Action of pyrazolopyridines as modulators of [3H]flunitrazepam binding to the gaba/benzodiazepine receptor complex of the cerebellum

The pyrazolopyridines etazolate (SQ 20009) and cartazolate (SQ 65396) have strong modulatory effects on the GABA/benzodiazepine receptor complex of rate cerebellum. Thus, etazolate and cartazolate directly stimulate [3H]flunitrazepam binding (with EC50 values of 1.2 microM and 0.3 microM respectively) by increasing the apparent affinity of [3H]flunitrazepam for its binding sites. Stimulation of [3H]flunitrazepam binding by pyrazolopyridines is dependent on the presence of certain anions like chloride, bromide, iodide, nitrite, nitrate but not fluoride, acetate, formate or sulfate. If is inhibited by bicuculline-methiodide, and by the "chloride channel drugs' picrotoxinin and IPTBO. isoTHAZ, a GABA analogue with GABA antagonist properties in vivo, fails to inhibit binding stimulated by etazolate but antagonizes [3H]flunitrazepam binding stimulated by GABA. The pyrazolopyridines have also indirect effects on benzodiazepine receptor binding since they enhance the apparent sensitivity of those GABA recognition sites which are coupled to benzodiazepine binding sites. Thus, in the presence of 10 microM etazolate, GABA and muscimol enhance [3H]flunitrazepam binding, with EC50 values of 109 nM and 12 nM respectively. This sensitization effect is partially dependent on the presence of chloride ions. The pyrazolopyridines facilitate also the stimulation of benzodiazepine receptor binding by beta-alanine and taurine and by the rigid and flattened GABA analogues THIP and piperidine-4-sulfonic acid. Taken together, these results suggest that the pyrazolopyridines modulate [3H]flunitrazepam binding by acting at a site closely related to GABA receptor-regulated chloride ion channels.     

Preferential protection of cortical type II benzodiazepine receptors by γ-aminobutyric acid during heat inactivation

Pharmacological and biochemical evidence supports the existence of a heterogeneous population of benzodiazepine (BDZ) receptors. One major tool which has been used to identify distinct receptor subtypes is a novel series of triazolopyridazines (TPZ) that recognize two subpopulations (types I and II) of BDZ binding sites. Earlier studies demonstrated that γ-aminobutyric acid (GABA) enhanced specific BDZ binding and protected a fraction of BDZ binding sites against heat inactivation. GABA/BDZ interactions were further investigated by studying the properties of those BDZ binding sites that are thermostable in the presence of 1.0 mM GABA when cortical membrane fragments are heated at 60°C. Saturation isotherms of ³H-flunitrazepam (³H-Flu) binding revealed a 90% and 60% diminution of binding sites in the absence and presence of 1 mM GABA, respectively. In the presence of 1 mM GABA, displacement of ³H-Flu binding with varied concentrations of CL 218,872, a TPZ, in heated cortical membrane preparations indicated a significant increase in the IC₅₀ value, with no effect on the Hill coefficient when compared to parallel control experiments with unheated membranes. In contrast, no significant differences in either Hill coefficients or IC₅₀ values were obtained when similar displacement experiments were performed utilizing diazepam, an agent that does not distinguish between BDZ receptor subtypes. Hofstee plots of CL 218,872 displacement of ³H-Flu binding were curvilinear in both heated and unheated membranes and could be resolved into two components by computer analysis. The relative affinity of CL 218,872 at the high-affinity (K_D1) or lowaffinity (K_D2) recognition sites was unchanged after heating the membrane preparations. However, after heating, the density of type I receptors (B_max1) was reduced 75%, while the number of type II receptors (B_max2) was decreased only 38%. These results are indicative of a preferential GABA-mediated protective effect on the thermal inactivation of type II BDZ recognition sites.     

