The development of type I and type II benzodiazepine receptors in the mouse cortex and cerebellum

The postnatal development of benzodiazepine (BDZ) receptors was monitored in Heterogeneous Stock (HS) mice, and the BDZ receptors were characterized and categorized into Type I and Type II receptors. When the number of 3H-Flu binding sites (Bmax) was assessed at weekly intervals after the birth of the animal, the number of sites in both the cortex and cerebellum increased significantly if the data was expressed as fmol/mg tissue. On the other hand, no significant change in 3H-Flu binding sites was evidenced in the cortex, and the number of 3H-Flu binding sites in the cerebellum decreased during postnatal development if Bmax values were expressed as fmole/mg protein. When receptor binding data was analyzed for the presence of Type I and Type II BDZ receptors, the changes in KD values for 3H-Flu binding development could be accounted for by changes in relative proportions of Type I and Type II receptors present in the cortex and cerebellum during the maturation process. Type II receptors predominated in both cortex and cerebellum at birth, and Type I receptors proliferated primarily during the first two weeks of postnatal life. In the cortex of adult mice there were approximately equal numbers of Type I and Type II BDZ receptors. In the cerebellum of adult mice, computer assisted analysis of binding data could not distinguish the presence of two distinct BDZ binding sites. However, Hill coefficients and overall binding constants determined from data on CL-218,872 displacement of 3H-Flu binding to cerebellar membranes indicated that cerebellar tissue from adult mice did contain a heterogeneous array of BDZ receptors.     

The separation of 3H-Benzodiazepine binding sites in brain and of benzodiazepine pharmacological properties

In addition to anxiolytic and anticonvulsant properties, benzodiazepines (BDZ) produce sedation, ataxia, and muscular relaxation. In general, it was difficult to separate these properties within this chemical class during the search for clinically useful anxiolytics; and when BDZ's were used to characterize ³H-BDZ binding sites they indicated only a single homogenous class of receptors. A new chemical series was discovered, triazolopyridazines (TPZ, prototype CL 218,872), which showed anticonflict activity in rats and monkeys without sedation or ataxia and inhibited ³H-BDZ binding in brain membranes with kinetic characteristics suggesting the presence of multiple BDZ receptors. High affinity and low affinity sites for the TPZ were demonstrated, the former designated at Type 1 and the latter as Type 2. Anatomical and in vivo studies have supported different distributions of each receptor in brain. Lately, the physical separation of discrete proteins which bind ³H-BDZ has been reported. The multiple receptors and the variety of endogeneous substances which have been proposed as modulators and ligands of the receptors might explain variability as well as selectivity in pharmacological properties in these drugs.     

Characteristics of type 1 and type 2 benzodiazepine receptors in the ovine brain

Analysis of the displacement of 3H-diazepam binding to membranes prepared from the ovine frontal cortex by the triazolopyradiazine CL218,872 yielded a Hill coefficient significantly below unity. By analogy with similar studies of this drug in rat brain this suggested the existence of Types 1 and 2 benzodiazepine receptors. The degree of displacement of 3H-diazepam by CL218,872 (200 nM, Type 1; 800 nM, Type 2) in homogenates of brain regions differed, the rank order being cerebellum greater than parietal cortex greater than frontal cortex congruent to temporal cortex congruent to hippocampus greater than striatum. Displacement of 3H-diazepam by CL218,872 was enhanced by 10(-5) M GABA in the striatum (at 200 nM and 800 nM CL218,872) and cerebellum (at 200 nM CL218,872). Benzodiazepine receptors in the ovine frontal cortex were least sensitive to CL218,872 (200 nM) in young fetuses (54-68 days gestation) and achieved adult levels of sensitivity by late gestation. Finally, the potency of CL218,872 to displace 3H-diazepam was not effected by the 3H-ligand concentration (0.5 nM or 5.0 nM), suggesting that Types 1 and 2 benzodiazepine receptors are not identical to the high and low affinity 3H-diazepam binding sites we have previously identified in the ovine brain.     

