Effects of the potential antidepressant dihydroergosine in rats forced to swim: influence on plasma corticosterone.

The potential antidepressant properties of the ergot alkaloid dihydroergosine (DHESN) were studied in rats forced to swim in a restricted space. DHESN (50 mg/kg, intraperitoneally) reduced the duration of immobility in rats forced to swim 1 hr after administration. This effect was still present after 48 hr, but 7 hr. In non-swimming rats, the same dose of DHESN increased the plasma corticosterone concentration when administered 1 hr prior to sacrifice, but was without effect 48 hr after administration. In rats forced to swim, DHESN elicited two opposite effects. One hr following a single administration, it increased plasma secretion of corticosterone, whereas administered 48 hr prior to forced swimming, it decreased plasma corticosterone. These results, along with our previous data, give evidence that DHESN might possess antidepressant properties.

Effect of dihydroergotoxine on the susceptibility of rats to convulsions produced by different convulsant agents.

The study was undertaken to test further whether diminished GABAergic transmission might be responsible for the increased susceptibility of rats to picrotoxin-induced convulsions. In rats kept individually in cages in a noise-free room, the time between the intraperitoneal injection of the convulsant agent and the onset of convulsions was measured. Acute and subacute treatment with low doses of dihydroergotoxine (0.01-1.0 mg/kg) increased the occurrence and decreased the latency of picrotoxin-induced convulsions. Acute administration of dihydroergotoxine, 1.0 mg/kg, caused convulsions in animals injected with the subconvulsive dose (3 mg/kg) of bicuculline and of 10.0 mg/kg dihydroergotoxine in animals injected with the subconvulsive dose (1.5 mg/kg) of strychnine. Some of the animals injected with the 100% convulsive dose of strychnine were protected by dihydroergotoxine pretreatment (1.0 mg/kg) as evidenced by the lower occurrence of convulsions and fewer animals dying, as well as by a delay in the appearance of convulsions at 10.0 mg/kg. These results together with the previous findings on the GABA system suggest that dihydroergotoxine potentiates the appearance of picrotoxin and bicuculline-induced convulsions by a diminution of GABAergic transmission.

Effect of diazepam on plasma corticosterone levels.

In light of the numerous but rather conflicting reports on the action of benzodiazepines upon the hypothalamo-hypophyseal-adrenal (HHA) axis activity, the effect of different doses of diazepam (0.1, 1.0 and 10.0 mg/kg) administered 15, 30, 60, 120 and 240 min before decapitation on plasma corticosterone level was studied in rats. While 0.1 mg/kg diazepam had no effect, 1.0 mg/kg diazepam decreased plasma corticosterone levels 30 and 60 min following drug administration. On the other hand, treatment with 10.0 mg/kg diazepam produced an increase in plasma corticosterone levels from 15-120 min following drug administration.

Sex differences in the sensitivity of CBA mice to convulsions induced by GABA antagonists are age-dependent.

The administration of the GABA-blocking agents picrotoxin and bicuculline to adult (2.5-3 months old) CBA/HZgr mice resulted in the appearance of convulsions, the occurrence and/or lethality of which was greater in males than in females. The latency of picrotoxin-induced convulsions was also shorter in male mice. Strychnine, a drug which induces convulsions by blocking glycine receptors was equally effective in producing convulsions in both male and female adult mice. Unlike adult mice, young (20 days old) or old (2 years old) mice fail to display sex differences following the picrotoxin administration. Accordingly, the observed sex differences in the sensitivity of CBA mice to administration of convulsive agents are specific for the GABA system and present only in sexually mature, but not in immature or old animals.

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.

Effect of dihydroergosine (DHESN) on the serotoninergic system and behaviour: is DHESN a new antidepressive agent?

Acute (50.0 mg/kg) and repeated (0.1-10.0 mg/kg) administration of dihydroergosine (DHESN) to rats over 5 days lowered the concentration of 5-HIAA in the brain. DHESN given acutely increased the brain 5-HT in p-CPA-treated animals and diminished the probenecid-induced increase in brain 5-HIAA. In pargyline-treated rats DHESN enhanced the 5-HT/5-HIAA ratio. DHESN administered to rats repeatedly over 5 days decreased the level of 5-HT in blood platelets, and in vitro at concentrations of 10(-4) M and 10(-3) M inhibited the uptake of [14C]-5-HT in platelets. DHESN (10.0-100.0 mg/kg) potentiated the 5-HT syndrome produced in rats by pargyline and 5-HTP. This potentiation was blocked with cyproheptadine but not with haloperidol. DHESN (1.0 and 10.0 mg/kg) lowered the locomotor activity of rats and 10.0 mg/kg DHESN also reduced the duration of immobility in rats forced to swim in a restricted space. The results indicate that DHESN, like antidepressants, decreases the turnover of serotonin in the brain and potentiates the 5-HT-mediated behaviour. This might suggest that the drug should be further investigated for its potential antidepressive properties.

