Development of beta-adrenergic receptors in normal and mutant mouse cerebellum

[3H]-dihydroalprenolol was used to examine the development of beta-adrenoceptors in the cerebellum of weaver, reeler, staggerer, and jimpy neurologically mutant mice and their normal counterparts. In normal animals the greatest increase in [3H] binding occurred during the postnatal interval of 10-15 days, and maximum values were obtained at about 3 weeks. Binding was saturable with an apparent dissociation constant of 0.26 nM, and the affinity of [3]-dihydroalprenolol for its receptor did not change significantly during development. [3H]-dihydroalprenolol binding was significantly reduced in weaver, reeler, and staggerer (14-22% of control values) at 20 days, but not in the jimpy mutant. These results are discussed in relation to the ontogeny of beta-adrenoreceptors with the known noradrenergic innervation of the cerebellum, and to the paucity of both granule neurons and myelination which occurs in the neurological mutants.     

Neuronal localization of cannabinoid receptors in the basal ganglia of the rat

Cannabinoid receptors have recently been characterized and localized using a high-affinity radiolabeled cannabinoid analog in section binding assays. In rat brain, the highest receptor densities are in the globus pallidus and substantia nigra pars reticulata. Receptors are also dense in the caudate-putamen. In order to determine the neuronal localization of these receptors, selective lesions of key striatal afferent and efferent systems were made. Striatal neurons and efferent projections were selectively destroyed by unilateral infusion of ibotenic acid into the caudate-putamen. The nigrostriatal pathway was selectively destroyed in another set of animals by infusion of 6-hydroxydopamine into the medial forebrain bundle. After 2- or 4-week survivals, slide-mounted brain sections were incubated with ligands selective for cannabinoid ([3H]CP 55,940), dopamine D1 3H]SCH-23390) and D2 ([3H]raclopride) receptors, and dopamine uptake sites ([3H]GBR-12935). Slides were exposed to 3H-sensitive film. The resulting autoradiography showed ibotenate-induced losses of cannabinoid, D1 and D2 receptors in the caudate-putamen and topographic losses of cannabinoid and D1 receptors in the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata at both survivals. Four weeks after medial forebrain bundle lesions (which resulted in amphetamine-induced rotations), there was loss of dopamine uptake sites in the striatum and substantia nigra pars compacta but no change in cannabinoid receptor binding. The data show that cannabinoid receptors in the basal ganglia are neuronally located on striatal projection neurons, including their axons and terminals. Cannabinoid receptors may be co-localized with D1 receptors on striatonigral neurons. Cannabinoid receptors are not localized on dopaminergic nigrostriatal cell bodies or terminals.     

Functional localization of cannabinoid receptors and endogenous cannabinoid production in distinct neuron populations of the hippocampus

The possible localization of cannabinoid (CB) receptors to glutamatergic and GABAergic synaptic terminals impinging upon GABAergic interneurons in the CA1 region of the rat hippocampus was examined using the electrophysiological measurement of neurotransmitter release in brain slices. Whereas activation of cannabinoid receptors via the application of the cannabinoid agonist WIN55,212-2 significantly and dose-dependently reduced evoked IPSCs recorded from interneurons possessing somata located in the stratum radiatum (S.R.) and stratum oriens (S.O.) lamellae, evoked glutamatergic EPSCs were unaffected in both neuronal populations. However, in agreement with previous reports, WIN55,212-2 significantly reduced EPSCs recorded from CA1 pyramidal neurons. Additional experiments confirmed that the effects of WIN55,212-2 on IPSCs were presynaptic and that they could be blocked by the CB1 receptor antagonist SR141716A. The involvement of endogenous cannabinoids in the presynaptic inhibition of GABA release was also examined in the interneurons and pyramidal cells using a depolarization-induced suppression of inhibition (DSI) paradigm. DSI was observed in CA1 pyramidal neurons under control conditions, and its incidence was greatly increased by the cholinergic agonist carbachol. However, DSI was not observed in the S.R. or S.O. interneuron populations, in either the presence or absence of carbachol. Whereas DSI was not present in these interneurons, the inhibitory inputs to these cells were modulated by the synthetic cannabinoid WIN55,212-2. These data support the hypothesis that cannabinoid receptors are located on inhibitory, but not excitatory, axon terminals impinging upon hippocampal interneurons, and that CA1 pyramidal neurons, and not interneurons, are capable of generating endogenous cannabinoids during prolonged states of depolarization.     

Observations on the cerebellum of normal-reeler mutant mouse chimera

The normal-reeler chimera mouse (+/+ in equilibrium with rl/rl) provides an experimental system in which an analysis of the migration of immature neurons in the cerebellum can be accomplished. In the present study, five chimera mice were produced from embryos of the wild-type control (C57Bl/6N) and the reeler mutant mouse (BALB/c) by the aggregation technique. The isozyme pattern of glucosephosphate isomerase (GPI) revealed that the brain tissue in the chimera contained both isozymes of the BALB/c (reeler) and C57Bl/6N (normal) strains, implying that internal mosaicism of the cerebellum truly existed. We found no abnormality in the cerebellum of the chimera mouse: the neuronal and glial subpopulations revealed no difference from those of the control. Such normalization of the cerebellum in the chimera suggests that the disturbance of neuronal migration in the reeler is attributable to an abnormal cell-to-cell interaction between migrating young neurons and the radial glial cells.     

