Positive GABA Modulators Research Articles

Selectivity in generalization to GABAergic drugs in midazolam-trained baboons

When barbiturates have been tested in animals trained to discriminate the intravenous benzodiazepine (Bz) anesthetic midazolam, squirrel monkeys and pigeons did not reliably generalize to barbiturates but rats did. To explore this unexpected phenomenon in another species and to extend the midazolam generalization profile to GABAergic compounds not previously tested, five baboons were trained to discriminate midazolam maleate (0.32 mg/kg iv) from saline under a two-lever procedure. In tests 10 min after dose delivery, the partial agonist imidazenil, the full agonist chlordiazepoxide, and the receptor-subtype-selective hypnotic zolpidem fully shared discriminative effects with midazolam. The barbiturate pentobarbital did so in only one of five baboons, and the intravenous anesthetic propofol failed to do so in the three baboons tested. Testing 1 min after dose delivery shifted midazolam and zolpidem curves to the left and increased generalization to propofol but not pentobarbital. Taken together with previous published data, partial or full agonism at the Bz binding site appears sufficient for midazolam-like discriminative effects in nonhuman primates, pigeons, and rodents, and modulation through the anesthetic site is sufficient in baboons. However, to date, positive modulation of GABA through the barbiturate site is not generally sufficient for this effect in nonhuman primates and pigeons although it is in rodents.

Methionine 286 in transmembrane domain 3 of the GABA A receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics

γ-Aminobutyric acid type A (GABA A) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA A receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA A receptor β 2 subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the β 2 subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the β 2(M286W) mutation resulted in a decrease in the ‘cut-off’ volume for propofol analog molecules to enhance GABA responses at GABA A α 1β 2γ 2s receptors. This suggests that mutation of M286 in the GABA A β 2 subunit alters the dimensions of a ‘binding pocket’ for propofol and related alkylphenol general anesthetics.

Early postnatal ethanol intubation blunts GABA A receptor up-regulation and modifies 3α-hydroxy-5α-pregnan-20-one sensitivity in rat MS/DB neurons

Previously we found postnatal binge-like ethanol exposure using an artificial-rearing method in the rat delayed developmental up-regulation of GABA A receptors (GABA ARs) in both medial septum/diagonal band (MS/DB) and cerebellar Purkinje neurons. In the present study, the impact of ethanol on developing GABA ARs in MS/DB neurons was further tested under conditions not requiring anesthesia or maternal deprivation. Nursing rat pups received ethanol (4.5–5.25 g/kg/day) on postnatal days (PD) 4–9, which was administrated manually by oral intragastric intubation. This treatment caused dose-dependent blunting of peak GABA A receptor whole cell currents in acutely dissociated MS/DB cells on PD 12–15. The threshold with oral intubation was slightly higher than previously observed for artificial-rearing (4.9 vs. 4.5 g/kg/day). The previously observed reduced sensitivity of GABA ARs to Zn 2+-inhibition after ethanol was not found with the intubation model. In studies only carried out using the intubation method, 3α-hydroxy-5α-pregnan-20-one (3α-OH-DHP) caused an allosteric concentration-dependent potentiation of currents activated by non-saturated concentrations of GABA. A bicuculline sensitive direct activation of GABA ARs also occurred with higher concentrations of 3α-OH-DHP alone. Ethanol intubation up-regulated allosteric neurosteroid potentiation with low concentrations of GABA, but did not change direct agonist actions of 3α-OH-DHP. Finally, 3α-OH-DHP did not prime ethanol insensitive GABA ARs to become sensitivity to acute ethanol potentiation. These results indicate ethanol consistently blunts postnatal GABA A receptor up-regulation across early postnatal binge-type ethanol exposure models and may increase positive modulation of GABA A receptors by endogenous neurosteroids.

Antinociceptive properties of neurosteroids II.: Experiments with Saffan ® and its components alphaxalone and alphadolone to reveal separation of anaesthetic and antinociceptive effects and the involvement of spinal cord GABA A receptors

Studies have shown that the steroid anaesthetic alphaxalone positively modulates γ-aminobutyric acid (GABA) receptors in vitro. It has also been reported that positive modulation of GABA A receptors in the rat spinal cord can produce antinociception in vivo. This present study looks at the interaction of an intraperitoneal injection (i.p.) of the steroid anaesthetic combination Saffan ® (alphaxalone 9 mg/ml, alphadolone acetate 3 mg/ml) with GABA A receptors in the spinal cord. Full recovery from anaesthesia induced by Saffan ® 2 ml/kg i.p., as assessed by the rotarod test, occurred after 28.78±0.86 min. Residual antinociceptive effects were assessed by application of electrical current at two skin sites (neck and tail) and also tail withdrawal from noxious heat. Residual antinociception was observed at both skin sites assessed by the electrical test but not when assessed by noxious heat. The antinociceptive effects in the tail but not the neck were suppressed by intrathecal administration of GABA A antagonists (bicuculline and SR-95531). In a separate group of experiments alphaxalone and alphadolone were given i.p. individually at the same doses that were given when formulated in Saffan ®. Alphaxalone produced sedative and anaesthetic effects with no antinociception. Alphadolone caused no sedation but it did cause antinociceptive effects equal in magnitude to those produced by Saffan ®. We conclude that Saffan ® produces antinociception in rats when given i.p. by an interaction with spinal GABA A receptors. Furthermore, this antinociception is due to the alphadolone content of the neurosteroid anaesthetic and not the alphaxalone.