THIP and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding

The effects of THIP and isoguvacine on 3H-flunitrazepam binding to washed membranes prepared from the cerebral cortex of adult rats have been examined. THIP, which has only minimal stimulatory effects on benzodiazepine (BZ) receptor binding, has been found to inhibit the stimulation induced by small concentrations (2 microM) of exogenous GABA. While isoguvacine stimulates BZ receptor binding, although to a smaller extent than GABA, it also antagonizes the stimulation of BZ receptor binding induced by GABA. Thus THIP and isoguvacine exhibit the properties of a partial agonist of GABA-stimulated BZ receptor binding.     

gamma-Hydroxybutyric acid binding sites: evidence for coupling to a chloride anion channel

The effect of eight anions, including chloride, on the binding of gamma-hydroxy[2,3-3H]butyric acid (GHB) to synaptosomal membranes of rat and human brain was ascertained, as was the effect of a number of other allosteric modulators of the GABA/benzodiazepine/picrotoxin complex. All ions which were active at the chloride ion channel, inhibited the binding of [3H]GHB in a dose-dependent manner, with maximum inhibition of binding being 60% of 300 mM concentration of anion. Inactive ions in this binding system included sulfate, acetate and fluoride, all impermeable to the chloride ion channel. The inhibition of binding was temperature-dependent, being abolished at 37 degrees C and was independent of the cation used. The binding of [3H]GHB was also enhanced by pentobarbital, picrotoxin and diazepam but unchanged in the presence of GABA, muscimol, bicuculline, baclofen or strychnine. These data raise the possibility that the epileptogenic effect of GHB may be modulated by an action on the chloride ion channel, that is tightly coupled to the GABA/benzodiazepine/picrotoxin and/or GHB receptor complex.     

Enhancement of benzodiazepine binding by a diaryltriazine,LY81067

Ly81067, a diaryltriazine, represents a new class of compounds to enhance [³H]flunitrazepam binding to membranes of rat cerebral cortex. The enhancement induced by LY81067 exceeds that induced by γ-aminobutyric acid (GABA) and is partially abolished by the GABA antagonists bicuculline and picrotoxin. Scatchard analysis on the saturable binding of [³H]flunitrazepam reveals that LY81067 at 10 μM increases mainly the affinity for the [³H]ligand, due to a reduced rate of dissociation of the receptor-bound [³H]flunitrazepam. A dependence on chloride anions was demonstrated in the enhancement of [³H]flunitrazepam binding by LY81067. These findings suggest that the diaryltriazine LY81067 enhances [³H]flunitrazepam binding by exerting its effect at or near the picrotoxinsensitive anion recognition sites of the GABA/benzodiazepine receptor complex.     

Picrate and niflumate block anion modulation of radioligand binding to the gamma-aminobutyric acid/benzodiazepine receptor complex

The organic anions picrate (2,4,6-trinitrophenol) and niflumate (2-[[3-(trifluoromethyl)phenyl]-amino]-3-pyridinecarboxylate) were examined for their effects on radioligand binding to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Neither organic anion produced the enhancement of [35S] t-butylbicyclophosphorothionate (TBPS) binding characteristic of anions (such as Cl- and Br-) known to permeate GABA-gated chloride channels. However, both picrate and niflumate potently (IC50 values between 66 and 531 and 30 and 155 microM, respectively) inhibited the effect of 100-200 mM concentrations of anions (I-, Br-, Cl-, SCN-, and F-) to increase the binding of [35S]TBPS to GABA-gated chloride channels. This inhibition resulted from a decrease in both the maximum number of binding sites and the apparent affinity (increased Kd) of [35S]TBPS. Niflumate was consistently more potent than picrate, but both organic anions exhibited the same sequence of relative potencies against smaller anions (I- greater than Br- greater than Cl- greater than SCN- greater than F-). This sequence was similar to that described for the relative permeabilities of these anions through GABA-gated chloride channels. Niflumate and picrate were potent inhibitors of Cl-, but not GABA-modulated radioligand binding to benzodiazepine receptors. These findings suggest that picrate and niflumate bind with high affinity at or near an anion binding site that may regulate the movement of anions through GABA-gated chloride channels and radioligand binding at this "supramolecular complex."     

Correlation between the inhibitory activities of calcium entry blockers on vascular smooth muscle constriction in vitro after K+-depolarisation and in vivo afterα 2-adrenoceptor stimulation

Summary The acetylcholine (ACh) release was studied in superfused, electrically-stimulated slices of guinea-pig cerebral cortex.Muscimol and 4,5,6,7-tetrahydroisoxazolo (5-4-c)-pyridin-3-ol (THIP), as well as exogenous GABA, reduced the electrically-evoked ACh release and enhanced its spontaneous outflow. Picrotoxin antagonized these effects.In addition, picrotoxin and ethanolamine-O-sulphate (EOS) caused opposite changes in transmitter outflow, suggesting the existence of an endogenous GABAergic control on the cholinergic nerve endings. Neither flurazepam 6.6×10^–6–3.3×10^–5 mol/l nor diazepam 3.3×10^–6–3.3×10^–5 mol/l by themselves affected ACh release.Diazepam prevented GABA-, muscimol- and EOS-induced changes in spontaneous and 1 Hz-evoked outflow. Ro 15-1788 3.3×10^–6 mol/l abolished diazepam antagonism vs exogenous GABA.The ineffectiveness of flurazepam and diazepam on normal release (i.e. the lack of potentiation vs the endogenous GABAergic control) supports the view that synaptic GABA receptors acting upon the cholinergic nerve endings are not coupled with Benzodiazepine receptors.The unexpected diazepam antagonism vs exogenous GABA and GABA-like compounds can be explained with an unusual Diazepam negative cooperation with extrasynaptic GABA receptors, possibly present on the cholinergic terminals.Thus, the rule of benzodiazepine-GABA synergism does not seem always tenable, at least at certain pre-synaptic sites.     

3H-dopamine uptake and 3H-haloperidol binding in striatum after administration of methyl mercury to rats

Methyl mercury inhibits dopamine (DA) and serotonin (5-HT) uptake by brain synaptosomes and decreases antagonist binding to striatal dopaminergic D₂ receptors in vitro. To assess the effects in vivo, adult rats were given methyl mercury, either as a single dose (10 mg/kg by gavage) or a cumulative total dose of 50 mg/kg in 2 weeks. The repeated dosing decreased body weight and caused neuromuscular dysfunction. In spite of this overt toxicity, neither ³H-DA uptake nor ³H-haloperidol binding changed in striatal synaptosomes. There were no significant alterations in ³H-5-HT uptake by hypothalamic synaptosomes or ³H-flunitrazepam binding in cerebellar synaptosomes. The results suggest that monoaminergic synapses and the benzodiazepine binding sites, associated with cerebellar GABA receptors, remain functionally normal at doses of methyl mercury that are otherwise toxic. The results also emphasize the importance of due care when extrapolating cellular or biochemical data to the level of the whole organism.     

Paradoxical blockade of a muscimol response by thip, a GABA agonist and also by GABA-transaminase inhibitors

In a particular strain of mice, relatively large doses of muscimol caused myoclonic jerks of high frequency. This muscimol response was blocked in a dose-dependent manner by a γ-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol-hydrate (THIP). Studies using γ-acetylenic GABA and γ-vinyl GABA demonstrated that the blockade of the muscimol-induced jerks caused by these irreversible GABA-transaminase inhibitors closely paralleled the elevation of brain GABA level. It seems that, in this particular strain of mice, muscimol, or one of its metabolites, possibly acts on certain specific binding sites in the central nervous system, eliciting myoclonic jerks and that these receptor sites are different from those to which GABA or THIP binds.

Earlier studies demonstrated the value of the muscimol-induced myoclonic jerks as an animal model for postanoxic action myoclonus. Based on this finding, it is proposed that both THIP and the GABA-transaminase inhibitors might prove to be of value in the management of this clinical condition.     

The GABA/benzodiazepine receptor chloride channel complex during repeated episodes of physical ethanol dependence in the rat

The GABA/benzodiazepine (BZ) receptor chloride channel complex was investigated during repeated episodes of ethanol intoxication and withdrawal in the rat; the intragastric intoxication technique was applied and the severity of intoxication, withdrawal and number of seizures were recorded. The following groups were studied after decapitation during withdrawal 10–16 h after the last ethanol feeding: A) isocalorically fed controls not receiving ethanol; B) isocalorical controls subjected to a single ethanol intoxication period; C) animals subjected to 15 intoxication-withdrawal episodes (spontaneous seizures); D) same as C, but without developing seizures. A radio receptor technique was applied in the characterization of the receptor complex comprising specific binding to the BZ-receptor, the chloride channel and the GABA receptor by ³H-diazepam, ³⁵S-TBPS and ³H-muscimol, respectively. The allosteric couplings among the components of the receptor complex were studied by ³H-diazepam and ³⁵S-TBPS binding enhancement tests involving muscimol, ZK 93423 and DMCM. Cortex, hippocampus and cerebellum were the brain regions studied. Except for a reduced specific binding of ³H-diazepam in cerebellum, there were no indications of changes in specific binding to any part of the receptor complex. The allosteric coupling of BZ and GABA receptors as well as chloride channel-BZ receptors were unchanged in all groups. It is notable that no changes at all could be related to number of intoxication-withdrawal episodes or to the development of seizures. Thus, the present study gave no indication that the GABA/benzodiazepine receptor chloride channel complex is directly involved in the augmentation of cerebral nervous system excitability (seizures) during repeated episodes of physical ethanol dependence.     

Alpha noradrenergic receptors in brain membranes: Sodium,magnesium and guanyl nucleotides modulate agonist binding

Summary Binding of [³H]clonidine to alpha noradrenergic receptors in rat brain is inhibited by monovalent cations (Na⁺>Li⁺>K⁺), stimulated by magnesium ion and inhibited by guanyl nucleotides. In the presence of 1 mM EDTA the receptors bind tritiated clonidine in a noncooperative fashion at a single site with a K _a(association constant) of 0.12 nM^–1. In the presence of magnesium the affinity of the receptors increases by a factor of two (K _a=0.23 nM^–1). The increase of affinity is attributed to a two-fold decrease in the dissociation rate constant. In the presence of sodium ions the concentration of binding sites is not changed but Scatchard plots are now curvilinear indicating either heterogeneity of the receptors or negative cooperativity in ligand binding. This effect of sodium ions is not influenced by the presence of magnesium. The conversion into the sodium-liganded state is rapid; it is complete within 60 s at 30° C.The effects of the guanyl nucleotides on clonidine binding are complex: In the presence saturating concentrations of sodium ions they cannot inhibit clonidine binding except when free magnesium (>1 mM) is present. Without added sodium and in the presence of 1 mM EDTA the rank order of potencies is: GDPGTP>Gpp(NH)p. In the presence of 10 mM magnesium the rank order is reversed: Gpp(NH)p GTPGDP. The apparent affinity of the nucleotides for inhibition of clonidine binding is also changed by magnesium. The affinity of Gpp(NH)p increases about 100-fold by addition of magnesium ion.     

Facilitation of specific benzodiazepine binding in rat brain membrane fragments by a number of anions

The ability of eight anions, in the concentration range 0–200 mM, to facilitate specific [³H]-diazepam binding to well washed membrane fragments prepared from whole rat brain (less medullapons) was investigated. All the anions studied produced a significant increase in specific [³H]-diazepam binding, though to different degrees, with the exception of isethionate which showed no effect at any concentration investigated. Scatchard analysis revealed that Br^? (50 mM) produced a significant increase in the affinity of the receptor for [³H]-diazepam with no change in the total number of sites available. and that with the additional presence of GABA (10 μM), the affinity was further increased, again with no changes in the number of sites available. Attempts to correlate the facilitatory effects of these anions on specific [³H]-diazepam binding with their ability to modify cortical inhibitory postsynaptic potentials which are thought to be chloride mediated produced no significant correlation; neither was there an obvious correlation between the facilitatory effects of these anions and their chaotropic properties. We must therefore conclude that the facilitatory effects of these anions on specific [³H]-diazepam binding cannot be adequately explained either by an intimate linkage between the benzodiazepine receptor and a chloride channel or by the chaotropic properties of these anions alone.     

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.     

Barbiturates allosterically inhibit GABA antagonist and benzodiazepine inverse agonist binding

Barbiturates and the related depressant drugs, etazolate and etomidate, inhibited both the binding of [3H]bicuculline methochloride (BMC) to gamma-aminobutyric acid (GABA) receptor sites and the binding of [3H] beta-carboline-3-carboxylic acid methyl ester (beta CCM) to benzodiazepine receptor sites in mammalian brain. These concentration-dependent effects were chemically specific and stereospecific in a manner correlating with the activity of barbiturates to enhance GABA responses in neurons and to enhance GABA and benzodiazepine receptor agonist binding in vitro. The barbiturate inhibition of [3H]BMC binding involved a decrease in affinity which at high concentrations of barbiturates results in an effective complete loss of detectable binding. The maximal inhibition of [3H] beta CCM binding involved a more modest decrease in affinity (increase in KD from 1.35 to 1.85 nM). The barbiturate inhibitions of both ligands could be reversed by picrotoxin, suggesting an indirect action at previously defined picrotoxin/barbiturate modulatory sites on the GABA-benzodiazepine receptor/chloride ion channel complex.     

3H-ACh release from guinea pig gallbladder evoked by GABA through the bicuculline-sensitive GABA receptor

Summary We investigated the effect of GABA on the spontaneous efflux of ³H-acetylcholine (ACh) from the isolated guinea pig gallbladder loaded with ³H-choline. Application of GABA (10^–5 M) caused a significant increase in the fractional rate of tritium efflux. This GABA-evoked efflux of ACh was inhibited by the perfusion of tetrodotoxin (10^–6 M) and Ca-free medium. Nipecotic acid (10^–4 M) did not affect the GABA-evoked release of ACh, indicating that ACh was not released by the entry of GABA into cholinergic nerve terminals. Bicuculline (10^–6 M) and furosemide (10^–6 M), the chloride ion channel blocker, inhibited the GABA-evoked ACh release. The application of muscimol (10^–5 M), but not baclofen (10^–5 M) also produced an increase in the fractional rate of ACh release. Thus, the GABA receptors involved in the increase of ACh release are bicuculline-sensitive. The GABA-evoked release of ACh was not altered by the perfusion with hexamethonium (10^–5 M), thus indicating the presence of GABA receptors on the postganglionic cholinergic neurons.These findings suggest that bicuculline-sensitive GABA receptors probably coupled to a Cl^– ionophore are present on postganglionic cholinergic neurons and are involved in the increase of ACh release in guinea pig gallbladder.This paper is part of a dissertation submitted by N. Saito to Kobe University School of Medicine, for the requirement of Doctor of Philosophy     

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.     

Pyrazolopyridines: Effect of structural alterations on activity at adenosine- and GABAA-receptors

A series of 4-substituted 1H-pyrazolo[3,4-b]pyridine- 5-carboxylic acid derivatives related in structure to the putative anxiolytics cartazolate, tracazolate, and etazolate were assessed for affinity at A₁- and A_2A-adenosine receptors and at GABA-, benzodiazepine-, and picrotoxinin- binding sites of the GABA_A-receptor-channel. None of the derivatives had markedly greater affinity at A₁-receptors than cartazolate (K_i-0.5 μM), but many had markedly lower affinity than cartazolate (K_i-1.5 μM) at A_2A-receptors. At the benzodiazepine-binding site of GABA_A-receptors some of the derivatives enhanced [³H]diazepam binding, as did cartazolate and GABA, some had no effect and some inhibited binding. Most of the derivatives inhibited binding of the benzodiazepine-antagonist [³H]Ro 15-1788. At the GABA-binding site, only a few of the derivatives inhibited binding of the antagonist [³H]SR-95531, as did GABA. At the picrotoxinin-binding site, many inhibited binding of [³⁵S]TBPS, but none were as potent as cartazolate or GABA. Analysis of the interactions indicates that stimulation of [³H]diazepam binding is allosteric and results from binding of the pyrazolopyridine at the GABA site or a subdomain of that site, while inhibition of [³H]Ro 15-1788 binding is competitive and due to binding at the benzodiazepine site. Inhibition of [³⁵S]TBPS binding at the picrotoxinin-channel site appears to be allosteric through the GABA site and/or by direct competition at the channel site. Alterations in structure markedly alter the affinities of pyrazolopyridines at such sites on the GABA_A-receptor-channel. Drug Dev. Res. 42:41–56, 1997. © 1997 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Parallel changes in the sensitivity of gamma-aminobutyric acid and noradrenergic receptors following chronic administration of antidepressant and GABAergic drugs. A possible role in affective disorders

Chronic treatment with the antidepressants imipramine or nomifensine, or the gamma-aminobutyric acid (GABAergic) agents, baclofen or THIP, produced a decrease in the Bmax for binding sites of GABAergic and noradrenergic receptors. Chronic treatment with imipramine or nomifensine produced a decrease in the Bmax of both binding of [3H]dihydroalprenolol and [3H]GABA receptors in the cerebral cortex and hippocampus. Chronic treatment with baclofen or THIP also produced a decrease in the Bmax of binding of [3H]dihydroalprenolol receptor in the cerebral cortex and hippocampus. These results suggest a possible link between the GABAergic and noradrenergic systems, which may be important in understanding the mechanism of action of antidepressant drugs, and suggests a possible role for GABA in affective disorders.     

A steroid derivative,R 5135, antagonizes the GABA/benzodiazepine receptor interaction

A novel steroid derivative, R 5135 (3α-hydroxy-16-imino-5β-17-aza-androstan-11-one) showed high affinity for both the GABA and glycine receptors in vitro. It also displaced [³H]diazepam from the benzodiazepine receptor in a rat cortex membrane preparation, but in this case a plateau occurred in the displacement curve at a concentration of R 5135 between 10^?7 and 10^?6 M, where binding was decreased by about 50 %. The “high affinity” component of R 5135 inhibition no longer appeared when the endogenous GABA concentration was reduced by extensive washing of the membrane preparation and it reappeared when GABA was re-introduced. Thus R 5135 behaves as a powerful antagonist of the GABA stimulation of [³H]diazepam binding, being 500 times more active than the GABA-antagonist bicuculline. The dual interaction between R 5135 and GABA and glycine receptors suggests that these may share some common structural feature or that they have overlapping specificity.     

3H-Flunitrazepam binding to bovine retina and the effect of GABA thereon

Bovine retinae were examined for their ability to bind ³H-flunitrazepam. Whole retinal homogenates revealed specific, high affinity, and saturable ³H-flunitrazepam binding (K_d, 1.1 nM; B_max, 19.5 fmoles/mg tissue). In washed membrane preparations, 10 μM GABA caused a dramatic decrease in the K_d (42 percent). The rank order of potency for the inhibition of ³H-flunitrazepam binding by different benzodiazepines was: clonazepam > clobazam ? Ro 5-4864. These results are supportive for the presence of benzodiazepine receptors in the bovine retina and indicate a likeness with the previously described benzodiazepine receptors of mammalian brain.