Preclinical and clinical pharmacology of TPA023B, a GABAA receptor α2/α3 subtype-selective partial agonist.

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABA(A) receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABA(A) subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABA(A) receptors as measured using an in vivo [(3)H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [(11)C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABA(A) receptor population for avoiding sedation in man.

MRK-409 (MK-0343), a GABAA receptor subtype-selective partial agonist, is a non-sedating anxiolytic in preclinical species but causes sedation in humans.

MRK-409 binds to α1-, α2-, α3- and α5-containing human recombinant GABA(A) receptors with comparable high affinity (0.21-0.40 nM). However, MRK-409 has greater agonist efficacy at the α3 compared with α1 subtypes (respective efficacies relative to the full agonist chlordiazepoxide of 0.45 and 0.18). This compound readily penetrates the brain in rats and occupies the benzodiazepine site of GABA(A) receptors, measured using an in vivo [(3)H]flumazenil binding assay, with an Occ(50) of 2.2 mg/kg p.o. and a corresponding plasma EC(50) of 115 ng/mL. Behaviourally, the α3-preferring agonist efficacy profile of MRK-409 produced anxiolytic-like activity in rodent and primate unconditioned and conditioned models of anxiety with minimum effective doses corresponding to occupancies, depending on the particular model, ranging from ∼35% to 65% yet there were minimal overt signs of sedation at occupancies greater than 90%. In humans, however, safety and tolerability studies showed that there was pronounced sedation at a dose of 2 mg, resulting in a maximal tolerated dose of 1 mg. This 2 mg dose corresponded to a C(max) plasma concentration of 28 ng/mL, which, based on the rodent plasma EC(50) for occupancy of 115 ng/mL, suggested that sedation in humans occurs at low levels of occupancy. This was confirmed in human positron emission tomography studies, in which [(11)C]flumazenil uptake following a single dose of 1 mg MRK-409 was comparable to that of placebo, indicating that occupancy of GABA(A) receptor benzodiazepine binding sites by MRK-409 was below the limits of detection (i.e. <10%). Taken together, these data show that MRK-409 causes sedation in humans at a dose (2 mg) corresponding to levels of occupancy considerably less than those predicted from rodent models to be required for anxiolytic efficacy (∼35-65%). Thus, the preclinical non-sedating anxiolytic profile of MRK-409 did not translate into humans and further development of this compound was halted.

Occupancy of human brain GABA(A) receptors by the novel α5 subtype-selective benzodiazepine site inverse agonist α5IA as measured using [¹¹C]flumazenil PET imaging.

GABA(A) receptor α5-selective inverse agonists enhance cognitive performance in pre-clinical species. However, a key aspect of the clinical development of such compounds is the demonstration that in man such compounds are devoid of the anxiogenic-like activity associated with non-selective inverse agonists such as FG 7142. The triazolophthalazine α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is an α5-selective inverse agonist which enhances cognitive performance in rodents and encouragingly in human Phase I Safety and Tolerability studies it was devoid of the anxiogenic-like activity associated with FG 7142. However, in order to appropriately interpret this latter observation, it was considered important to demonstrate that the absence of anxiogenic-like activity occurs at significant levels of receptor occupancy. Consequently, the occupancy of human brain GABA(A) receptors was measured using [¹¹C]flumazenil positron emission tomography in three healthy normal young male volunteers following a single oral dose of 2 mg α5IA. One hour after dosing, mean occupancy levels were 53% and this fell to 16% by 8 h post-dose, with the plasma α5IA concentration corresponding to 50% occupancy being 10 ng/mL. These data clearly show that an α5-selective inverse agonist is not associated with anxiogenic-like side effects at doses that give ~50% occupancy.