[3H]CL 218,872, a novel triazolopyridazine which labels the benzodiazepine receptor in rat brain

We examined the specific binding of [³H]CL 218,872, a novel triazolopyridazine to benzodiazepine receptors in the rat cerebral cortex. Two binding sites with K_D values of about 10–30 nM and 200–600 nM were demonstrated. A number of benzodiazepine anxiolytics inhibited [³H]CL 218,872 binding in a manner which correlates with the potency of these drugs for inhibiting [³H]benzodiazepine binding. Our initial studies show that [³H]CL 218,872 can label the benzodiazepine receptors and may prove to be a useful probe for studying benzodiazepine heterogeneity and regulation.     

A synthetic non-benzodiazepine ligand for benzodiazepine receptors: A probe for investigating neuronal substrates of anxiety

CL 218,872 is the first non-benzodiazepine to selectively displace brain specific ³H-diazepam binding with a potency comparable to that of the benzodiazepines. Like the benzodiazepines, CL 218,872 increased punished responding in a conflict situation and protected against the convulsions induced by pentylenetetrazole. These three pharmacological properties are highly predictive of anxiolytic activity. Unlike the benzodiazepines, however, CL 218,872 was relatively inactive in tests designed to measure effects on neuronal systems which utilize GABA, glycine and serotonin as transmitters. Furthermore, CL 218,872 was relatively free of the ataxic and depressant side effects commonly associated with the benzodiazepines. Because of this high degree of selectivity, CL 218,872 may represent a new probe for investigating neuronal substrates of anxiety.     

Brain uptake and distribution of the potential memory enhancer CL 275,838 and its main metabolites in rats: relationship between brain concentrations and in vitro potencies on neurotransmitter mechanisms

The kinetics, brain uptake and distribution of CL 275,838, a potential memory enhancer, and its main metabolites (II and IV) were evaluated in rats after intraperitoneal doses of 5, 10 and 20 mg/kg. Brain maximum concentrations (C_max) of the three compounds after pharmacologically active doses were then related to the in vitro concentrations affecting some monoaminergic and amino acid receptor sites to examine the relative importance of these neurotransmitter systems in the pharmacological actions of CL 275,838. After 10 mg/kg CL 275,838, the unchanged compound rapidly entered the brain and distributed almost uniformly in various regions inside the blood-brain barrier. Its disappearance from brain and plasma was almost parallel with a comparable elimination half-life (t_1/2) of about 2 h. Metabolite II entered the brain and equilibrated with plasma more slowly than the parent compound, achieving mean C_max (0.2 µM) within 3 h of dosing. Metabolite IV was rapidly detected in rat brain but hardly amounted to 10% (0.1 µM) of the parent compound C_max (1 µM). There was a linear relationship between dose and plasma and brain concentrations of the three compounds up to 20 mg/kg CL 275,838. At micromolar concentrations the parent compound had affinity for serotonin (5-HT) uptake sites, 5-HT₂ and dopamine (DA₂) receptors. Only at much higher concentrations than those achieved in vivo after pharmacologically active doses did it increase the binding of³H-glutamate to NMDA (N-methyl-d-aspartate) receptors. Metabolite II had a similar neurochemical profile. Metabolite IV had no affinity for these neurotransmitter systems, except for benzodiazepine (BDZ) receptor sites where it interacts with micromolar affinity behaving, however, as an agonist as determined by the GABA ratio. Although the effect on NMDA is compatible with favourable effects on memory, it is unlikely that it is involved in the ability of CL 275,838 to attenuate the impairment of a passive avoidance task caused by drugs or aging in rats.     

Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors

Brain-specific binding sites have been isolated on synaptosomal membrane fragments which recognize pharmacologically active benzodiazepines (BDZ's) and triazolopyridazines (TPZ's). While early evidence indicated the existence of a single homogeneous class of BDZ binding sites, more recent biological and pharmacological studies support the notion of BDZ receptor multiplicity. We now propose that two biochemically distinct BDZ receptors exist in brain which are responsible for the mediation of different pharmacological activities. Type I BDZ receptors display a high affinity for both BDZ's and TPZ's, are not coupled to GABA receptors or to chloride ionophores, and are the sites which mediate anxiolytic actions. Type II BDZ receptors display a high affinity for BDZ's, display a low affinity for TPZ's, are coupled to GABA receptors and/or chloride ionophores, and are the sites which mediate pharmacological effects other than anxiolytic activity.     

