v. infusion of convulsants started 15 min thereafter. The latency to the onset of several convulsant signs and death was measured, and the doses of convulsants producing convulsions and death were calculated. Additional experiments included mice swimming at room temperature, and those which were stressed repeatedly (twice a day for four consecutive days, plus one stressful procedure on the fifth day). Swim stress increased the dose needed to produce convulsant signs and death after bicuculline, picrotoxin, pentylenetetrazole, strychnine and 4-aminopyridine, while kainic acid-induced convulsions were not affected. Using picrotoxin infusion, the effect of swimming in room temperature water was less than the effect of swimming in 18-19 degrees C water. In addition, the effect of repeated stress was less than the effect of acute stress on picrotoxin-induced convulsions. The results demonstrate that acute swim stress lowers the convulsive potency of GABA-related and some GABA-unrelated convulsants. Repeatedly stressed animals develop tolerance to anticonvulsive effect of swim stress.

Anticonvulsive effect of swim stress in mice.

To explore the possible involvement of glucocorticoids in the previously observed anticonvulsive effect of swim stress, mice were, prior to administration of convulsants, subjected to treatments that diminish or enhance plasma corticosterone levels. Aminoglutethimide, the inhibitor of steroid synthesis, failed to modify convulsant doses of picrotoxin, but enhanced threshold doses of pentylenetetrazole producing myoclonus and death, both in unstressed and stressed animals. The same drug prevented the effect of stress on pentylenetetrazole-induced running bouncing clonus (RB clonus) and abolished the appearance of tonic hindlimb extension (THE). Doses of kainic acid producing convulsions and death were not affected by stress, but they were enhanced by aminoglutethimide. Corticosterone administration could not imitate the effect of swim stress. Finasteride, a 5 alpha-reductase inhibitor, did not interfere with the effect of stress on picrotoxin-induced convulsions. Swim stress failed to modify the binding of the convulsant t[3H]-butylbicycloorthobenzoate [3H]TBOB, to washed mouse forebrain membranes. The results confirmed an anticonvulsant effect of swim stress against convulsions produced by GABA-related convulsants, but they do not support the hypothesis suggesting the involvement of glucocorticoids or neurosteroids in this effect.

Beta-1 adrenoceptor antagonists potentiate the anticonvulsive effect of swim stress in mice.

To explore the possible involvement of beta adrenoceptor antagonists in the previously observed anticonvulsive effect of swim stress, the mice were, prior to administration of convulsants, pre-treated with propranolol (a non-selective beta adrenoceptor antagonist), betaxolol (a selective beta-1 adrenoreceptor antagonist), or ICI 118,551 (a selective beta-2 adrenoreceptor antagonist). In control unstressed animals, only propranolol [10 mg/kg, intraperitoneally (ip)] produced a significant change. It enhanced the threshold dose of picrotoxin producing tonic hindlimb extension. However, in swim-stressed animals, propranolol enhanced doses of picrotoxin producing tonic hindlimb extension and death, while betaxolol (20 mg/kg, i.p.) enhanced doses of picrotoxin producing running/bouncing clonus, tonic hindlimb extension and death. Pre-treatment with ICI 118,551 (4 mg/kg, i.p.) failed to affect doses of picrotoxin producing convulsions and death. The results demonstrate that blockade of beta-1 adrenoceptors potentiates the anticonvulsant effect of swim stress against convulsions produced by picrotoxin, a noncompetitive GABA(A) receptor antagonist.

Effects of adrenalectomy and gonadectomy on sex and stress-induced differences in bicuculline-induced convulsions.

The effects of adrenalectomy, gonadectomy and combined adrenalectomy plus gonadectomy on the previously described sex-dependent anticonvulsive effect of swim stress were studied in rats. The convulsive signs (myoclonic twitch, generalized convulsions, tonic hindlimb extension) were produced by constant i.v. infusion of gamma-aminobutyric acid(A) (GABA(A)) antagonist bicuculline, which started 15 min after termination of swim stress (10-min swim at 18-19 degrees C). Adrenalectomy decreased the threshold doses of bicuculline producing the first myoclonic twitch and the onset of generalized convulsions only in females. In adrenalectomized females, but not in males, swim stress enhanced the threshold dose of bicuculline producing generalized convulsions, but, unlike in adrenal-intact animals, it failed to enhance the dose of bicuculline producing tonic hindlimb extension. In gonadectomized stressed and unstressed animals all sex differences disappeared, and swim stress enhanced in both sexes only the threshold doses of bicuculline producing tonic hindlimb extension. Adrenalectomized plus gonadectomized animals displayed clear sex differences in doses of bicuculline necessary to produce all the convulsive signs. In the same animals swim stress postponed, especially in females, the onset of the first myoclonic twitch and generalized convulsions, but not the onset of tonic hindlimb extension. In summary, our results suggest that hormones of the adrenal and gonadal glands are only partly responsible for decreased susceptibility, especially of female rats, to the GABA(A) antagonist bicuculline. Moreover, they have demonstrated that stress produces a gender-specific anticonvulsive effect even in the animals completely deprived of steroid hormones of peripheral origin.