Alpha2-containing GABA(A) receptors are involved in mediating stimulant effects of cocaine.

alpha2 subunit-containing GABA(A) receptors are involved in incentive learning associated with cocaine, and in cocaine addiction. Deletion of alpha2-containing receptors abolishes cocaine-induced behavioural sensitisation (BS), while selective activation of alpha2 receptors, achieved using Ro 15-4513's agonist properties in alpha2(H101R) mice, induced BS. Here, we investigate further the mechanisms underlying Ro 15-4513-induced behavioural sensitisation in alpha2(H101R) mice. alpha2(H101R) mice sensitised to Ro 15-4513 (10 mg/kg) showed an enhanced stimulant response to cocaine (10 mg/kg). In contrast, cocaine (10 mg/kg)-sensitised alpha2(H101R) mice did not show enhanced sensitivity to the stimulant effects of Ro 15-4513 (1, 3 and 10 mg/kg), suggesting that the neural adaptations underlying Ro 15-4513 induced BS are related to, but not identical with those associated with cocaine-induced plasticity. Secondly, we investigated whether alpha2-containing receptors are involved in mediating the ability of BZs to facilitate cocaine-induced activity. The non-selective (i.e., alpha1, alpha2, alpha3 and alpha5 subtype) benzodiazepine GABA(A) receptor agonist midazolam (10 and 30 mg/kg) potentiated cocaine (10 mg/kg) hyperactivity in wildtype mice, but not in alpha2(H101R) mice, in which alpha2-containing receptors are insensitive to benzodiazepines. To determine where alpha2 receptors are localised we compared BZ-insensitive sites between wildtype (alpha4 and alpha6) and alpha2(H101R) (alpha2, alpha4 and alpha6) mice, using quantitative autoradiography to estimate [(3)H]Ro 15-4513 binding in the presence of 10 muM diazepam. alpha2 receptors were found in projection areas of the mesolimbic dopamine pathway including accumbens, central amygdala, and basolateral amygdala as well as CA1 and CA3 areas of the hippocampus. The involvement of the alpha2-containing receptor in mediating BZ's potentiating effect on cocaine hyperactivity suggests that the locomotor stimulant effects of BZs and psychostimulants may be mediated by a common neural system, but the lack of cross sensitisation to Ro 15-4513 in cocaine-sensitised alpha2(H101R) mice, suggests that this form of BS may occur downstream of plastic events underlying cocaine sensitisation.

Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain.

BACKGROUND AND PURPOSE: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. EXPERIMENTAL APPROACH: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. KEY RESULTS: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. CONCLUSIONS AND IMPLICATIONS: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.

Both alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm.

Mice with point-mutated alpha2 GABAA receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in unconditioned models of anxiety. We investigated the role of the alpha2 GABAA subtype in a model of conditioned anxiety. alpha2(H101R) and wildtype mice were trained in a conditioned emotional response (CER) task, in which lever-pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a conditioned stimulus (CS+) that predicted footshock. The ability of diazepam, ethanol and pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. Diazepam (0, 0.5, 1, 2, 4 and 8 mg/kg) induced a dose-dependent anxiolytic-like effect in wildtype mice. At high doses, diazepam (2, 4 and 8 mg/kg) was sedative in alpha2(H101R) mice. Analysis of the anxiolytic properties of nonsedative diazepam doses (0.5 and 1 mg/kg), showed that alpha2(H101R) mice were resistant to the anxiolytic effects of diazepam. Equivalent anxiolytic properties of pentobarbital (20 mg/kg) and ethanol (1 and 2 g/kg) were seen in both genotypes. These findings confirm the critical importance of the alpha2 GABAA subtype in mediating BZ anxiolysis. However, as a compound, L-838417, with agonist properties at alpha2, alpha3 and alpha5-containing receptors, gave rise to anxiolytic-like activity in alpha2(H101R) mice in the CER test, alpha3-containing GABA receptors are also likely to contribute to anxiolysis. Observations that alpha2(H101R) mice were more active, and displayed a greater suppression of lever pressing in response to fear-conditioned stimuli than wildtype mice, suggests that the alpha2(H101R) mutation may not be behaviourally silent.

An inverse agonist selective for alpha5 subunit-containing GABAA receptors improves encoding and recall but not consolidation in the Morris water maze.

