Rabbit Cerebellum Research Articles

Binding of dynorphin A and related peptides to κ- and μ-opioid receptors: sensitivity to Na + ions and Gpp(NH)p

We have examined the effects of Na + ions and 5′-guanylyl imidodiphosphate (Gpp(NH)p) on the equilibrium binding of dynorphin A and of a series of related agonist and antogonist peptides to κ- and μ-opioid receptors in guinea pig (κ) and rabbit (μ) cerebellum membrane preparations. The binding to κ sites of dynorphin A and of the peptides displaying agonist properties was strongly inhibited in the presence of 120 mM NaCl and 50 μM Gpp(NH)p. In contrast, a somewhat lower sensitivity to the inhibitory effect of the two allosteric effectors was observed for the analogues of the series showing antagonist properties. The same general behavior, but more marked, was observed at μ sites, at both μ- and κ-opioid receptors. The peptides had biochemical properties (binding sensitivity vs. insensitivity to sodium ions and guanine nucleotides) that correlated well with their biological activities (agonist vs. antagonist) previously determined in in vitro pharmacological bioassays.

Cellular localization of heat shock gene expression in rabbit cerebellum by in situ hybridization with plastic-embedded tissue.

The major cell types in rabbit cerebellum which engage in the expression of a heat shock gene (hsp70) after hyperthermia were identified. This required in situ hybridization on thin sections derived from plastic-embedded tissue. All classes of cerebellar neurons which were examined (Purkinje, granule, and stellate cells) responded by induction of hsp70 mRNA within 1 hr after hyperthermia. Prominent induction of hsp70 mRNA was also observed in oligodendroglia in the deep white matter.

Comparison of neuronal activity in the rabbit motor cortex and cerebellum during food-acquisition

Comparison of neuronal activity in the rabbit motor cortex and cerebellum during food-acquisition

Scavenger effect of elastase on calcifications in liver, kidney and central nervous system tissues of rabbits with atherosclerosis induced by cholesterol-rich diet.

The inhibitory effect of elastase on calcification in liver, kidney and the central nervous system (CNS) was studied in rabbits with atherosclerosis induced by a cholesterol-rich diet. A total of 25 male rabbits were divided into six groups. In the first three groups, rabbits received a standard diet, a 1.5% cholesterol-rich diet or a 1.5% cholesterol-rich diet with intraperitoneal elastase (450 IU/kg.day) for 3 months. In the second three groups, rabbits received a standard diet, a 0.67% cholesterol-rich diet or a 0.67% cholesterol-rich diet with intraperitoneal elastase (450 IU/kg.day) for 6 months. Tissue calcium levels were then determined by neutron activation analysis. Tissue lipid levels were also measured. Dietary cholesterol increased serum lipid levels, atherosclerotic lesions and calcium levels in the CNS, but not in the liver or kidney. Calcium levels in the cerebellum and spinal cord of rabbits receiving the 0.67% cholesterol-rich diet plus elastase were significantly lower than those of rabbits receiving the 0.67% cholesterol diet alone. Elastase had a gradual inhibitory effect on the increase in calcium levels in CNS of cholesterol-induced atherosclerotic rabbits, reduced the increase in serum lipids and decreased the incidence of atherosclerotic lesions.

The nature of memory

Memory, and its failings, are surely the most important factors in determining human behaviour. In humans and the higher animals the memory process is manifestly so complex as to defy direct experimental investigation. However, the snail Hermissenda crassicornis, with a nervous system many orders of magnitude simpler, has been shown to be capable of associative learning, and investigation of its cellular basis throws light on the mechanisms governing the memory process generally, and, more specifically, as now being revealed in the rabbit hippocampus and cerebellum.

N,N-diallyl-tyrosyl substitution confers antagonist properties on the kappa-selective opioid peptide [D-Pro10]dynorphin A(1-11).

