Cerebellar Membrane Preparations Research Articles

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: Importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA A-receptor ligand [ 3H]TBOB to rat forebrain and cerebellar membrane preparations with IC 50 (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [ 3H]flunitrazepam and [ 3H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [ 3H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [ 3H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC 50 values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.

Pharmacological characterization and anatomical distribution of the dopamine transporter in the mouse cerebellum

We studied the binding parameters, the pharmacological profile and the anatomical distribution of the dopamine transporter in the mouse cerebellum by using the specific dopamine uptake antagonist [(3)H]GBR12935 and an antidopamine transporter monoclonal antibody. Competition experiments in cerebellar and striatal membrane preparations showed that [(3)H]GBR12935 binds to a specific binding site, sensitive to dopamine and low concentrations of mazindol. The affinity of dopamine for the cerebellar binding site was one order of magnitude lower than the affinity for the striatal binding site. Saturation experiments in cerebellar membrane preparations and thin frozen sections showed that the affinity of [(3)H]GBR12935 for this binding site is similar to its affinity for the striatal dopamine transporter. Saturable binding was lobule specific and in general was higher in the molecular layer compared to the granule cell layer. The immunohistochemical signal was mostly concentrated in the Purkinje cell layer and the cerebellar nuclei. The results suggest that the cerebellar dopamine transporter is similar but not identical to the striatal dopamine transporter and that it is present in the mouse cerebellum in a lobule and lamina specific pattern.

Separation of cannabinoid receptor affinity and efficacy in delta-8-tetrahydrocannabinol side-chain analogues.

1. The activities of a number of side-chain analogues of delta-8-tetrahydrocannabinol (Delta(8)-THC) in rat cerebellar membrane preparations were tested. 2. The affinities of each compound for the CB(1) receptor were compared by their respective abilities to displace [(3)H]-SR141716A and their efficacies compared by stimulation of [(35)S]-GTPgammaS binding. 3. It was found that the affinities varied from 0.19+/-0.03 nM for 3-norpentyl-3-[6'-cyano,1',1'-dimethyl]hexyl-Delta(8)-THC to 395+/-66.3 nM for 5'-[N-(4-chlorophenyl)]-1',1'-dimethyl-carboxamido-Delta(8)-THC. 4. The efficacies of these compounds varied greatly, ranging from the very low efficacy exhibited to acetylenic compounds such as 1'-heptyn-Delta(8)-THC and 4'-octyn-Delta(8)-THC to higher efficacy compounds such as 5'-(4-cyanophenoxy)-1',1'-dimethyl-Delta(8)-THC and 5'-[N-(4-aminosulphonylphenyl)]-1',1' dimethyl-carboxamido Delta(8)-THC. All agonist activities were antagonized by the CB(1)-selective antagonist SR141716A. 5. It was found that a ligand's CB(1) affinity and efficacy are differentially altered by modifications in the side-chain. Decreasing the flexibility of the side-chain reduced efficacy but largely did not alter affinity. Additionally, the positioning of electrostatic moieties, such as cyano groups, within the side-chain also has contrasting effects on these two properties. 6. In summary, this report details the characterization of a number of novel Delta(8)-THC analogues in rat cerebellar membranes. It provides the first detailed pharmacological analysis of how the inclusion of electrostatic moieties in the side-chain and also how alteration of the side-chain's flexibility may differentially affect a CB(1) cannabinoid receptor ligand's affinity and efficacy.

Evidence that the σ 1 receptor is not directly coupled to G proteins

Sigma (σ) receptors have been implicated in psychosis, cognition, neuroprotection, and locomotion in the central nervous system. The signal transduction mechanisms for σ receptors have not been fully elucidated. In this study, we examined the possible coupling between σ 1 receptors and heterotrimeric guanine nucleotide-binding proteins (G proteins) in rodent brain. In σ 1 receptor-rich cerebellar membrane preparations, the competitive binding curves of two σ 1 agonists, (+)pentazocine and 1 S,2 R-(−)- cis- N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737), were unaffected by the addition of 10 μM guanosine-5′- O-(γ-thio)-triphosphate (GTPγS). Neither (+)pentazocine (1–100 μM) nor BD737 (0.01–10 μM) stimulated GTPase activities significantly above basal levels in agonist-stimulated GTPase activity assays in cerebellar membranes. Furthermore, when using the method of agonist-stimulated [ 35S]GTPγS binding as assessed by autoradiography, we did not observe significant stimulation of [ 35S]GTPγS binding in rat brain sections by either (+)pentazocine or BD737. The above results demonstrate that the σ 1 receptor is not likely be directly coupled to G proteins.

A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4, 5)P3). Cellular levels of PtdIns(3,4,5)P3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32PO4, extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P3, are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P4. The latter is measured by its ability to displace [32P]Ins(1,3,4,5)P4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.

Ibuprofen inhibits the metabolism of the endogenous cannabimimetic agent anandamide.