RATIONALE: Compounds selective for the GABAA receptors containing an alpha5 subunit have been reported to enhance performance in the hippocampally mediated delayed-matching-to-position version of the Morris water maze, in which reduction in the time required to find a hidden platform relative to an initial trial is used as an index of learning and memory. OBJECTIVE: In the present study, we have used one such compound, alpha5IA-II, to examine whether these effects occur during the encoding, consolidation or recall phases of this paradigm. METHODS: alpha5IA-II was administered in the absence or presence of the benzodiazepine site antagonist flumazenil, so as to limit its action to periods associated with encoding, consolidation and recall. Drug doses and timings of administrations were defined using occupancy data derived from an in vivo [3H]flumazenil binding assay. Similar experiments were carried out to study the memory-disruptive properties of chlordiazepoxide (CDP). RESULTS: The trial 1 to trial 2 difference was increased when alpha5IA-II was given before either trial 1 or trial 2, indicating an effect on the encoding and recall phases, respectively, of learning and memory. Conversely, alpha5IA-II had no effect on performance when given immediately after trial 1, suggesting that it had no effect on the consolidation phase. In contrast to the facilitation of performance produced by the alpha5-selective inverse agonist alpha5IA-II given during the encoding and recall but not the consolidation phase, the non-selective agonist CDP impaired performance when given during the encoding and recall phases, whilst having no effect on the consolidation phase. CONCLUSIONS: These data further highlight the cognition-enhancing properties of GABAA alpha5-selective inverse agonists and define the functional specificity of these effects in terms of encoding and recall processes in the Morris water maze.

An inverse agonist selective for alpha5 subunit-containing GABAA receptors enhances cognition.

Alpha5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has inverse agonist efficacy selective for the alpha5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABA(A) receptors containing an alpha5 subunit. In a mouse hippocampal slice model, alpha5IA significantly enhanced the burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that alpha5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, alpha5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice alpha5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, alpha5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABA(A) alpha5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

Rodent pharmacokinetics and receptor occupancy of the GABAA receptor subtype selective benzodiazepine site ligand L-838417.

The pharmacokinetics of L-838417, a GABAA receptor subtype selective benzodiazepine site agonist, were studied in rats and mice after single oral (p.o.), intraperitoneal (i.p.) and intravenous (i.v.) doses. In both species L-838417 was well absorbed following i.p. administration and whilst in rats p.o. bioavailability was good (41%), in mice it was negligible (<1%), precluding this as a route of administration for mouse behavioural studies. Despite having a similar volume of distribution (ca 1.4 l/kg) in rats and mice, L-838417 was cleared at twice liver blood flow in mice (161 ml/min/kg) and moderately cleared in rats (24 ml/min/kg), potentially explaining the poor oral bioavailability in mice. Finally, as a pharmacodynamic readout the benzodiazepine binding site occupancy was determined in rats (0.3-3 mg/kg, p.o.) and mice (1-30 mg/kg, i.p.) using a [3H]Ro 15-1788 in vivo binding assay. Although the resulting dose-occupancy relationship for both species demonstrated a dose-dependent increase in receptor occupancy, appreciable variability was observed at low dose levels, potentially making interpretation of behavioural responses difficult. In contrast, a clear relationship between plasma and brain concentrations and receptor occupancy were determined suggesting the observed dose-occupancy variability is a consequence of the pharmacokinetic properties of L-838417. The plasma and brain concentrations required to occupy 50% of the benzodiazepine binding sites were similar in both rats (28 ng/ml and 33 ng/ml g, respectively) and mice (63 ng/ml and 53 ng/ml g, respectively), with a non-linear concentration response observed with increasing doses of L-838417. These studies demonstrate the importance of utilizing pharmacokinetic/receptor occupancy data when interpreting pharmacodynamic responses at a given dose.

GABA(A) alpha 1 subunit knock-out mice do not show a hyperlocomotor response following amphetamine or cocaine treatment.