1. In the search for kappa-opioid antagonists, we have designed two N,N-diallyl substituted analogues of the kappa-selective peptide [D-Pro10]dynorphin A (1-11)(DPDYN). In this study, we have examined (i) the binding properties of N,N-diallyl-DPDYN (analogue 1) and N,N-diallyl-[Aib2,3]DPDYN (analogue 2) at the three main types (mu, delta, kappa) of opioid binding sites, (ii) their binding sensitivity to Na+ ions (120 mM NaCl) and guanine nucleotide (50 microM Gpp(NH)p) at mu- and kappa-binding sites and (iii) their biological activity in two pharmacological bioassays specific for mu- and kappa-(guinea-pig ileum) and kappa-(rabbit vas deferens) opioid receptors. 2. Steric hindrance resulting from incorporation of two bulky allyl groups at the tyrosal nitrogen atom greatly altered the binding properties of DPDYN. A dramatic fall in apparent affinity for the three types (mu, delta, kappa) of site as well as selectivity for kappa-sites was observed for the two N,N-diallyl-substituted peptide analogues. 3. At kappa-sites of guinea-pig cerebellum and mu-sites of rabbit cerebellum, N,N-diallyl-substitution led to a complete loss of binding sensitivity to the inhibitory effect of 120 mM NaCl + 50 microM Gpp(NH)p compared to the high sensitivity of DPDYN. This may therefore suggest that the N,N-diallyl-DPDYN analogues are endowed with opioid antagonist properties. 4. No agonist activity of the analogues was observed in guinea-pig myenteric plexus and rabbit vas deferens organ preparations. In contrast, both of the diallyl-substituted peptides displayed similar antagonist properties against the kappa-agonist DPDYN in both preparations. In the guinea-pig ileum, the affinities of the antagonist peptides against the mu-agonist Tyr-D-Ala-Gly-MePhe- NH(CH2)20H(DAGOL) were approximately half that observed against DPDYN. 5. These results show that N,N-diallyl-tyrosyl substitution leads to analogues of DPDYN which act in vitro as pure opioid antagonists and exhibit a reasonable affinity at, but a weak selectivity for, the K-opioid receptors.

Parasagittal zonal pattern of olivo-nodular projections in rabbit cerebellum

Injection of horseradish peroxidase (HRP) into the nodulus of the rabbit retrogradely labeled cells in 5 subdivisions of the contralateral inferior olive. Localized HRP injections at each of the medial, intermediate and lateral parts of the nodulus revealed 6 longitudinal zones, two zones in each part, projected differentially from the 5 olivary subdivisions: (i) the medial-most zone projected from the β nucleus, (ii) the lateral zone of the medial part and (iii) the lateral zone of the intermediate part both from the dorsal cap, (iv) the medial zone of the intermediate part from the ventrolateral outgrowth, (v) the medial zone of the lateral part from the dorsomedial cell column, and (vi) the lateral-most zone from the rostrolateral part of the medial accessory olive. A complication is that the zones (v) and (vi) seemed to cover the dorsal lamina of the nodulus, but not the ventral lamina. The lateral part of the ventral nodulus seemed to be projected from the dorsal cap and may therefore be a lateral extension of zone (iv). There was an indication that the rostral-most area of the medial accessory olive also projects to the nodulus, but a specific receptive zone for this projection was unclear. The present results suggest that the small lateral area of the nodulus previously found to be involved in cardiovascular control is projected from the rostrolateral part of the medial accessory olive and/or the dorsomedial cell column.

Cell type-specific expression of protein kinase C isozymes in the rabbit cerebellum.

Monoclonal antibodies raised against rabbit brain protein kinase C were classified into at least three types, MC-1a, MC-2a, and MC-3a, which selectively interact with hydroxyapatite column chromatographically resolved isozymes type I, II, and III of protein kinase C, respectively, determined on the basis of immunoblotting experiments. Immunoprecipitating analysis revealed that MC-1a, -2a, and -3a interacted with protein kinase C expressed in the brain and MC-2a and -3a, but MC-1a immunoprecipitated the protein kinase from rabbit spleen. Immunocytochemical localization of the protein kinase isozymes in the rabbit cerebellum and spleen was studied using these monoclonal antibodies. Staining with MC-1a was restricted to Purkinje cells of the rabbit cerebellum, and there was no reaction product in the Purkinje cells with either MC-2a or MC-3a. Cells in the granular layer of the cerebellum were labeled with MC-2a, and oligodendroglia in the white matter of the cerebellum was stained with MC-3a. The rabbit spleen contained no product reacting with MC-1a, but MC-2a and MC-3a reacted with lymphocytes in the mantle zone and the periarterial lymphatic sheath of the white pulp, respectively. These results suggest that expression of the protein kinase C isozyme in the rabbit cerebellum may depend on the cell type.

Fine distribution of γ-aminobutyric acid, glutamic acid decarboxylase, and glutamic acid in the rabbit cerebellum

The fine distribution of GABA, glutamic acid decarboxylase, and glutamic acid within each layer of the rabbit cerebellar cortex was determined with microanalytical methods. The greatest glutamic acid decarboxylase activity and the highest GABA concentration were found in the Purkinje cell layer. In the distribution of GABA and glutamic acid decarboxylase the peak of glutamic acid decarboxylase activity was more pronounced than that of GABA; the concentration of glutamic acid did not show much variation between each layer.

Modulation of binding at opioid receptors by mono- and divalent cations and by cholinergic compounds.