[3H]t-butylbicycloorthobenzoate binding to recombinant alpha1beta2gamma2s GABA(A) receptor.

The interaction of several selected compounds with the binding of the cage convulsant t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to membranes isolated from human embryonic kidney (HEK) 293 cells stably transfected with alpha1beta2gamma2s subtype of GABA(A) receptors was studied. Scatchard analysis of binding data revealed the existence of a single type of binding site for [3H]TBOB with a Kd of 47.06+/-4.06 nM and a Bmax value of 6.72+/-0.52 pmol/mg protein. GABA, thiopental, TBOB, picrotoxin and the neurosteroid dehydroepiandrosterone sulfate displaced concentration-dependently the binding of [3H]TBOB to this recombinant receptor. Dehydroepiandrosterone sulfate reversed the 5 microM GABA-induced inhibition of specific [3H]TBOB binding. It is concluded that membranes isolated from HEK 293 cells stably transfected with alpha1beta2gamma2s subunits exhibit specific high-affinity [3H]TBOB binding. The potency of drugs to inhibit [3H]TBOB binding mainly corresponded to that observed for the inhibition of the binding of cage convulsants to the native receptors or to transiently transfected HEK 293 cells.

Sex differences in the response to GABA antagonists depend on the route of drug administration.

Sex differences in the responses to two GABA-related convulsants (bicuculline, picrotoxin) were studied in rats and mice following intraperitoneal (i.p.) and intravenous (i.v.) drug administration. Following i.p. administration male and female rats were equally sensitive to bicuculline, while female rats were more sensitive to picrotoxin. After i.v. infusion the threshold doses of bicuculline and picrotoxin producing running/bouncing clonus (RB clonus) were significantly lower in male than in female rats, i.e. male rats were more sensitive to both convulsants than females. Following i.p. administration, at some doses female mice were more sensitive to bicuculline and male mice to picrotoxin, although ED50 values between the sexes were not significantly different. After i.v. infusion, doses of bicuculline producing RB clonus and death were significantly lower in male than in female mice, i.e. male mice were more sensitive to bicuculline. The two sexes of mice were equally sensitive to i.v. administration of picrotoxin. While sex and species differences obtained following i.p. drug administration could presumably be explained by differences in pharmacokinetics, the i.v. route of drug administration is suggested as a reliable technique in the studies of sex and species differences in pharmacodynamics.

Sex differences in bicuculline-induced convulsions: interaction with stress and ligands of benzodiazepine binding sites.

The response to i.v. administration of bicuculline and its interaction with the benzodiazepine agonist diazepam and antagonist flumazenil were studied in male and female handling stressed and swim stressed rats. Both handling stressed and swim stressed male rats needed less bicuculline to produce myoclonic twitch and running/bouncing (RB) clonus than females. Besides, a lower dose of bicuculline produced tonic hindlimb extensor convulsion (THE) in male than in female swim stressed rats. Flumazenil failed to affect seizure thresholds for bicuculline either in handling stressed or in swim stressed animals. Sex differences remained present after diazepam pre-treatment as well. While diazepam enhanced doses of bicuculline producing all three convulsive signs similarly in both handling and swim stressed rats (141-162%), swim stress had the lowest anticonvulsive effect for the onset of myoclonic twitch (110% in males and 117% in females) and the highest for THE (148% in males and 188% in females). The anticonvulsive effect of diazepam was not sex-dependent, while the anticonvulsive effect of swim stress was greater in female than in male rats. The results suggest that greater sensitivity of male rats to bicuculline and the anticonvulsive effect of swim stress do not result from the release of endogenous modulators of benzodiazepine binding sites.

Regional differences in NaCl-induced increase of the potency of bicuculline to displace [3H]muscimol binding.

It has been shown that the potency of bicuculline to displace [3H]muscimol binding to crude brain membranes can be enhanced markedly by different anions. This study shows that although bicuculline alone was a more potent displacer of [3H]muscimol binding in cortical than in cerebellar membranes, the NaCl (250 mM)-induced leftward shift of the bicuculline inhibition curve of [3H]muscimol binding was considerably higher in cerebellum than in cortex. The some concentration of NaCl failed to affect either the affinity or the density of cortical and cerebellar [3H]muscimol binding sites. The results suggest that sodium chloride is able to reveal regional differences in bicuculline potency.

