GABA/BZ-and NMDA-receptor interaction in digoxin-induced convulsions in rats.

Digoxin (7.5 micrograms icv) induced 'pop-corn' type of convulsions and 100% mortality. The GABA-ergic agents produced varying degree of protection against digoxin-induced neurotoxicity. Diazepam (4 mg/kg) offered significant protection whereas pentobarbital (5 mg/kg) and baclofen (5 mg/kg) markedly reduced per cent mortality, but ethanol (2 g/kg), progabide (50 mg/kg) and muscimol (0.5 mg/kg) as well as GABA (50 mg/kg) could not offer significant protection in doses used. GABA-ergic agonists; GABA, baclofen, diazepam and pentobarbital when administered along with MK-801 (0.5 mg/kg) a non-competitive NMDA antagonist, a potentiation of anticonvulsant action of MK-801 was observed. MK-801 showed potent anticonvulsant profile in dose range (0.25-1 mg/kg) studied. A synergistic influence of Mg2+ and K+ ions on NMDA receptor antagonism was also observed. A role of GABA-ergic facilitation and NMDA antagonism as a potential anticonvulsant approach in digoxin-induced convulsions in rats has been suggested.     

Risperidone resets the circadian clock in mice

Risperidone is an atypical antipsychotic that is active at multiple dopamine and serotonin receptor subtypes. Based on its high affinity for serotonin receptors, we predicted that it might reset circadian rhythms in a nocturnal rodent. We report temporally differentiated and differential effects of various doses of risperidone on the voluntary locomotor activity rhythm in the Indian field mice, Mus booduga. Risperidone (0.5 mg/kg) elicited phase delays at phases between CT (circadian time) 12 to CT18 and CT0 to CT3, and phase advances at CT6, CT9 and CT21. However, mice injected at CT6 showed maximum advances (1.299 ± 0.286 h), whereas at CT15 showed maximum delays (?1.514 ± 0.312 h). Increasing the dose beyond 0.5 mg/kg at maximally responsive CTs (CT6 and CT15) resulted in progressively smaller but significant shifts. Thus, 0.5 mg/kg is the optimal dose in this species. The fact that risperidone resets the circadian rhythm in a mammal can be extended to clinical studies and used for optimal adjustment of the circadian rhythm in mental disorders. Conversely, risperidone administration for various treatments must be carefully timed to prevent unwanted phase shifts in patients.     

Role of the GABA-ergic systems of the brain in manifesting the activating effect of diazepam]

It was shown in experiments on rats that the selective blocker of GABA receptors bicuculline (2 mg/kg) does not decrease the activating effect of diazepam as to the reaction of self-stimulation. The GABA-mimetic muscimol (0.5 and 1 mg/kg) had no effect on self-stimulation rate, while in the dose of 2 mg/kg causing behavioral changes produced a powerful decrease in it (by 93.3%). During the combined administration of diazepam and muscimol (1 mg/kg and 0.5 mg/kg, respectively) no potentiation of diazepam effect was observed. It is suggested that diazepam-induced facilitation of the reaction of self-stimulation is not due to the alteration in the activity of GABA-ergic processes.     

Central administration of muscimol phase-shifts the mammalian circadian clock

Summary The suprachiasmatic nucleus (SCN) of the hypothalamus contains a neural oscillatory system which regulates many circadian rhythms in mammals. Immunohistochemical evidence indicates that a relatively high density of GABAergic neurons exist in the suprachiasmatic region. Since intraperitoneal injections of the benzodiazepine, triazolam, have been shown to induce phase shifts in the free-running circadian rhythm of locomotor activity in the golden hamster, the extent to which microinjections of muscimol, a specific agonist for gamma-aminobutyric acid (GABA), may cause phase-shifts in hamster activity rhythms was investigated. Stereotaxically implanted guide cannulae aimed at the region of the SCN were used to deliver repeated microinjections in individual animals. A phase-response curve (PRC) generated from microinjections of muscimol revealed that the magnitude and direction of permanent phase-shifts in the activity rhythm were associated with the time of administration. The PRC generated for muscimol was characterized by maximal phase-advances induced 6 h before activity onset and by maximal phase-delays which occurred 6 h after activity onset. The PRC for muscimol had a shape similar to a PRC previously generated for the short-acting benzodiazepine, triazolam. Single microinjections of different doses of muscimol given 6 h before activity onset induced phase-advances in a dose-dependent fashion. Histological analysis revealed that phase shifts induced by the administration of muscimol were associated with the proximity of the injection site to the SCN area. These data indicate that a GABAergic system may exist within the suprachiasmatic region as part of a central biological clock responsible for the regulation of the circadian rhythm of locomotor activity in the golden hamster.Abbreviations CT circadian time - GABA gamma-aminobutyric acid - OC optic chiasm - PRC phase-response curve - SEM standard error of mean - SCN suprachiasmatic nuclei - T track - IIIV third ventricle     

GABA influences the development of the ventromedial nucleus of the hypothalamus.