Biochemical evidence that 2-phenyl-4[(4-piperidinyl) ethyl]quinoline, a quinoline derivative with pure anticonflict properties, is a partial agonist of benzodiazepine receptors

The atypical profile of 2-phenyl-4[2-(4-piperidinyl) ethyl]quinoline (PK 8165), a quinoline derivative with pure anticonflict properties, seems to be due to the fact that this compound is a partial agonist of benzodiazepine receptors. The drug PK 8165 is a competitive inhibitor of benzodiazepine binding sites with a Hill coefficient near unity. Opposite to 3-methyl-6-(3-trifluoromethylphenyl)2,4-triazolo(4,5-b)pyridazine (CL 218,872) it was unable to discriminate between BZ1 and BZ2 receptors in sections of brain. However, modulation by gamma-aminobutyric acid (GABA) and the effect of photolabelling by flunitrazepam on the affinity of PK 8165 indicated that GABA or photolabelling shifts of PK 8165 were between full agonists and antagonists. By itself PK 8165 was unable to modify the levels of cGMP in the cerebellum, but potentiated the lowering of levels of cGMP by diazepam and did not present antagonistic properties of this effect.     

Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA_A) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [³H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [³H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC₅₀ = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [³H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA_A receptors containing α6 and δ subunits. In contrast, [³H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC₅₀ ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [³H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [³H]EBOB binding with EC₅₀ ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC_50,1 ~ 16 nM and EC_50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABA_A receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA_A receptor populations.     

Generalization between benzodiazepine- and triazolopyridazine-elicited discriminative cues

Using a milk reinforced two-lever operant procedure, rats were trained to discriminate 3 mg/kg chlordiazepoxide (CDP) from saline. Following this, generalization experiments were conducted with the triazolopyridazine CL 218,872, a synthetic non-benzodiazepine (BDZ) ligand for the BDZ receptor. CL 218,872 produced CDP lever selection in a dose related fashion and thus generalized to the standard CDP treatment. However, this generalization was antagonized by the concurrent administration of pentylenetetrazol or amphetamine, but not by strychnine or bicuculline. Also, there was evidence for cross tolerance for a sedative effect between CDP and CL 218,872.     

Hypothalamic GABAA receptor blockade modulates cerebral cortical systems sensitive to acute stressors

Pharmacologic blockade of GABA binding sites in the hypothalamus elicits a pattern of physiological and behavioral arousal. The latter outcome implicates a perturbation in the neural functioning of higher brain centers. The effect that hypothalamic GABA_A receptor modulation has on the function of cerebral cortical neural substrates linked with responses to stressors was assessed using microinfusion of bicuculline methiodide (BMI) into the medial hypothalamus of freely moving, handling habituated rats. BMI led to rapid increases in frontal cortical dopamine (DA) utilization (calculated from the sum of the levels of the DA metabolites, homovanilic and dihydroxyphenylacetic acids, divided by DA levels) resembling that identified following restraint-induced stress. Also, cortical GABA_A receptor function [using chloride (Cl^–) enhancement of³H-flunitrazepam (Flu) binding as an index] was disrupted; i.e. there was a loss of typical Cl^– enhancement of³H-Flu binding in animals after BMI infusions. However, placing animals in restraint after BMI infusion reversed the effects of BMI, with both DA utilization and Cl^– facilitated³H-Flu binding similar to control basal values. Muscimol infusions in separately prepared animals did not alter either frontal cortical DA utilization or GABA_A receptor function. The present results implicate GABA in the hypothalamus as gating activity of cortical systems involved in sensation of and/or responses to stressors. These findings may have important implications for effects of autonomic arousal on neural substrates involved in mediating stress responses.     