Influence of gender and gonadectomy on bicuculline-induced convulsions and on GABAA receptors.

The response to IV administration of GABAA receptor antagonist bicuculline was studied in young (30 days) and in adult gonad-intact or gonadectomized male and female rats. The properties of GABAA receptors, obtained from cortex and cerebellum 30 days following gonadectomy, and the affinity of muscimol and bicuculline for cortical and cerebellar GABA binding sites were also studied. While young rats failed to show sex differences, the threshold doses of bicuculline producing the first myoclonic twitch and running/bouncing clonus (RB clonus) were lower in adult male than female rats. Fifteen days after gonadectomy or sham operation male rats needed less bicuculline to the onset of myoclonic twitch and RB clonus than identically treated females, while orchidectomized rats needed more bicuculline to the onset of tonic hindlimb extension than all other groups examined. All sex differences disappeared 30 days following gonadectomy. At the same time, in males gonadectomy decreased the affinity and enhanced the density of cortical 3H-muscimol binding sites. In female rats, gonadectomy only decreased the affinity of cortical GABAA receptors. Only regional but not sex differences were observed in the affinity of muscimol and bicuculline for GABAA receptors. Sex differences in the threshold doses of bicuculline-producing convulsions do not correlate either with the properties of cortical and cerebellar GABAA receptors or with the affinity of bicuculline for the same binding sites.

Gonadal hormones and picrotoxin-induced convulsions in male and female rats.

The sensitivity to the GABA-blocking agent picrotoxin was studied in young and adult male and female rats, in rats treated with gonadal hormones and in gonadectomized male and female rats. Picrotoxin was equipotent in producing convulsions in male and female 20-day-old rats. Adult females tended to be more, while adult males were considerably less sensitive to picrotoxin than young rats. Picrotoxin was equipotent in displacing t-[3H]butylbicycloorthobenzoate ([3H]TBOB) binding to crude cortical and cerebellar membranes from male and female rat brain. Chronic treatment of male rats, beginning with 30 days of age, with estradiol benzoate enhanced their sensitivity to picrotoxin, while an analogous treatment of female rats with testosterone propionate was ineffective. Thirty days following castration adult male rats had shorter latencies to the appearance of picrotoxin-induced convulsions and a higher incidence of death. Ovariectomy in females failed to modify the sensitivity to picrotoxin. The results suggest that gonadal hormones have a crucial role in the development of sex related differences in the response of rats to picrotoxin and presumably to other GABA-related drugs. When developed, the male type of reactivity appears to depend more, and the female type less on the presence of circulating hormones in the blood.

Effects of diazepam on conflict behaviour and on plasma corticosterone levels in male and female rats.

The anxiolytic properties of diazepam and its effects on plasma corticosterone levels were compared in male and female, water deprived rats exposed to the punished (0.8 mA) drinking procedure. The effects of diazepam on unpunished licking, tested under familiar or unfamiliar conditions, and on the lick latency were also studied and a comparison between the two sexes was made. Both punished and unpunished drinking were less in females than in males. In both sexes, a clear anticonflict effect, i.e. a much greater effect on punished than on unpunished drinking, was obtained with 2 and 4 mg/kg, but not with 1 mg/kg, of diazepam i.p. Plasma corticosterone levels were higher in water deprived females than in males. Following the punished and unpunished drinking procedure, plasma corticosterone levels were found to have decreased more in female than in male rats, especially after administration of 1 mg/kg of diazepam. Diazepam had similar anticonflict effects in rats of both sexes but had a greater suppressive effect on the plasma corticosterone levels in female rats. There was no correlation between the anxiolytic effects of diazepam and its effect on the plasma corticosterone levels. When testing was done under unfamiliar conditions, the latency to licking was greater in female than in male rats and diazepam (1, 2 and 4 mg/kg) increased this latency in both sexes. The results suggest sex differences in the neuroendocrine, but not in the anxiolytic, effects of diazepam.

Sex differences in conflict behaviour and in plasma corticosterone levels.

The levels of plasma corticosterone and conflict behaviour were followed in male and female water deprived Wistar rats exposed to the punished (0.2 or 0.8 mA) drinking test. The unpunished drinking, performed under familiar or unfamiliar conditions, and plasma corticosterone levels of these male and female rats were determined. Plasma corticosterone was elevated in water-deprived rats compared to rats under normal conditions. In all cases plasma corticosterone levels were considerably higher in water deprived females than in males. The highest levels in both sexes were obtained following drinking punished with 0.8 mA shocks. While the unpunished drinking did not differ between the two sexes, the punished drinking was significantly lower in females than in males. Although female rats displayed less punished licks and had higher plasma corticosterone levels than males, there was no correlation between the two parameters.