The region that becomes the ventromedial nucleus of the hypothalamus (VMH) is surrounded by cells and fibers containing immunoreactive gamma-aminobutyric acid (GABA) by embryonic day 13 (E13), several days before the nucleus emerges in Nissl stains. As GABA plays many roles during neural development, we hypothesized that it influences VMH development, perhaps by providing boundary information for migrating neurons. To test this hypothesis we examined the VMH in embryonic mice in which the beta3 subunit of the GABA(A)-receptor, a receptor subunit that is normally highly expressed in this nucleus, was disrupted by gene targeting. In beta3 -/- embryos the VMH was significantly larger, and the distribution of cells containing immunoreactive estrogen receptor-alpha was expanded compared to controls. Using in vitro brain slices from wild-type C57BL/6J mice killed at E15 we found that treatment with the GABA(A) antagonist bicuculline increased the number of cells migrating per video field analyzed in the VMH. In addition, treatment with either bicuculline or the GABA(A) agonist muscimol altered the orientation of cell migration in particular regions of this nucleus. These data suggest that GABA is important for the organization of cells during VMH formation.     

Effect of GABA agonists on the neurotoxicity and anticonvulsant activity of benzodiazepines

Progabide (50 mg/kg, i.p.), a GABA receptor agonist, significantly decreases the median minimal neurotoxic dose (TD50) of clobazam, chlordiazepoxide, and diazepam; the receptor binding of these substances is highly enhanced by muscimol. Progabide has no significant effect on the TD50 of clonazepam and triazolam; the receptor bindings of these substances is either only slightly enhanced or not altered by muscimol. Progabide also significantly decreases the median antimaximal electroshock dose (MES ED50) of all the benzodiazepines tested. However, progabide has no effect on the median antipentylenetetrazol dose (PTZ ED50) of the benzodiazepines. Likewise, THIP (2.5 mg/kg, i.p.) significantly decreases the TD50 of chlordiazepoxide but not that of triazolam. THIP significantly decreases the MES ED50 of chlordiazepoxide and triazolam but has no effect on the PTZ ED50 of these two substances. The above data suggest that benzodiazepine receptors linked to GABA receptors contribute to the minimal neurotoxicity and anti-MES activity but not to the anti-PTZ activity of benzodiazepines.     

Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats

In vivo pharmacological effects of ramelteon (TAK-375), a novel, highly MT1/MT2-selective receptor agonist, were studied in rats to determine ramelteon's ability to reentrain the circadian rhythm after an abrupt phase advance. Experiments were also conducted to assess the potential cognitive side effects of ramelteon and its potential to become a drug of abuse. After an abrupt 8-h phase shift, ramelteon (0.1 and 1 mg/kg, p.o.) and melatonin (10 mg/kg, p.o.) accelerated reentrainment of running wheel activity rhythm to the new lightdark cycle. Ramelteon (3-30 mg/kg, p.o.) and melatonin (10-100 mg/kg, p.o.) did not affect learning or memory in rats tested by the water maze task and the delayed match to position task, although diazepam and triazolam impaired both of the tasks. Neither ramelteon (3-30 mg/kg, p.o.) nor melatonin (10-100 mg/kg, p.o.) demonstrated a rewarding property in the conditioned place-preference test, implying that MT1/MT2 receptor agonists have no abuse potential. In contrast, benzodiazepines and morphine showed rewarding properties in this test. The authors' results suggest that ramelteon may be useful for treatment of circadian rhythm sleep disorders without adverse effects typically associated with benzodiazepine use, such as learning and memory impairment, and drug dependence.     