A measure of the metabolism of anandamide, an endogenous cannabimimetic agent, by rat cerebellar membrane preparations was obtained by following the time-dependent reduction in potency of this compound towards inhibition of binding of the high-affinity cannabinoid agonist ligand [3H]WIN 55212-2 to cannabinoid receptors. Thus for example, incubation of the membranes with 100 nM anandamide for 0, 10 and 30 min. prior to addition of [3H]WIN 55212-2 and phenylmethylsulphonyl fluoride (to inhibit the activity of anandamide amidase, thereby blocking further anandamide metabolism during the binding assay) produced 57 +/- 3, 38 +/- 5 and 19 +/- 7% inhibition, respectively, of [3H]WIN 55212-2 binding. This time-dependent effect was blocked by ibuprofen but not by acetyl salicylic acid, sulindac, acetaminophen or to any significant extent by ketoprofen and naproxen. Preliminary experiments using a direct assay of anandamide amidase with [14C]anandamide as ligand gave an IC50 value for ibuprofen of approximately 400 microM. The potency of ibuprofen as an inhibitor of anandamide metabolism was of the same order of magnitude as required for inhibition of cyclooxygenase-2 in cell-free systems and of the peak plasma concentrations of this drug following a 2 x 200 mg dose regimen. It is concluded that following therapeutic doses of ibuprofen, the metabolism of anandamide may be affected.

Effects of the anticonvulsant losigamone and its isomers on the GABAA receptor system.

We conducted in vitro studies to clarify the possible involvement of GABAA receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+ losigamone) and AO-294 (- losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, < or = 100 microM losigamone did not affect the specific binding of [3H]GABA, [3H]flunitrazepam, or [35S]t-butyl-bicyclophosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10(-8)-10(-5) M, stimulated 36Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10(-5) M potentiated the effect of a suboptimal concentration of exogenous GABA 10(-5) M on 36 Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS-induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating 36Cl influx in the spinal cord neurons by a GABA-sensitive mechanism and at a side distant from the GABA channel.

Food restriction in female Wistar rats. II. β-adrenoceptor density in the cerebellum and in the splenic lymphocytes

The effect of diet restriction, applied on an every-other-day schedule from 3.5 months of age on, has been investigated on the β-adrenoceptor density in the cerebellum and in the splenic lymphocytes of old female Wistar rats. Comparing animals 6 months and 24 months old fed ad libitum, a 75% age-dependent reduction in specific binding of the agonist dihydroalprenolol was observed in cerebellar membrane preparations, while the β-adrenoceptor density of lymphocytes remained unaltered. Diet restriction induced a partial recovery of the age-related decrease of this parameter in the cerebellum without affecting the receptor density of lymphocytes. Present results suggest that undernutrition delays the appearance of those alterations related to aging.

β-Adrenergic receptor binding in brain of alcoholics

The binding of agonists and antagonists to β-adrenergic receptors in brain tissue obtained postmortem in nonalcoholic controls and matched intoxicated and sober alcoholics was measured to assess the state of the receptors and their coupling to adenylate cyclase. Binding of antagonist, iodocyanopindolol, to cerebral cortical and cerebellar membrane preparations was not different in alcoholics compared to that in controls, suggesting that the number of β-adrenergic receptors was not affected by chronic ethanol ingestion. Agonist binding data, however, indicated the loss of the high-affinity agonist binding state of the β-adrenergic receptor, representing the receptor-guanine nucleotide binding protein (G s) complex. Such changes were observed in cerebral cortex but not in cerebellum of intoxicated alcoholics. These data suggest that cerebral cortical β-adrenergic receptors are uncoupled from adenylate cyclase in these subjects. In cerebral cortical and cerebellar membranes of sober alcoholics both the high- and low-affinity agonist binding sites were observed. These findings are similar to those seen in animal studies and suggest that the effect of chronic ethanol ingestion on β-adrenergic receptor-adenylate cyclase coupling is brain region specific and reversible with abstinence. Ethanol-induced changes in the coupling of receptors to adenylate cyclase may contribute to the physiological and behavioral manifestations of alcohol abuse.

Sensitive and Specific Radioreceptor Assay for Calcitonin Gene‐Related Peptide

Abstract Calcitonin gene-related peptide is a 37-amino-acid neuropeptide which has a direct action on the heart and is an extremely potent vasodilator. Immunoreactive calcitonin gene-related peptide is present in plasma, cerebrospinal fluid and in other tissues. We have previously established the specific binding characteristics of this neuropeptide to the cerebellar membranes. This paper describes a sensitive, rapid and specific radioreceptor assay for the measurement of receptor-active calcitonin gene-related peptide in tissues and in plasma extracts using cerebellar membrane preparations as the binding agent. This assay should prove a valuable tool in studies of the physiology of this vasoactive neuropeptide and of its involvement in disease.