Comparison of the effects of chlordiazepoxide and CL 218,872 on serum corticosterone concentrations in rats

Fifteen minute exposure to a novel environment plus 120 dB sound stimulation produced a three-fold increase in serum corticosterone concentrations in rats. A low dose of intraperitoneally (IP) administered chlordiazepoxide (CDP) (5 mg/kg) attenuated this response, whereas a higher dose (20 mg/kg) elevated corticosterone concentrations in rats not subjected to sound stress. Parallel results were obtained after intracerebroventricular (ICV) drug administration, with a low dose of CDP (5 g) reducing the sound stress response and higher doses (25 and 50 g) increasing corticosterone concentrations in unstressed animals. Thus, despite the presence of benzodiazepine (BDZ) receptors at every level of the hypothalamic-pituitary-adrenocortical axis, it appears that BDZs alter the activity of this system via an interaction with BDZ receptors in brain. CL 218,872 (2.5–20 mg/kg), a novel non-BDZ anxiolytic compound, did not attenuate the corticosterone elevation produced by sound stimulation, and also failed to alter baseline corticosterone concentrations in unstressed animals. The fact that CL 218,872 is a selective agonist for brain Type I BDZ receptors suggests that BDZs are not influencing corticosterone secretion through an interaction with this BDZ receptor subtype. Furthermore, these results indicate that stress (as measured by pituitary-adrenocortical activation) can be dissociated from anxiety (as measured by conflict paradigms), thus challenging the validity of the corticosteroid stress test as a screening procedure for anxiolytic activity.     

Solubilization and characterization of the cholecystokininB binding site from pig cerebral cortex

Cholecystokinin (CCK) binding sites were solubilized from pig cerebral cortical membranes with digitonin (2%, w/v) in the presence of Na⁺ (120 mM) and Mg²⁺ (5 mM). Scatchard plot transformation of equilibrium binding data obtained with ¹²⁵I-CCK-8S gave an apparent dissociation constant (K_d) of 0.6 nM, comparable to that obtained in membranes in the presence of these cations. Hill coefficients close to unity suggested the presence of a single population of receptor sites. Competitive inhibition studies with pentagastrin, gastrin(1–17)S and the CCK_A receptor antagonist L-364,718 indicated that the solubilized receptor sites were of the B-type (CCK_B), with the same pharmacological profile as that observed in membranes. Optimal specific binding of ¹²⁵I-CCK-8S to membrane-bound and solubilized receptors was obtained in the presence of divalent cations. Both the membrane-bound and the solubilized receptor activity were attenuated by guanylyl-imidodiphosphate (Gpp(NH)p) indicating that the brain CCK_B receptors are coupled to G proteins.     

Is upregulation of benzodiazepine receptors a compensatory reaction to reduced GABAergic tone in the brain of stressed mice?

Effects of various forms of stress on the GABA_A receptor-chloride ionophore complex in the brain of NMRI mice were investigated. Male albino mice were subjected to stress by placing them on small platforms (SP; 3.5 cm diameter) surrounded by water for 24 h. This experimental model contains several stress factors like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. As additional stress control groups we used animals subjected to isolation, large platform (9.0 cm diameter) and repeated swimming stress. SP stress induced an increase in the number of cortical benzodiazepine (BDZ) receptors and a reduction in the GABA-stimulated ³⁶C1^– uptake by brain microsacs, whereas none of these changes could be observed in animals exposed to isolation, swimming or large platform stresses. Furthermore, the amount of GABA-induced stimulation of [³H]flunitrazepam binding was reduced in cortical brain membranes of SP-stressed animals, an effect due to fact that these animals dispayed an increase in the basal [³H]flunitrazepam binding, whereas the absolute level of maximally enhanced BDZ binding in the presence of GABA did not differ from those found in controls. Neither basal [³H]muscimol binding or thiopentone sodium-induced stimulation of [³H]flunitrazepam binding were changed in any group of stressed mice. It is proposed that the observed upregulation in the number (B _max ) of cortical BDZ receptors in SP-stressed mice may represent a compensatory response to a stress-induced attenuation of GABAergic neurotransmission.     