The GABA(A) receptor system provides the major inhibitory control in the CNS, with the alpha 1 beta 2 gamma 2 subunit combination being the most abundant and widely distributed form of the receptor. The alpha1 subunit knock-out (alpha1 KO) mice had a surprisingly mild overt phenotype, despite having lost approximately 60% of all GABA(A) receptors. The alpha1 KO mice had normal spontaneous locomotor activity, but were more sensitive to the sedating/ataxic effects of diazepam than wildtype (WT) mice. Pharmacological modulation of dopamine and N-methyl-D-aspartate (NMDA) receptors also produced altered responses in alpha1 KO mice compared with WT mice. As expected, the NMDA receptor antagonist MK801, amphetamine and cocaine increased locomotor activity in WT mice. Although MK801 increased locomotor activity in alpha1 KO mice, amphetamine and cocaine induced stereotypy not hyperlocomotion. Binding studies showed no gross changes in the total number of D1, D2 or NMDA receptors. Furthermore, pre-pulse inhibition of acoustic startle and the effects of cocaine in conditioned place preference were similar in both alpha1 KO and WT mice, indicating selective rather that global changes in response to dopaminergic agents. These data demonstrate subtle changes in behaviours mediated by neurotransmitters other than GABA in alpha1 KO mice and suggest that compensation may have occurred beyond the GABAergic system.

Loss of the major GABA(A) receptor subtype in the brain is not lethal in mice.

The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant gamma-aminobutyric acid(A) receptors determined using (36)Cl ion flux.

Inhibitory gamma-aminobutyric acid (GABA)(A) receptors are subject to modulation at a variety of allosteric sites, with pharmacology dependent on receptor subunit combination. The influence of different alpha subunits in combination with beta3gamma2s was examined in stably expressed human recombinant GABA(A) receptors by measuring (36)Cl influx through the ion channel pore. Muscimol and GABA exhibited similar maximal efficacy at each receptor subtype, although muscimol was more potent, with responses blocked by picrotoxin and bicuculline. Receptors containing the alpha3 subunit exhibited slightly lower potency. The comparative pharmacology of a range of benzodiazepine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABA(A) receptors (alpha1, alpha2, alpha3, alpha5), alpha5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at alpha3beta3gamma2s with nonselective agonist chlordiazepoxide was greater than at alpha1, alpha2, or alpha5 (P < 0.001). The presence of an alpha4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower alpha4 affinity, and FG8205 displayed similar efficacy. Most striking were the lack of affinity/efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 +/- 12%), CGS8216 (56 +/- 6%), CGS9895 (65 +/- 6%), and the weak partial inverse agonist Ro15-4513 (87 +/- 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5alpha-pregnan-3alpha-ol-20-one, but the type of alpha subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at alpha4beta3gamma2s (226 +/- 10% increase in the EC(20) response to GABA) than any other modulator. The rank order of potentiation for pregnanolone was alpha5 > alpha2 > alpha3 = alpha4 > alpha1, for loreclezole alpha1 = alpha2 = alpha3 > alpha5 > alpha4, and for pentobarbital alpha4 = alpha5 = alpha2 > alpha1 = alpha3.

Density and pharmacology of alpha5 subunit-containing GABA(A) receptors are preserved in hippocampus of Alzheimer's disease patients.

The anatomical localization and pharmacology of alpha5 subunit-containing GABA type-A receptors in the human hippocampal formation of Alzheimer's disease patients were studied with an alpha5 receptor selective ligand, [3H]L-655,708 and compared to age-matched human controls. Autoradiographic analyses revealed a heterogeneous distribution of [3H]L-655,708 binding sites in CA1-CA3 areas with high levels in stratum oriens, stratum pyramidale and stratum radiatum contrasting with low levels in stratum lacunosum. The highest quantity of alpha5 receptors was found in the molecular layer of the dentate gyrus. This pattern of expression was identical in both hippocampus from control and Alzheimer's disease subjects. Quantitative studies demonstrated that the number of [3H]L-655,708 binding sites is well preserved in Alzheimer's disease with only a moderate reduction (25-30%) in the CA1 subfield and entorhinal cortex. Furthermore, saturation and competition experiments with [3H]L-655,708 and representative benzodiazepine site ligands revealed that alpha5 receptors in Alzheimer's hippocampus have an alpha5beta2/3gamma2 pharmacology and structure as in control human brain.Overall, the data reported here provide evidence for a specific expression and relative sparing of alpha5 subunit-containing gamma-aminobutyric acid type-A receptors in the hippocampus of Alzheimer's patients.