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH4Cl and KCl, inhibit the equilibrium binding (25 degrees C) of the selective mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin, the selective delta-agonist [3H]-[D-Pen2,D-Pen5]enkephalin and the selective kappa-agonist [3H]-dynorphin A (1-9). Choline chloride inhibits the binding of the mu- and kappa-agonists but not of the delta-agonist; the choline derivative, methacholine, inhibits also the binding of the delta-agonist. Binding of the delta-agonist is potentiated by CaCl2, MgCl2 and MnCl2; these salts inhibit binding of the kappa-agonist. As far as binding of the mu-agonist is concerned, MgCl2 and MnCl2 may potentiate or inhibit whereas CaCl2 is only inhibitory. The binding of the mu-antagonist [3H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the mu-type; the binding of the mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the mu-antagonists [3H]-naloxone and [3H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the kappa-type; they are particularly effective for assays of kappa-receptors when the selective kappa-agonist [3H]-dynorphin A (1-9) is used as ligand.

Differences of binding characteristics of non-selective opiates towards μ and δ receptor types

[ 3H]ET (etorphine), which is considered either as an “universal” ligand or a μ agonist, interacts with identical affinities K D = 0.33 – 0.38 nM to hybrid cells and rabbit cerebellum, pure δ and μ-enriched opioid receptor preparations, respectively. In rat brain tissue, [ 3H]ET binding is inhibited by DAGO (Tyr-D-Ala-Gly-(Me)-Phe-Gly-ol), a μ selective agonist, in a competitive manner without apparent modification of the maximal number of sites. Furthermore, even at a DAGO concentration (300 nM) which should be sufficient to block [ 3H]ET interaction with μ sites, no variation in the total capacity of the tritiated ligand is observed. In contrast, DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), a δ-preferential agonist, blocks [ 3H]ET binding in rat brain at a concentration able to saturate δ-sites. At higher concentrations, where DTLET cross reacts with μ-sites, this ligand exhibits similar properties to those of DAGO. These data are very different from those obtained with [ 3H]EKC (ethylketocyclazocine), another “universal” ligand, the binding properties of which are easily explained by the occurence in rat brain tissue of independent sites exhibiting pharmacological profiles of μ, δ and κ sites. Our results underline the possible misinterpretation of binding data obtained by using [ 3H] etorphine as a non selective ligand.

Effects of cations on binding, in membrane suspensions, of various opioids at mu-sites of rabbit cerebellum and kappa-sites of guinea-pig cerebellum.

At the mu-sites of rabbit cerebellum, NaCl, LiCl, KCl, choline chloride and MnCl2 were tested for potentiation and inhibition of the binding of several opioids. Naloxone, (-)-bremazocine and diprenorphine are mu-antagonists in pharmacological assays and their binding is potentiated by the lower concentrations and inhibited by the higher concentrations of NaCl. The binding of the agonists [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]-dihydromorphine is inhibited. MnCl2 potentiates the binding of the agonist [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin but not the binding of the antagonists. The thresholds of inhibition and slopes of the dose-response curves for inhibition by MnCl2 and LiCl vary. This finding may indicate that potentiating effects of MnCl2 and LiCl are masked by simultaneous inhibition. At the kappa-sites of guinea-pig cerebellum, NaCl, KCl and MnCl2 inhibit the binding of [3H]-dynorphin A (1-8), [3H]-dynorphin A (1-9), [3H]-(-)-bremazocine, [3H]-tifluadom, and [3H]-diprenorphine. NaCl also causes a small potentiation of the binding of [3H]-diprenorphine, which is a kappa-agonist in the guinea-pig myenteric plexus but a kappa-antagonist in the rabbit vas deferens. The slopes of the inhibitory dose-response curves and the thresholds of inhibition vary with the different ligands. Therefore some potentiating effects may have been masked. The results support the view that NaCl, and perhaps LiCl, but not KCl and choline chloride, potentiate the binding of mu-antagonists but not the binding of mu-agonists. It is not yet possible to decide whether, at the kappa-site, there is a similar differentiation of the binding of agonists and antagonists.

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin (LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opiod binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.

Differential regulation of two molecular forms of a mu-opioid receptor type by sodium ions, manganese ions and by guanyl-5'-yl imidodiphosphate.

The rabbit cerebellum contains a very high proportion (up to 80%) of mu-opioid receptor sites (Meunier, J.C., Kouakou, Y., Puget, A. and Moisand, C., Mol. Pharmacol. 24, 23-29, 1983). A membrane fraction derived therefrom is labeled either with the opioid agonist, 3H-etorphine or with the opioid antagonist, 3H-diprenorphine, and solubilized with digitonin. Centrifugation of the soluble extracts in linear sucrose gradients reveals that bound 3H-etorphine sediments faster than does bound 3H-diprenorphine: 12S vs 10S. Pre-incubation of membranes and radioligand in the presence of 120 mM NaCl results in considerably decreased recovery of the 3H-agonist in 12S form while recovery of the 3H-antagonist in 10S form is substantially increased. The opposite situation is observed when the membranes have been prelabeled with radioligand in the presence of 1 mM MnCl2. Guanyl-5'-yl imidodiphosphate, a metabolically stable structural analog of GTP is found to selectively reduce recovery of labeled 12S receptors while it does not affect that of labeled 10S receptors. These data indicate that the mu-opioid receptor from rabbit cerebellum is capable of existing in two forms which differ in apparent molecular size: an "antagonist" (10S) form of apparent Mr approximately 230,000 which is stabilized in the presence of sodium ions and an "agonist" (12S) form of apparent Mr approximately 300,000 which, unlike the antagonist one, is sensitive to guanyl-5'-yl imidodiphosphate. It is thought that the form of larger apparent size represents the mu-opioid receptor associated with a guanine nucleotide regulatory protein.