γ- l-Glutamyl- l-aspartate induces specific deficits in long-term memory and inhibits [ 3H]glutamate binding on hippocampal membranes

γ- l-Glutamyl- l-aspartic acid (γ-LGLA) has been isolated from Datura stramonium; its structure has been determined and it was then synthesized. In male Swiss mice intraperitoneal administration of the natural peptide (125 nmol/kg) or of the synthetic peptide (25–2500 nmol/kg) 24 h after acquisition of a Y-maze avoidance task induced a dose-dependent deficit in retention performance 48 h later. γ-LGLA had no effect on locomotor activity or emotional reactivity at the doses used. Separate or simultaneous administration of aspartate or glutamate (each at 250 nmol/kg) had no effect on learning retention, indicating that deficit induced by γ-LGLA was specific to the peptide. γ-LGLA impaired learning acquisition in a time-dependent manner when administered juom 3 min to 24 h post-training, but had no effect when administered 3 days following acquisition. γ-LGLA administered just after the training session did not affect retention performance during the first 3 h, but suppressed the retention improvement observed in control animals from 6 to 24 h after acquisition; this deficit was still evident 7 days after the treatment. γ-LGLA partially inhibited l-[ 3H]glutamate binding on crude hippocampal or striatal membrane preparations; this inhibition was not observed on cerebellar membrane preparations. These results suggest a specific action of γ-LGLA on excitatory amino acid systems which may be responsible for its effects on learning retention.

Alterations in cerebral glutamic acid decarboxylase and 3H-flunitrazepam binding during continuous treatment of rats for up to 1 year with haloperidol, sulpiride or clozapine.

Rats were treated continuously for 12 months with therapeutically equivalent doses of haloperidol (1.4-1.6 mg/kg/day), sulpiride (102-109 mg/kg/day) or clozapine (24-27 mg/kg/day) and examined for alterations in brain glutamic acid decarboxylase (GAD) and 3H-flunitrazepam binding. Administration of haloperidol, but not sulpiride or clozapine, for 6 or 12 months increased striatal GAD activity. None of the drug treatments altered nigral GAD activity when examined after 1, 3, 6, 9 or 12 months administration. The number of specific 3H-flunitrazepam binding sites (Bmax) in striatal membrane preparations were not altered by 12 months administration of haloperidol, sulpiride or clozapine. Surprisingly, Bmax for 3H-flunitrazepam binding to cerebellar membrane preparations was decreased by 12 months administration of all drug treatments. The dissociation constant (Kd) for 3H-flunitrazepam binding in striatal and cerebellar preparations was not altered. The ability of GABA (0.25-100 microM) alone, and in conjunction with sodium chloride (200 mM), to stimulate specific 3H-flunitrazepam binding in striatal and cerebellar preparations was unaltered by haloperidol, sulpiride or clozapine administration for 12 months. The selective effect of haloperidol, but not sulpiride or clozapine, treatment on striatal GAD activity parallels the ability of haloperidol, but not sulpiride or clozapine, to induce striatal dopamine receptor supersensitivity in the same animals. The actions of haloperidol may reflect its greater ability to induce tardive dyskinesia compared to sulpiride or clozapine.

Ivermectin interactions with benzodiazepine receptors in rat cortex and cerebellum in vitro.

The anthelmintic macrolide, ivermectin, enhances the binding of benzodiazepine agonist ( [3H]-diazepam) and antagonist ( [3H] beta-carboline ethyl ester) ligands to rat cortical and cerebellar membrane preparations. Enhancement of benzodiazepine agonist binding is partially additive with that of gamma-aminobutyric acid (GABA) and is inhibited by etazolate, bicuculline, and the steroid GABA antagonist R5135. Ivermectin-stimulated benzodiazepine antagonist binding is enhanced by bicuculline and inhibited by GABA and etazolate. The modulatory effects of bicuculline are chloride-dependent. The stimulatory effects of ivermectin, while quantitatively different in cortex and cerebellum, are qualitatively similar in both brain regions and are reduced in the presence of chloride. Ivermectin effects on benzodiazepine ligand binding to the benzodiazepine receptor complex and the differences in the effects of GABA, bicuculline, and R5135 on ivermectin-stimulated agonist and antagonist binding may provide evidence for distinct differences in the recognition sites for the two classes of benzodiazepine receptor ligand and their interactions with other components of the receptor complex.

Kainic acid receptor sites in the cerebellum of nervous, Purkinje cell degeneration, reeler, staggerer and weaver mice mutant strains

[ 3H]Kainic acid binding to crude cerebellar membrane preparations from mice nervous, Purkinje cell degeneration, reeler, staggerer and weaver mutant strains and their corresponding wildtype strains was determined. Specific kainate binding was strongly reduced in staggerer mutants, less in reeler and weaver mutation and was little affected by the nervous and Purkinje cell degeneration mutation. It is concluded that in the cerebellum kainate receptor sites are associated with the parallel fiber-Purkinje cell contacts and that kainate binding sites are most likely localized on the presynaptic side.