Changes in [3H]zolpidem and [3H]Ro 15-1788 binding in rat globus pallidus and substantia nigra pars reticulata following a nigrostriatal tract lesion.

Changes in GABA(A) receptor alpha(1) subunit gene expression occur in the globus pallidus and substantia nigra pars reticulata following lesions of the nigrostriatal tract. To determine whether these changes are translated at the protein level, we performed quantitative autoradiography with the alpha(1) selective ligand, [3H]zolpidem, and the non-selective benzodiazepine site ligand, [3H]Ro 15-1788. Binding of both [3H]zolpidem and [3H]Ro 15-1788 was significantly increased in the substantia nigra pars reticulata (13. 5+/-4.1 and 26.3+/-2.9%, respectively) and significantly reduced in the globus pallidus (20.9+/-0.8 and 18.3+/-1.3%, respectively). These changes in alpha(1) subunit protein expression may help to compensate for the pathological changes in GABAergic activity that occur after striatal dopamine depletion.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Kindling induced by pentylenetetrazole in rats is not directly associated with changes in the expression of NMDA or benzodiazepine receptors.

Repeated injections of a subconvulsant dose of pentylenetetrazole (PTZ, 30 mg/kg IP three times weekly for 13 injections) in Wistar and hooded Lister rats resulted in kindled seizures, the extent of which varied between strains. Wistar rats achieved stage 4 of clonic-tonic seizures, whereas hooded Lister rats only reached stage 2 of convulsive waves axially through the body. Rats were killed 10 days after their final injection, and radioligand binding was used to measure the expression of NMDA receptors in cortex and hippocampus using [3H]MK-801 and [3H]L-689,560, the latter binding specifically to the NR1 subunit. [3H]Ro 15-1788 measured expression of GABA(A)-benzodiazepine binding sites containing alpha1, alpha2, alpha3, or alpha5 subunits. Specific analysis of GABA(A) receptors containing the alpha5 subunit, which are preferentially localized in the hippocampus, was assessed with [3H]L-655,708. In the cortex, there was no effect of strain or treatment on the K(D) or B(max) of any of the ligands. Similarly, there was no effect of strain or treatment on hippocampal [3H]L-689,560 or [3H]Ro 15-1788 binding. However, in the hippocampus there was a significant, albeit modest, effect of treatment on the B(max) of [3H]MK-801 binding and the B(max) and K(D) of [3H]L-655,708 binding, i.e., PTZ-treated rats had fewer [3H]MK-801 and [3H]L-655,708 binding sites (NMDA and alpha5-containing GABA(A) receptors, respectively), but, these reductions were significant only in the relatively seizure-insensitive hooded Lister strain. This suggests that the increased susceptibility to kindling in Wistar rats is not directly related to alterations in the expression of NMDA or GABA(A) receptors.

Preferential coassembly of alpha4 and delta subunits of the gamma-aminobutyric acidA receptor in rat thalamus.