Comparative aspects of cerebellar [ 3H]flunitrazepam and [ 3H]GABA binding

1. 1. [ 3H]Flu and [ 3H]GABA binding has been studied in mice and Rabbit cerebellum (Ce). 2. 2. The B max of [ 3H]Flu binding in Ce membranes is similar in all mice strains examined and Rabbit. However, in Ce homogenate there are significant differences in both k d and B max. 3. 3. Subcellular distribution shows higher [ 3H]Flu binding in the nuclear than in the membranous fraction. However, the [ 3H]GABA binding is lower in the nuclear than the membranous fraction.

Analysis of newborn rabbit cerebellum by monoclonal antibody

Analysis of newborn rabbit cerebellum by monoclonal antibody

Na + ions and Gpp(NH)p selectively inhibit agonist interactions at μ- and κ-opioid receptor sites in rabbit and guinea-pig cerebellum membranes

Rabbit and guinea-pig cerebellum membrane preparations contain a high proportion (> 80%) of μ- and of κ-opioid binding sites, respectively. These preparations were therefore used to compare the regulation of binding of μ- and of κ-opioid agonists and antagonists by sodium ions and by guanyl-5′-yl imidodiphosphate. We report here that Na + ions, Gpp(NH)p and most efficiently, the two agents in association selectively inhibited binding of opioid agonists not only in the μ preparation (rabbit cerebellum) but also in the κ preparaion (guinea-pig cerebellum). These allosteric effectors did not inhibit equilibrium binding of antagonists (naloxone, Mr 2266 or diprenorphine) in the two preparations. Taken together these results suggest that occupancy either of the μ-receptor by a μ-agonist or of the κ-receptor by a κ-agonist may be accompanied by similar if not identical molecular events. They also suggest a method to rapidly screen newly designed drugs as μ- or κ-opioid agonists or antagonists.

Glutamate decarboxylase activities in single vertebrate neurons.

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.

Opiate receptor binding profile in the rabbit cerebellum and brain membranes

The equilibrium dissociation constants ( K d ) and maximal binding capacities of tritiated dihydromorphine (DHM), [ d-Ala 2, d-Leu 5]enkephalin (DADLE), ethylketocyclazocine (EKC) and human β-endorphin (β-EP) in rabbit cerebellum and brain membranes have been investigated. Binding of tritiated DHM and DADLE was adequately described by a single affinity class of binding sites, while that of EKC required two affinity sites. Binding of tritiated β-EP was also consistent with a single affinity class of binding sites by Scatchard analysis, but inhibition of the binding with type selective opiate receptor ligands revealed multiple sites. Sequential displacement of a broad spectrum opiate ligand. diprenorphine (DIP), by type selective ligands showed that cerebellum membranes are relatively rich in μ (40%) and deficient in κ (12%) binding sites, while brain membranes are relatively rich in κ (32%) and deficient in μ (12%) binding sites. β-EP displaces 88 and 73% of tritiated DIP from cerebellum and brain membranes, respectively, suggesting multiple sites of β-EP binding.

Types of opioid receptors: relation to antinociception.

The endogenous opioid peptides are derived from three large precursors. Pro-opiocortin and proenkephalin yield [Met]enkephalin, carboxy-extended [Met]enkephalins and [Leu]enkephalin. The fragments of prodynorphin are all carboxy-extended [Leu]enkephalins. Three approaches are of importance for an analysis of the physiological functions of the different endogenous opioid peptides. First, since these peptides interact with more than one of the mu-, delta- and kappa-binding sites and thus with their receptors, it is necessary to synthesize peptides or non-peptides, which bind to only one of the sites. As far as narcotic analgesics are concerned, morphine fulfils these conditions since it interacts almost exclusively with the mu-receptor. Secondly, antagonists are required that are selective for only one of the opioid receptors, even when used in high concentrations. Finally, it is important to find circumscribed areas in the nervous system that possess only one type of opioid receptor. It is now known that in the rabbit cerebellum the opioid receptors are almost exclusively of the mu-type whereas in the guinea-pig cerebellum they are almost exclusively of the kappa-type.