Dihydroergosine: anticonflict effect in rats and enhancing effects on [3H]muscimol binding in the human brain post mortem.

The anticonflict activity of the ergot alkaloid, dihydroergosine, a drug which binds to 5-hydroxytryptamine1 (5-HT1) receptors and to gamma-aminobutyric acidA (GABAA) receptor-associated Cl- ionophore, was studied in water-deprived rats. In vitro effects of this drug on [3H]muscimol and [3H]flunitrazepam binding to the crude synaptosomal pellet of the human frontal cortex post-mortem were also investigated. Dihydroergosine, given 2 h prior to testing, enhanced drinking under punished (0.8 mA) conditions, and diminished it under unpunished conditions. The mechanism of this effect was (-)-propranolol- and pindolol-insensitive and picrotoxin-sensitive. Flumazenil either failed to affect, or at a higher dose (10 mg/kg), counteracted the dihydroergosine-induced enhancement of punished drinking. This dose of flumazenil was itself anxiogenic. Dihydroergosine had mild sedative and analgesic properties. Low concentrations of dihydroergosine (10 nM to 100 microM) enhanced the binding of [3H]muscimol but not of [3H]flunitrazepam. The results suggest that dihydroergosine may possess anxiolytic properties presumably mediated by its specific action at the GABA/benzodiazepine/chloride channel complex.

Inhibitory effect of diazepam on the activity of the hypothalamic-pituitary-adrenal axis in female rats.

The acute intraperitoneal administration of anxiolytic diazepam (2 mg/kg) inhibits the activity of the hypothalamic-pituitary-adrenal (HPA) axis, i.e., it decreases the concentration of adrenocorticotropic hormone (ACTH) and corticosterone in female rats. This fall of ACTH and corticosterone levels was reversed by an antagonist of central benzodiazepine receptors-flumazenil. The antagonist of peripheral benzodiazepine receptors-PK 11195, failed to affect diazepam-induced decrement of plasma ACTH and corticosterone levels. The suppressed HPA function obtained after diazepam administration was also antagonized by bicuculline, an antagonist of GABA recognition sites, and by picrotoxin, a drug that blocks the GABA-A receptor associated chloride channel. These results suggest that central benzodiazepine receptors, the part of GABA-A macromolecular complex, are involved in diazepam-induced inhibition of the activity of the HPA axis.

Possible antidepressant dihydroergosine preferentially binds to 5-HT1B receptor sites in the rat hippocampus.

The binding affinity of a possible antidepressant drug, dihydroergosine, for various 5-HT1 receptor subtypes was studied in the hippocampal rat brain membranes. Dihydroergosine displaced the binding of [3H]5-HT to the whole population of hippocampal 5-HT1 receptors with high affinity (Ki = 4.8 nM). The displacement curve was shallow and the slope factor less than unity, suggesting the interaction of dihydroergosine with multiple binding sites. When 8-OH-DPAT (100 nM) + chlorpromazine (500 nM), CGS 12066 B (200 nM) + ritanserin (500 nM), and (+/-)pindolol (1 microM) were included to block 5-HT1A + 5-HT1C, 5-HT1B + 5-HT1C, and 5-HT1A + 5-HT1B receptor subtype respectively, the competition studies have shown that under these selective conditions dihydroergosine binds with the highest affinity for 5-HT1B (Ki = 0.48 nM), with 8.7 times lower affinity for 5-HT1A (Ki = 4.2 nM) and with a moderate affinity for 5-HT1C (Ki = 156 nM) receptor subtype. While our previous studies suggested that dihydroergosine stimulates 5-HT1A and inhibits 5-HT2 receptors, this study suggests that the high affinity of this drug for 5-HT1B receptors should not be neglected.

Effect of ergot-alkaloid dihydroergosine on the immune reaction and plasma corticosterone in rats.

Ergot-alkaloid dihydroergosine, a potential antidepressive and anxiolytic drug, suppressed humoral and cellular immune reaction in rats, if given in a single injection (2-100 mg/kg) 1 hour after immunization. Plasma corticosterone levels were decreased upon assay of the immune response, 5 or 8 days following treatment.

Dihydroergotoxine modulation of the GABAA receptor-associated Cl- ionophore in mouse brain.

Dihydroergotoxine non-competitively displaced the binding of t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to crude synaptosomal membranes from the mouse brain (cerebrum minus cortex), and gamma-aminobutyric acid (GABA) (10 microM) enhanced the displacement potency of dihydroergotoxine in a bicuculline-sensitive manner. The same ergot compound prolonged pentobarbital-induced sleeping in mice and diminished the convulsive potency of picrotoxin in the same animal species. The results are indicative of the positive coupling between GABA and dihydroergotoxine.