Ethanol-induced limb defects in mice: effect of strain and Ro15-4513

It is now thought that ethanol exerts many of its behavioral effects in the CNS by interaction with the gamma-aminobutyric acid (GABA) receptor, and it has been shown that the benzodiazepine reverse agonist Ro15-4513 reverses some of the CNS effects produced by ethanol. The hypothesis was tested that ethanol exerts its teratogenic effects through interaction with a putative embryonic GABA receptor by determining whether Ro15-4513 reverses ethanol-induced forelimb ectrodactyly in C57BL/6 mice. First, pregnant C57BL/6 dams were injected twice i.p. with ethanol (2.9 g/kg body weight, 4 hr apart) on day 10 of gestation: 49% of the fetuses were resorbed or dead and 46% of the survivors showed forelimb ectrodactyly. In contrast, when SWV mice were treated with ethanol, embryolethality was only 11.9% and no forelimb ectrodactyly was observed. In a second experiment, when ethanol (2.6 g/kg x 2) was administered to C57BL/6 mice, 34% resorptions and 31% forelimb ectrodactyly were observed. Ectrodactyly induced by ethanol was primarily of the forelimb and exclusively postaxial. Ethanol produced an unusual forelimb defect in a small number of instances where there was a postaxial autopod reduction defect coupled with a preaxial zeugopod reduction defect. Ro15-4513 administered alone (50 mg/kg x 2) was neither embryolethal nor teratogenic in C57BL/6 mice. To attempt to reverse the teratogenic effect of ethanol, dams that were injected 5 min before each ethanol administration with Ro15-4513 (0.5, 1, 2.5, 5, 10 mg/kg twice) showed no significant change in frequency of forelimb ectrodactyly compared to embryos treated with ethanol alone. However, resorptions increased significantly to 77% and 62% with the 5 and 10 mg/kg doses of Ro15-4513. Thus there appears to be an embryolethal interaction of Ro15-4513 with ethanol. Nevertheless, since Ro15-4513 did not reverse the teratogenic effect induced by ethanol, these results do not support the hypothesis that the teratogenic mechanism of ethanol is mediated through a putative embryonic GABA receptor.     

Antinociceptive action of GABA-mimetic agent--N-phthaloyl GABA

N-phthaloyl GABA (P-GABA), a nonselective GABA-ergic drug, showed positive analgesic response in four different models in mice, viz-tail immersion, tail clip, hot plate and writhing-induced by acetic acid. Antinociceptive ED50 (ip in mice) of P-GABA was lowest in tail immersion method (ED50 = 24.27, mg/kg). Though pethidine (10 mg/kg, ip) significantly potentiated the antinociceptive action of P-GABA (20 mg/kg, ip), pretreatment of naloxone (5 mg/kg, im) did not influence the same. Pretreatment with atropine (10 mg/kg, im), picrotoxin (0.08 mg/kg) and 3-mercaptopropionic acid (2 mg/kg) reduced the antinociceptive action of P-GABA significantly. But pretreatment with bicuculline (0.4 mg/kg), a specific GABA antagonist, did not reduce the antinociceptive action of P-GABA.     

Triazolam and Phase-Shifting Acceleration Re-Evaluated

In two experiments, triazolam (2.5 and 1.5 mg/animal) failed to significantly enhance the rate of re-entrainment of hamsters (Mesocricetus auratus) to an 8-hr advance of their light-dark cycle. Evidently the phase-shifting effects of triazolam are not robust. The animals did not run much in their wheels in response to the drug in these two experiments. In a third experiment, triazolam (0.5 and 2.5 mg/animal) produced phase advances of activity rhythms of hamsters in the dark. In this experiment, running in response to the drug was greater. Hamsters given triazolam but confined to their nest boxes over the next few hours did not show phase shifts. The phase-shifting effects of triazolam (when they do occur) appear to be mediated through activity increases. Triazolam-treated hamsters became ataxic in all three of these experiments. Suggestions that triazolam may be useful in ameliorating rhythm disturbances in people should be treated with a caution.     

Differential effects of pentobarbital and ethanol in mice

The duration of the loss of righting reflex (RR) after ethanol, 4 g/kg, intraperitoneally (i.p.), was significantly longer in “long-sleep” (LS) than in “short-sleep” (SS) mice. This effect was shown to be correlated with differences in brain sensitivities to ethanol. In contrast, pentobarbital sodium (PB), 50 mg/kg, i.p., produced a significantly longer loss of RR in SS than in LS mice. The PB concentrations in the brain were the same in both mouse strains at the time of RR recovery suggesting equal sensitivities of the central nervous systems to PB. The rates of disappearance of PB from the blood were the same in both strains, but the apparent volume of distribution of PB in the LS strain was greater than in SS mice.In addition, C57BL/6J mice were found to be more sensitive than DBA/2J mice to PB, 50 mg/kg. In contrast, C57BL mice are known to be less sensitive than the DBA strain to ethanol. The PB concentration in the brain of DBA mice at the recovery of the RR was significantly greater than in C57BL mice. The apparent volumes of distribution of PB were not different in the two strains, but the rate of disappearance of PB from the blood of C57BL mice was significantly greater than for the DBA strain. In conclusion, factors which govern the brain sensitivities of selected mouse strains to ethanol and pentobarbital may not be equivalent.     