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.

Effect of diazepam on brain neurotransmitters, plasma corticosterone, and the immune system of stressed rats.

Rats were treated with injections of diazepam (1 or 10 mg/kg) and stressed by restraint lasting 3 hours. This was performed once or, in animals immunized with sheep erythrocytes, repeatedly for 4 consecutive days. After repeated stress and/or diazepam treatment, the levels of brain noradrenalin decreased in all treated groups. Although both treatments (stress and diazepam) diminished the 5-hydroxytryptamine (5-HT)/5-hydroxyindoleacetic acid (5-HIAA) ratio, treatment with either dose of diazepam prevented the stress-induced fall of this ratio. The activity of hypothalamic glutamate decarboxylase, the enzyme taking part in GABA synthesis, was affected neither by the acute nor by repeated stress and/or diazepam treatment. The levels of plasma corticosterone were enhanced in all stressed rats, with and without drug. This finding was in accordance with the enhanced weights of adrenal glands in repeatedly stressed rats. The tendency to a corticosterone rise after repeated treatment with diazepam, 10 mg/kg, coincided with the enhanced weights of adrenal glands in these animals. The plaque-forming cell (PFC) response was reduced in all stressed animals and in animals treated with diazepam, 10 mg/kg. Accordingly, high doses of diazepam given repeatedly to rats are immunosuppressive, achieving this effect presumably by an enhancement of glucocorticoid secretion. Neither the low nor the high doses of diazepam affect the stress-induced enhancement of hypothalamohypophysial-adrenal axis activity and consecutive immunosuppression.

Suppression of immune response in rats by stress and drugs interfering with metabolism of serotonin.

Rats immunized with sheep erythrocytes were stressed by repeated restraint and/or treated with a precursor of serotonin (5-hydroxytryptophan, 5-HTP) or with an inhibitor of serotonin synthesis (parachlorophenylalanine, PCPA). As expected, repeated stress reduced the plaque-forming cell (PFC) response. Treatment with 5-HTP also reduced the PFC response, and potentiated the immunosuppressive effect of stress. This was accompanied by increased metabolism of serotonin in the brain, as indicated by increased concentration of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cerebral tissue. Treatment with PCPA also suppressed the PFC response, but this suppression was accompanied by decreased levels of brain serotonin and of 5-HIAA. Plasma corticosterone levels were elevated, reaching statistical significance, in rats treated with PCPA. Both drugs suppressed the in vitro PFC response of peripheral blood lymphocytes. Thus, although 5-HTP and PCPA altered serotonin metabolism in the brain in a diametrically opposite manner, their effects on the immune response were the same. Putative central effects of the drugs on serotoninergic regulation of the immune response were apparently obscured by their effects on corticosterone secretion as well as by their direct effects on immunocompetent cells.

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.

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.

Serotoninergic neurons: long-lasting activation by a possible antidepressant dihydroergosine.

The duration of the stimulating effect of one single i.p. injection of a possible antidepressant dihydroergosine (50 mg/kg) on the 5-HT syndrome was studied in rats. Dihydroergosine produced a very pronounced stimulation of the 5-HT syndrome which was still present after 1-144 h (6 days). The results suggest that dihydroergosine produces a long-lasting stimulation of serotoninergic neurons.

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.

Dihydrogenated ergot compounds bind with high affinity to GABAA receptor-associated Cl- ionophore.

The binding of t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to crude synaptosomal membranes of the mouse brain (cerebrum minus cortex) in the presence of dihydroergotoxine, dihydroergosine, dihydroergotamine and gamma-aminobutyric acid (GABA) was studied in vitro. [3H]TBOB binding was inhibited by all drugs used. The rank order of potency was dihydroergotoxine greater than GABA greater than dihydroergosine greater than dihydroergotamine. This suggests that dihydrogenated ergot compounds, especially dihydroergotoxine, possess appreciable binding activity (comparable to that of benzodiazepines and barbiturates) at the GABAA receptor-associated C1- ionophore.

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.

[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.

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.

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.

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.

Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine.

Dihydroergosine (50 and 100 mg/kg) enhanced the incidence of bicuculline (3 mg/kg)-induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline (2.5 mg/kg) to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline (4 mg/kg)-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of 3H-muscimol, the drug was able to diminish and to augment the IC50 of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited 3H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of 3H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of 3H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.