Pharmacological study of rat thalamic gamma-aminobutyric acidA (GABAA) receptors revealed the presence of two distinct populations, namely, diazepam-sensitive and diazepam-insensitive [3H]Ro15-4513 binding sites accounting for 94 +/- 2% (1339 +/- 253 fmol/mg protein) and 6 +/- 2% (90 +/- 44 fmol/mg protein) of total sites, respectively. Thalamic diazepam-insensitive sites exhibited a pharmacology that was distinct from diazepam-sensitive sites but comparable to that of the alpha4beta3gamma2 subtype of the GABAA receptor stably expressed in L(tk-) cells. Immunoprecipitation experiments with a specific anti-alpha4-antiserum immunoprecipitated 20 and 7% of total thalamic [3H]muscimol and [3H]Ro15-4513 sites, respectively. Combinatorial immunoprecipitation using antisera against the alpha4, gamma2, and delta subunit revealed that alpha4delta- and alpha4gamma2-containing receptors account for 13 +/- 2 and 8 +/- 3% of [3H]muscimol sites from thalamus, respectively. It also indicated that all delta subunits coexist with an alpha4 subunit in this brain region. In conclusion, our results show that in rat thalamus both alpha4betagamma2 and alpha4betadelta subtypes are expressed but alpha4betadelta is the major alpha4-containing GABAA receptor population.

Regional differences in the inhibition of mouse in vivo [3H]Ro 15-1788 binding reflect selectivity for alpha 1 versus alpha 2 and alpha 3 subunit-containing GABAA receptors.

The benzodiazepines flunitrazepam, diazepam, and Ro 15-1788 and the beta-carboline DMCM bind with equivalent affinity to the benzodiazepine binding site of GABAA receptors containing different alpha subunits (i.e., alpha 1, alpha 2, alpha 3, or alpha 5); whereas, the triazolopyridazine CL 218,872 and imidazopyridine zolpidem have higher affinity for alpha 1 subunit-containing GABAA receptors. In the present study, the in vivo binding of [3H]Ro 15-1788 in mouse cerebellum and spinal cord was used to establish the occupancy of the benzodiazepine binding site of GABAA receptors containing primarily alpha 1 and alpha 2/alpha 3 subunits, respectively. Thus, the nonselective compounds flunitrazepam, diazepam, and DMCM all produced a similar inhibition of binding in cerebellum and spinal cord (respective ID50 values of 0.2 to 0.3 mg/kg, 2 mg/kg, and 10 mg/kg i.p.); whereas, the alpha 1 selective compounds CL 218,872 and zolpidem were more potent at inhibiting [3H]Ro 15-1788 binding in the cerebellum (ID50 values 4.5 mg/kg and 10 mg/kg i.p.) compared to the spinal cord (ID50 values 12 mg/kg and > 30 mg/kg i.p.). Thus, the reduction of in vivo f[3H]Ro 15-1788 binding in tissues containing alpha 1 and alpha 2/alpha 3 receptor populations reflects the in vitro affinities of subtype selective compounds and should help to interpret the behavioral profile of such compounds.

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain.

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.

Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer.

Gamma-aminobutyric acid (GABA) dose dependently increased extracellular acidification rate in Ltk cells stably expressing human recombinant alpha1beta3gamma2 GABA(A) receptors but had no effect in non-transfected controls. Cells seeded at 1 x 10(5) cells/cup, with 4-5 days induction, had basal acidification rates of 105+/-2 microVs(-1) at 37 degrees C (mean+/-standard error of mean, n=37). GABA responses had a characteristic time-course with an initial alkalinisation followed by a peak of acidification, which was optimized by increasing agonist exposure from 15 s to 25-30 s. The maximum concentration of GABA tested (100 microM) produced a 40+/-2% increase over basal acidification rate (n=3), with an EC50 of 15.5 microM and a Hill slope of 1.5. Responses were specifically antagonized by bicuculline and could be modulated by benzodiazepine ligands with varying efficacies. Full benzodiazepine agonists flunitrazepam (1 microM) and zolpidem (10 microM) significantly potentiated the response to 10 microM GABA by 124+/-15% (n=7) and 117+/-23% (n=3), respectively. The partial agonist bretazenil (100 nM) produced a 45+/-13% (n=3) potentiation whilst the inverse agonist DMCM (10 microM) (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) inhibited the response to 20 microM GABA by 53+/-5%. The microphysiometer offers an alternative functional measure for GABA(A) receptors with the sensitivity to measure subtle modulatory effects of benzodiazepine site ligands and to determine their relative efficacy.