GABA influences the development of the ventromedial nucleus of the hypothalamus

The region that becomes the ventromedial nucleus of the hypothalamus (VMH) is surrounded by cells and fibers containing immunoreactive gamma‐aminobutyric acid (GABA) by embryonic day 13 (E13), several days before the nucleus emerges in Nissl stains. As GABA plays many roles during neural development, we hypothesized that it influences VMH development, perhaps by providing boundary information for migrating neurons. To test this hypothesis we examined the VMH in embryonic mice in which the β3 subunit of the GABA_A‐receptor, a receptor subunit that is normally highly expressed in this nucleus, was disrupted by gene targeting. In β3 ?/? embryos the VMH was significantly larger, and the distribution of cells containing immunoreactive estrogen receptor‐α was expanded compared to controls. Using in vitro brain slices from wild‐type C57BL/6J mice killed at E15 we found that treatment with the GABA_A antagonist bicuculline increased the number of cells migrating per video field analyzed in the VMH. In addition, treatment with either bicuculline or the GABA_A agonist muscimol altered the orientation of cell migration in particular regions of this nucleus. These data suggest that GABA is important for the organization of cells during VMH formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 264–276, 2001     

A pharmacological analysis of the GABA-ergic system of the primordial hippocampus in the frog Rana temporaria]

The data on evoked potentials (EP) in the frog primordial hippocampus during the surface application of gamma-aminobutyric acid (GABA), beta-alanine and aminooxyacetic acid in the region of EP recording are presented. These results in the aggregate with the earlier described effects of picrotoxin, bicuculline, muscimol, baclofen (on EP) and GABA (on intracellular potentials) permit to suggest the presence of GABA-ergic inhibitory system and two types of GABA receptors in the frog primordial hippocampus.     

Effects of muscimol and flurazepam on the sleep EEG in the rat

In order to assess the possible role of GABA receptor function in the hypnotic property of benzodiazepines, we have examined the sleep EEG in rats given the GABA agonist muscimol, alone and in combination with flurazepam. Muscimol 0.05 and 0.1 mg/kg IP failed to alter sleep latency or total sleep time, and did not interact with the sleep-enhancing properties of flurazepam 20 mg/kg IP. These observations, in conjunction with a previous study of bicuculline, suggest that the hypnotic property of benzodiazepines may not be mediated by alteration of GABAergic activity.     

Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6 mice

The chromosome aberration assay of metaphase bone marrow cells was used to study the clastogenic effects of acrylamide, cyclophosphamide, dioxidine, and their combinations with Verapamil (a calcium antagonist) in male BALB/C and C57BL/6 mice. Verapamil gavage at single (5 mg/kg) and repeated doses (2.5 and 5 mg/kg five times at 24-h intervals) significantly enhanced the clastogenic activity of acrylamide (50 and 100 mg/kg intraperitoneally) in BALB/C mice; in C57BL/6 mice, this effect was only observed when they received Verapamil at doses of 2.5 mg/kg for 5 days. Verapamil administered repeatedly (2.5–10 mg, gavage) significantly increased the clastogenic activity of cyclophosphamide (10 mg/kg intraperitoneally) in C57BL/6 mice. In BALB/C mice, this effect of Verapamil was only observed at a dose of 10 mg/kg (gavage). When injected intraperitoneally at a single dose of 0.1–0.4 mg/kg, Verapamil significantly enhanced the clastogenic activity of cyclophosphamide in mice of both strains. This calcium antagonist produced identical effects when administered to BALB/C mice intraperitoneally (2.5 and 5 mg/kg) and by gavage (5 mg/kg) and to C57BL/6 mice intraperitoneally (5 and 10 mg/kg) and by gavage (2.5 mg/kg). Repeated administration of Verapamil (at all doses tested) promoted the clastogenic effect of dioxidine (100 mg/kg intraperitoneally) on C57BL/6 mice, having no such influence on BALB/C mice. These results demonstrate the co-clastogenic activity of Verapamil in mice and suggest that its specific manifestations depend on the dose, method, and route of drug administration and the genotype of test animals.     

Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6 mice

The chromosome aberration assay of metaphase bone marrow cells was used to study the clastogenic effects of acrylamide, cyclophosphamide, dioxidine, and their combinations with Verapamil (a calcium antagonist) in male BALB/C and C57BL/6 mice. Verapamil gavage at single (5 mg/kg) and repeated doses (2.5 and 5 mg/kg five times at 24-h intervals) significantly enhanced the clastogenic activity of acrylamide (50 and 100 mg/kg intraperitoneally) in BALB/C mice; in C57BL/6 mice, this effect was only observed when they received Verapamil at doses of 2.5 mg/kg for 5 days. Verapamil administered repeatedly (2.5-10 mg, gavage) significantly increased the clastogenic activity of cyclophosphamide (10 mg/kg intraperitoneally) in C57BL/6 mice. In BALB/C mice, this effect of Verapamil was only observed at a dose of 10 mg/kg (gavage). When injected intraperitoneally at a single dose of 0.1-0.4 mg/kg, Verapamil significantly enhanced the clastogenic activity of cyclophosphamide in mice of both strains. This calcium antagonist produced identical effects when administered to BALB/C mice intraperitoneally (2.5 and 5 mg/kg) and by gavage (5 mg/kg) and to C57BL/6 mice intraperitoneally (5 and 10 mg/kg) and by gavage (2.5 mg/kg). Repeated administration of Verapamil (at all doses tested) promoted the clastogenic effect of dioxidine (100 mg/kg intraperitoneally) on C57BL/6 mice, having no such influence on BALB/C mice. These results demonstrate the co-clastogenic activity of Verapamil in mice and suggest that its specific manifestations depend on the dose, method, and route of drug administration and the genotype of test animals.     

Involvement of NMDA receptor in low-frequency magnetic field-induced anxiety in mice

It had been reported that exposure to extremely low-frequency magnetic field (ELFMF) induces anxiety in human and rodents. Anxiety mediates via the activation of N-methyl-d-aspartate (NMDA) receptor, whereas activation of γ-aminobutyric acid (GABA) receptor attenuates the same. Hence, the present study was carried out to understand the contribution of NMDA and/or GABA receptors modulation in ELFMF-induced anxiety for which Swiss albino mice were exposed to ELFMF (50?Hz, 10?G) by subjecting them to Helmholtz coils. The exposure was for 8?h/day for 7, 30, 60, 90 and 120 days. Anxiety level was assessed in elevated plus maze, open field test and social interaction test, on 7th, 30th, 60th, 90th and 120th exposure day, respectively. Moreover, the role of GABA and glutamate in ELFMF-induced anxiety was assessed by treating mice with muscimol [0.25?mg/kg intraperitoneally (i.p.)], bicuculline (1.0?mg/kg i.p.), NMDA (15?mg/kg i.p.) and MK-801 (0.03?mg/kg i.p.), as a GABA_A and NMDA receptor agonist and antagonist, respectively. Glutamate receptor agonist exacerbated while inhibitor attenuated the ELFMF-induced anxiety. In addition, levels of GABA and glutamate were determined in regions of the brain viz, cortex, striatum, hippocampus and hypothalamus. Experiments demonstrated significant elevation of GABA and glutamate levels in the hippocampus and hypothalamus. However, GABA receptor modulators did not produce significant effect on ELFMF-induced anxiety and elevated levels of GABA at tested dose. Together, these findings suggest that ELFMF significantly induced anxiety behavior, and indicated the involvement of NMDA receptor in its effect.     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Study of the hereditary differences in the cyclic nucleotide content of the blood serum in mice after exposure to stress and administration of phenazepam

Blood plasma content of cAMP and cGMP in C57BL/6, BALB/c mice and their reciprocal F1-hybrids has been studied at rest, upon exposure to stress induced in an open field technique and phenazepam injection at a dose of 0.05 and 0.1 mg/kg. Interstrain differences in baseline content and changes of nucleotide concentration in conditions of stress have been revealed. F1-hybrids inherit initial correlation between nucleotide content and the type of cAMP changes of C57BL/6 mice and cGMP changes of BALB/c mice. Phenazepam injection to C57BL/6 and BALB/c mice was shown to produce specific shifts in blood plasma cyclic nucleotide content.