Ethopharmacology of the Antidepressant Effect of Clonazepam in Diabetic Rats

Diabetes-associated depression may occur due to changes in the quality of life imposed by treatment, or may be a consequence of the biochemical changes accompanying the disease. It was our objective to evaluate the behaviors of diabetic rats through an animal model of depression, and determine if a positive GABA modulator agent, clonazepam, is an effective antidepressant. Wistar male rats were submitted to the forced-swimming test after 26 days of the induction of diabetes with streptozotocin (60 mg/kg). Test and retest days analyzed with an ethological approach. Clonazepam (control, 0.25, 0.5, and 1.0 mg/kg) was administered IP 24, 5, and 1 h before the retest. Diabetic rats presented longer immobility duration during test and retest of forced swimming. Diabetic rats dived significantly less during the test. Clonazepam 0.25 and 0.5 mg/kg decreased immobility of diabetic rats with no consequences on the behaviors of nondiabetic rats. These results demonstrate that diabetic rats present more intense depressive-like behavior, such as immobility and lack of interest in exploring the environment, when exposed to the forced-swimming test. It is possible that decreased GABA function is involved in depression associated with diabetes, because a benzodiazepine partially counteracts these changes without modifying blood glucose and glycogen parameters.

Differential modulatory actions of the volatile convulsant flurothyl and its anesthetic isomer at inhibitory ligand-gated ion channels

A challenge for theories of general anesthesia is the existence of compounds predicted to be anesthetics but which, instead, do not produce anesthesia and often elicit other behavioral effects such as convulsions. This study focused on flurothyl (bis[2,2,2-trifluoroethyl] ether), a potent volatile convulsant, and its anesthetic isomer, ‘iso-flurothyl’ (1,1,1,3,3,3-hexafluoro-2-methoxypropane). The effects of flurothyl and iso-flurothyl were studied using the whole-cell patch-clamp technique on agonist-activated chloride currents in human GABA A, glycine, and GABA C ρ 1 receptors expressed in HEK 293 cells. GABA A and glycine receptors are promising molecular targets for the actions of inhaled ether general anesthetics. Flurothyl acted as a non-competitive antagonist at GABA A α 2β 1 and α 2β 1γ 2s receptors, but had no effect at glycine α 1 receptors. Flurothyl had biphasic actions on GABA responses at GABA C ρ 1 receptors. In contrast, iso-flurothyl enhanced (‘potentiated’) submaximal agonist responses at GABA A and glycine receptors, but had no effect on GABA responses at GABA C ρ 1 receptors. Point mutations in GABA A and glycine receptor subunits, which have been previously shown to abolish potentiation of agonist responses by the ether anesthetics enflurane and isoflurane, also ablated potentiation of agonist responses by iso-flurothyl. These same mutations in the GABA A receptor had only modest effects on the inhibitory actions of flurothyl. GABA A receptors with mutations conferring insensitivity to antagonism by picrotoxin were still inhibited by flurothyl, suggesting that picrotoxin and flurothyl antagonize GABA responses by distinct sites or mechanisms of action. In summary, antagonism of GABA A receptors is likely to account for the convulsant effects of flurothyl, while the general anesthetic actions of iso-flurothyl, like those of other ether anesthetics, may be related to positive modulation of GABA A and/or glycine receptors.

Effect of ganaxolone in a rodent model of cerebral hematoma.

Therapy with gamma-aminobutyric acid (GABA) agonists appears to improve outcome after experimental hematoma but with unacceptable side effects. We looked to synthetic GABA agonists, or positive GABA modulators, widely developed as anticonvulsants and anxiolytics, to find compounds that may be effective. Ganaxolone is a synthetic neuroactive steroid that positively modulates GABA. We sought to determine whether ganaxolone was beneficial using a model of intracerebral hematoma. We stereotaxically injected varying doses of bacterial collagenase into the caudate nucleus of rats to induce blood-brain barrier failure and hematoma formation. Four hours later, we administered intravenously 15 or 30 mg/kg ganaxolone (n=23 each group), 20 mg/kg pregnanolone (n=21), or vehicle (n=30). Forty-eight hours after collagenase injection, we rated each animal using a standard rodent neurological examination. The ratings were compared with the amounts of injected collagenase using the quantal bioassay procedure. Other sets of animals were tested later for visuospatial learning. Brains were then prepared for histomorphometry, and brain volumes were estimated. We found that ganaxolone 30 mg/kg significantly increased the ED(50) in the bioassay, for a potency ratio of 1.8+/-0.41 compared with vehicle (P<0.05). Ganaxolone 15 mg/kg and pregnanolone did not affect neurological outcome. Ganaxolone 30 mg/kg did not clearly improve visuospatial learning several weeks after hemorrhage. Ganaxolone exhibited a weak effect on cerebral volumes 48 hours after stroke, but 3 months after hemorrhage no such effect could be detected. Ganaxolone improves neurological outcome 48 hours after intracerebral hematoma but not visuospatial learning several weeks after intracerebral hematoma. Histological evidence of damage was reduced at 48 hours but not at 3 months.

Discriminative stimulus effects of the endogenous neuroactive steroid pregnanolone

Naive male Sprague-Dawley rats were trained to discriminate the endogenous neuroactive steroid pregnanolone (5.6 mg/kg) from saline. Three positive modulators of the GABA A receptor complex substituted for pregnanolone: the neuroactive steroid allopregnanolone (1.0–10.0 mg/kg), the barbiturate pentobarbital (3.0–17.0 mg/kg), and the benzodiazepine diazepam (0.3–3.0 mg/kg). In contrast, buspirone, a 5-HT 1A-mediated anxiolytic, failed to substitute up to rate-suppressing doses (1.0–5.6 mg/kg). The present experiment demonstrated the ability of an endogenous neuroactive steroid to function as a discriminative stimulus. Moreover, these results suggest that the discriminative stimulus effects of pregnanolone are mediated via positive modulation of GABA A receptors.