Certain GABA mimetics reduce the affinity of anions required for benzodiazepine binding in a GABA-reversible way

Binding of ³H-flunitrazepam to specific sites on rat-brain membranes is anion-dependent. In the absence of added ions, binding is reduced to less than 10% of maximum, and eight anions (Tris salts) were found to increase binding in a concentration-dependent and saturable manner with affinity constants (Kd) ranging from 0.75 mM (phosphate) to 4.5 mM (chloride). The GABA mimetic THIP (200 μM) increases these Kd values from three- to 10-fold, depending on the anion. The GABA mimetics isoguvacine and piperidine-4-sulfonic acid (P4S) increase the Kd values for chloride ion eight- to 10-fold, while amino-propanesulfonate and imidazole acetate produce smaller (2- to 4-fold) increases. These effects of THIP on anion affinities are reversed in a competitive manner by GABA and muscimol. Although there is no correlation between the ability of the anions to enhance ³H-flunitrazepam binding and their ability to substitute for chloride ion electrophysiologically, it seems possible that the ions bind to sites similar to the chloride channels associated with GABA receptors. All benzodiazepine receptors are probably coupled indirectly to GABA receptors through anion binding sites.     

Effects of Ethanol on Recombinant Rat GABAA Receptors: [35S]t-Butylbicyclophosphorothionate ([35S]TBPS) Binding Study

Abstract: To determine the roles of the alternatively spliced short and long forms of the γ2 subunit in the effect of ethanol on the GABA_A receptor function, picrotoxin-sensitive [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding was studied in recombinant rat α1β2γ2 and α6β2γ2 receptors expressed in human embryonic kidney 293 cells. Ethanol (10–500 mM) in the absence of added GABA had only minor effects on [³⁵S]TBPS binding irrespective of the γ2 splice variant, its effects being greater in α6β2γ2 than in α1β2γ2 receptors. Ethanol (100 mM) decreased the binding in all four subunit combinations at various concentrations of GABA, again an effect independent of the γ2 variant. The two γ2 variants had different effects on GABA modulation of the binding, with the long γ2 variant decreasing the efficiency of GABA inhibition in α6β2γ2 receptors and enhancing the biphasic GABA stimulation and inhibition in α1β2γ2 receptors. The findings confirm the importance of the α subunits in the allosteric interactions between the convulsant binding site and other effector sites, which can be modified only to a minor extent by the type of the γ2 splice variant.     

Benzodiazepine receptors: preferential stimulation of Type 1 receptors by pentobarbital

The binding of [3H]flunitrazepam (FLU) to Type 1 and Type 2 benzodiazepine receptors in rat cerebellum and cerebral cortex was differentiated by the addition of 200 nM CL218,872 which preferentially displaces [3H]FLU from Type 1 receptors. Type 1 but not Type 2 receptor binding was significantly stimulated by 1 mM sodium pentobarbital.     

Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies

The binding characteristics of [³H]gabapentin, the radiolabelled analogue of the novel anticonvulsant gabapentin (1-(aminomethyl)cyclohexaneacetic acid) were studied using purified synaptic plasma membranes prepared from rat cerebral cortex. In 10 mM HEPES buffer [³H]gabapentin bound to a single population of sites with high affinity (K_D = 38 ± 2.8 mM) with a maximum binding capacity of 4.6 ± 0.4 pmol/mg protein, reaching equilibrium after 30 min at 20°C. This novel site was unique to the central nervous system with little or no specific [³H]gabapentin binding being measurable in a range of peripheral tissues. Binding was potently inhibited by a range of gabapentin analogues and 3-alkyl substituted γ-aminobutyric acid (GABA) derivatives although GABA itself and the selective GABA_B receptor ligand baclofen, were only weakly active. Gabapentin itself (IC₅₀ = 80 nM) and 3-isobutyl GABA (IC₅₀ = 80 nM) which also has anticonvulsant properties, showed the highest affinity for the binding site. Of a wide range of other pharmacologically active compounds only the polyamines spermine and spermidine influenced [³H]gabapentin binding, with both compounds producing a maximum of 50% inhibition of specific binding. Magnesium ions produced a similar pattern of inhibition but the effect of the polyamines and magnesium ions were not additive. The data provide evidence for the existence in brain of a novel binding site that may mediate the anticonvulsant effects of gabapentin and other potential anticonvulsant compounds.     

Pharmacology of Morphine and Morphine-3-glucuronide at Opioid,Excitatory Amino Acid,GABA and Glycine Binding Sites

Abstract: Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of ³H-MK801 (non-competitive antagonist) and ¹²⁵I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of ³H-muscimol (GABA receptor agonist), ³H-diazepam and ³H-flunitrazepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABA_A, receptors. Mor-phine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of ³H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors.