Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.     

Autoradiographic Analysis of GABAA Receptors in μ-Opioid Receptor Knockout Mice

Anatomical evidence indicates that γ-aminobutyric acid (GABA)-ergic and opioidergic systems are closely linked and act on the same neurons. However, the regulatory mechanisms between GABAergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are changes in GABA_A receptors in mice lacking μ-opioid receptor gene. The GABA_A receptor binding was carried out by autoradiography using [³H]-muscimol (GABA_A), [³H]-flunitrazepam (FNZ, native type 1 benzodiazepine) and [³⁵S]-t-butylbicyclophosphorothionate (TBPS, binding to GABA_A-gated chloride channels) in brain slices of wild type and μ-opioid receptor knockout mice. The binding of [³H]-FNZ in μ-opioid receptor knockout mice was significantly higher than that of the wild type controls in most of the cortex and hippocampal CA1 and CA2 formations. μ-Opioid receptor knockout mice show significantly lower binding of [³⁵S]-TBPS than that of the wild type mice in few of the cortical areas including ectorhinal cortex layers I, III, and V, but not in the hippocampus. There was no significant difference in binding of [³H]-muscimol between μ-opioid receptor knockout and wild type mice in the cortex and hippocampus. These data indicate that there are specific regional changes in GABA_A receptor binding sites in μ-opioid receptor knockout mice. These data also suggest that there are compensatory up-regulation of benzodiazepine binding site of GABA_A receptors in the cortex and hippocampus and down-regulation of GABA-gated chloride channel binding site of GABA_A receptors in the cortex of the μ-opioid receptor knockout mice.     

Effects of pentobarbital tolerance to and dependence on GABAB receptor binding

Effects of pentobarbital pellet implantation on [³H]baclofen binding in the frontal cortex of cerebellum of rat brains were examined. In the frontal cortex, pentobarbital tolerance caused an increase in the number of binding sites (B_max) without changing their affinity (K_D). Twenty-four hours after withdrawal of the pentobarbital pellets, there was a significant increase in the K_D and B_max values. Cerebellar binding, in contrast, was not significantly changed in any of the treatment groups. Addition of 1 mM of pentobarbital directly to binding assays using cortical membrane produced as increase in K_D without a change in B_max.In vitro, pentobarbital affected neither the K_D nor the B_max in the cerebellar [³H]baclofen binding. These results suggest that like the GABA_A receptor, [³H]baclofen binding to the GABA_B receptor in rat frontal cortex was affected by pentobarbital tolerance and dependence, and that there are regional differences in the properties of the GABA_B receptor.     

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.     

Benzodiazepine receptor sites in the chick optic lobe: Development and pharmacological characterization

To investigate the, interaction between -aminobutyric acid (GABA) and benzodiazepine (BZD) receptor sites during development, the time-course of appearance of flunitrazepam (FNZ) binding sites and their pharmacological characterization were studied in developing chick optic lobe. At the earliest stage examined, embryonic day (Ed) 12, the receptor density was 30.9 % (0.05±0.01 pmol/mg protein) of that found in the chick optic lobes of adult chicks. The adult value was achieved on Ed 16 (0.16±0.01 pmol/mg protein). After this stage there was a sharp and transient increase in specific [³H]FNZ binding of about two-fold reaching a maximal value between hatching and the postnatal day (pnd) 2 (0.33±0.01 pmol/mg protein). Scatchard analysis at different stages of development revealed the presence of a single population of specific FNZ binding sites. The increase in [³H]FNZ binding during development was due to a large number of binding sites while their affinity remained unchanged. Competition experiments in the chick optic lobe revealed that the order of potency for displacement of specific [³H]FNZ binding paralleled the pharmacological potency of the BZDs tested. The IC₅₀ s for clonazepam, flunitrazepam, Ro 15-1788 and chlordiazepoxide were 3.02, 4.30, 0.32, and 4778.64 nM respectively. Ro 5-4864, a potent inhibitor of BZD binding to peripheral tissues, had no effect on specific [³H]FNZ binding indicating that only central BZD binding sites are present in the chick optic lobe. The peak of maximal expression of BZD receptor sites precedes in 5–6 days the peak of GABA receptor sites indicating a precocious development of BZD receptor sites. The different appearance of both peaks may represent important events during development probably related to synaptogenesis.     

Changes in [3H]Forskolin Binding to Adenylate Cyclase and [3H]Phorbol Dibutyrate Binding to Protein Kinase C in Pentobarbital Tolerant/Dependent Rats

These studies were designed to examine the effect of chronic administration of pentobarbital on activity of adenylate cyclase (AC) and protein kinase C (PKC) in the rat brain by autoradiography. Recently, it has been suggested that the phosphorylation of specific proteins may be involved in the development of physical dependence. An experimental model of barbiturate tolerance and dependence was developed using i.c.v. infusion of pentobarbital (300 g/ 10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]forskolin binding were elevated (28–67%) in cortex, thalamus, dentate gyrus, hippocampal CA3 and cerebellum of the pentobarbital withdrawal animals, while these changes were not observed in tolerant rats. The levels of [³H]phorbol dibutyrate binding were highly elevated (38–65%) in the region of cortex, caudate putamen, septum, thalamus, dentate gyrus, and cerebellum of rats withdrawal from pentobarbital. These results show that the levels of AC and PKC were significantly elevated in pentobarbital withdrawal rats, and suggest that the levels of AC and PKC are altered in a region-specific manner during pentobarbital withdrawal.     

Effects of pentobarbital tolerance and dependence on convulsant and GABAA receptor antagonist binding.

Experiments were performed which examined the effects of pentobarbital tolerance and dependence on GABAA receptor antagonist binding. In rats implanted with pentobarbital pellets for 7 days, followed by 24 hours of withdrawal, there was a significant decrease in the latency of TBPS-induced seizures and an increase in [35S]TBPS binding in the frontal cortex. The pentobarbital tolerant rats had a significant increase in the low affinity KD of [3H]SR95531 binding. Removal of the pellets for 24 hours caused a reversal of the effect on the low affinity KD and caused a decrease in the number of low affinity binding sites. In vitro addition of pentobarbital to binding assays produced a decrease in the number of high affinity [3H]SR95531 binding sites without changing low affinity binding. In the cerebellum, the binding in none of the treatment groups was significantly different from placebo. These observations suggest that pentobarbital tolerance and withdrawal cause changes in the properties of the GABAA receptor antagonist binding site which are different from those caused by in vitro exposure to the drug.     

Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [³H]quinuclidinyl benzilate, for muscarinic M₁ receptors with [³H]pirenzepine, for muscarinic M₂ receptors with [³H]oxotremorine-M, and for nicotinic receptors with (–)-[³H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [³H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [³H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients B_max values for specific [³H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)-[³H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [³H]oxotremorine-M and (–)-[³H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [³H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.     

Bicuculline Up-Regulation of GABAA Receptors in Rat Brain

Effects of acute and subacute administration of bicuculline on [3H]muscimol, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to various brain regions were studied in Sprague-Dawley rats. Acute administration of bicuculline affected neither the KD nor the Bmax of the three receptor sites. In rats treated subacutely with bicuculline (2 mg/kg, i.p., daily for 10 days), [3H]muscimol binding was increased in the frontal cortex, cerebellum, striatum, and substantia nigra. Scatchard analysis revealed that subacute treatment of rats with bicuculline resulted in a significantly lower KD of high-affinity sites in the striatum and in a significantly lower KD of high- and low-affinity sites in the frontal cortex. In the cerebellum, two binding sites were apparent in controls and acutely treated animals; however, only the high-affinity site was defined in subacutely treated animals, with an increase in the Bmax value. Triton X-100 treatment of frontal cortical membranes eliminated the difference in [3H]muscimol binding between control and subacute bicuculline treatments. On the other hand, [3H]muscimol binding was significantly increased in the cerebellum from bicuculline-treated animals even after Triton X-100 treatment. The apparent Ki of bicuculline for the GABAA receptor was also decreased in the frontal cortex and the striatum following the treatment. However, subacute administration of bicuculline affected neither the KD nor the Bmax of [3H]flunitrazepam and [35S]TBPS binding in the frontal cortex and the cerebellum. These results suggest that GABAA receptors are up-regulated after subacute administration of bicuculline, with no change in benzodiazepine and picrotoxin binding sites.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Role of NMDA Receptors in Pentobarbital Tolerance/Dependence

Effects of continuous pentobarbital administration on binding characteristics of [³H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300g/10l/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [³H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10l/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [³⁵S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

[3H]Propyl β-Carboline-3-Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass

Abstract: Ethyl β-carboline-β-carboxylate (β-CCE) is a mixed-type inhibitor of [³H]flunitrazepam ([³H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [³H]Propyl β-carboline-3-carboxylate ([³H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [³H]PrCC was used as radioligand. However, we observed that [³H]PrCC labelled only 57% of [³H]FNM binding sites in rat hippocampus (B_max values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [³H]FNM binding was inhibited by β-CCE indicate that β-CCE and some other β-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [³H]PrCC. We conclude that [³H]PrCC at low concentration (0.3–0.4 × 10^-9 M) labels a subclass of benzodiazepine receptors, BZ₁, while another class, BZ₂ receptors, are not labelled by [³H]PrCC when filtration assays are used. By parallel determinations of the proportion between [³H]FNM and [³H]PrCC binding we calculated the percentage of BZ₁ receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Diazepam-Sensitive Specific Binding of Phenytoin in Rat Brain

Abstract

[³H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with K_d values of 32 ± 5 nM and 8.5 ± 1.1 μM, respectively. Similar enhancement of [³H]phenytoin binding was observed when diazepam was replaced by Ro 5–4864 (4″-chlorodiazepam) which is selective for the ‘peripheral’ type benzodiazepine binding sites. In contrast, neither the ‘central’ type receptor selective agonist clonazepam nor the antagonist Ro 15–1788 enhanced [³H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the ‘peripheral’ type binding site for its selective affinity for Ro 5–4864. However, judging from the micromolar concentrations required for the enhancement of [³H]phenytoin binding, they appear unlikely to be the same ‘peripheral’ type binding sites as measured by [³H]Ro 5–4864 binding (K_d approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

Regionally Selective and Age-Dependent Alterations in Benzodiazepine Receptor Binding in the Genetically Dystonic Hamster

Abstract: Previous pharmacological studies have indicated that impairment of GABAergic transmission may be involved in the pathophysiology of dystonia in the mutant dt^sz hamster, i.e., a genetic animal model for idiopathic dystonia. In the present experiments, the kinetic constants of [³H]flumazenil binding to the benzodiazepine site of the GABA_A receptor were calculated from equilibrium binding measurements in various brain regions of genetically dystonic hamsters and age-matched controls. Because dystonia in mutant dt^sz hamsters is transient and disappears after ～60–70 days of age, [³H]flumazenil binding was studied at the age of maximum severity of dystonia (30–40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In mutant hamsters with the maximum severity of dystonia, receptor affinity of [³H]flumazenil was increased in olfactory bulb,striatum, tectum, and cerebellum, as exemplified by significantly decreased dissociation constants (K_D) in these regions. An increased number of binding sites (B_max) were seen in striatum and frontal cortex but not in the other eight regions studied in this regard. All these changes in [³H]flumazenil binding disappeared in parallel with dystonia, implicating a causal relationship between altered benzodiazepine receptor binding and dystonia in mutant dt^sz hamsters. In view of the antidystonic effect of benzodiazepines, such as diazepam, and recent neurochemical findings indicating impaired function of the GABA-gated Cl^? channel in dystonic hamsters, the present data might be interpreted as up-regulation of benzodiazepine receptors in response to impaired GABAergic function. Furthermore, the present data represent the first evidence that GABA_A receptors are altered in the basal ganglia in idiopathic (primary)dystonia.     

Rat retinal benzodiazepine receptors are controlled by visual cortical mechanisms

[³H]flunitrazepam binding was assayed in retinae of 25-day-old rats subjected either to unilateral enucleation at day 15, to intracranial unilateral cutting of the optic nerve at day 17, or to unilateral ablation of the visual cortex at day 17 postnatally.Unilateral enucleation resulted in an enhanced [³H]flunitrazepam binding in the retina of the remaining eye by 23% (P < 0.002, two-tailed Student t-test) as compared to unoperated controls.In rats with one optic nerve cut shortly before the optic chiasm, benzodiazepine binding in the retina of the lesioned side was significantly higher by 20.4 ± 7.6% (P < 0.02, N = 10, paired test) in comparison to that in the retina with the intact optic nerve.Unilateral visual cortex ablation resulted in a 13% decrease (P < 0.02) in [³H]flunitrazepam binding in the retina contralateral to the brain lesion.In the lesioned rats of all three groups, the retinal benzodiazepine receptors were no longer capable of being modified by light/dark adaptation as is observed in normal rats. Our data suggest that (i) rat retinal benzodiazepine receptors are under a control from the visual cortex, and (ii) the benzodiazepine receptors of both eyes seem to be mutually tuned, presumably via a cortico-retinal feedback loop and an interhemispheric cortico-cortical information transfer.     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.