Neuropathological study on cerebellum of macular mutant mouse heterozygote

The hemizygote of the macular mutant mouse is clinically, biochemically and neuropathologically similar to a patient with Menkes kinky hair disease. The heterozygote of this mutant mouse was biochemically and neuropathologically examined. The copper content in the brain decreased in comparison with that in the normal littermate, although it was more than that in the hemizygote. In the Golgi study, abnormal Purkinje cells with somal sprouts, thick stem dendrites and dendritic focal swellings, which were seen in the hemizygote, were not observed in the heterozygote. Ultrastructurally, abnormal mitochondria were seen in the Purkinje cells in the anterior and middle cerebellar lobe of the heterozygote. Histochemically, cytochrome c oxidase activity decreased, especially at the anterior lobe in the cerebellar cortex of the heterozygote. This activity, as indicated by staining intensity, was in between that in the normal littermate and that in the hemizygote. The heterozygote did not show a mosaic pattern in the distribution of these neuropathological changes, although this mutant mouse shows x-linked recessive inheritance. Thus, our results lead to the conclusion that the neuropathological changes observed in this mutant mouse do not result directly from an abnormal gene in the Purkinje cell, but from the secondary effects subsequent to presumptive copper deficiency.     

Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus

The expression of CB2 cannabinoid receptors (CB2-Rs) in the brain and their neuronal function has now attracted research interest, since we and others have demonstrated the presence of CB2-Rs in neuronal and glial cells in the brain. In this study, we show the subcellular distribution of CB2-Rs in neuronal, glial, and endothelial cells in the rat hippocampus using immunohistochemical electron microscopy. Brain sections from the hippocampus were immunolabeled for CB2-R, visualized, and analyzed by electron microscopy. We found that in neurons, CB2-R immunoreactivity is present in the cell body as well as in large and medium-sized dendrites. In the soma, the CB2-R labeling is associated with the rough endoplasmic reticulum and Golgi apparatus demonstrating that CB2-Rs are synthesized by hippocampal neurons. CB2-R labeling in dendrites was observed in the cytoplasm and associated with the plasma membrane near the area of synaptic contact with axon terminals indicating a postsynaptic distribution of these receptors. In CB2-R immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. These results provide the first ultrastructural evidence that CB2-Rs are mainly postsynaptic in the rat hippocampus.     

Cytoarchitectonic abnormalities in hippocampal formation and cerebellum of dreher mutant mouse.

The laminated structures in the hippocampal formation and cerebellum of homozygous dreher mice were compared to their littermates and to C57BL/6J mice in Nissl- and myelin-stained preparations. In the dreher dentate gyrus, ectopic granule cells were situated in the molecular layer, and frequently there was either partial or complete absence of the infrapyramidal limb of the granule cell layer. In the dreher hippocampus, the cells of the pyramidal cell layer in area CA3 formed widely dispersed arrangements, and there were ectopically situated pyramidal cells in the stratum radiatum and stratum oriens. In the dreher cerebellum, 3 abnormal patterns were observed: (1) disruptions of foliation with normal cytoarchitectonic structure, (2) foliation with a mixture of normal laminated structure and abnormal laminated structure, and (3) almost complete absence of the cerebellum. In abnormal folia exhibiting the second or third pattern, islands consisting of agglomerations of both granule cells and Purkinje cells or just granule cells were observed. The neuronal heterotopias and cytoarchitectonic disorganization observed in the present study are apparently secondary to disruption of cell proliferation and neuronal migration produced directly or indirectly by the dreher mutation. In addition, the fact that the phenotypic abnormalities in homozygous dreher mice produces different abnormal morphologies in different specimens may be useful for analyzing the development of the hippocampal formation and cerebellum.     

Binding and uptake of putative neurotransmitters in mutant mouse cerebellum and cerebellar reaggregat

γ-Amino butyric acid (GABA) appears to be the inhibitory transmitter of various cerebellar interneurons, while glutamate has been suggested as the excitatory transmitter of granule cells. In this study we have determined the developmental profile from day 2 to day 60 for the Na^a-independent (presumably post-synaptic receptor) binding of [³H]GABA (K_d = 34 nM) and [³H]kainic acid, a glutamate analog (K_d = 53 nM. Both binding activities increase steadily to adult levels with kainic acid achieving this more rapidly. Cerebellar cells (7 day) cultured as reaggregates for 14 days showed an increase in GABA binding and glutamate uptake compared to the starting tissue but did not achieve the levels seen in vivo, while kainic acid binding and GABA uptake remained low. Binding and uptake measurements were performed in neurological mutants. Agranular mutants, weaver, reeler and staggerer demonstrated as expected, marked decreases in total GABA binding and glutamate uptake into synaptosomes, and to a lesser extent GABA uptake. In addition total kainate binding was depressed. In comparison, Purkinje cell degeneration mutants showed only a small decrease in kainic acid binding. These results suggest that kainic acid binding does not identify the post-synaptic glutamate receptor.     

Catecholamines in mutant mouse cerebellum: fluorescence microscopic and chemical studies.

Catecholamine-containing fibers have been examined in the cerebella of normal and hypocerebellar mutant mice using Falck-Hillarp and glyoxylic acid histofluorescence techniques. The amounts of norepinephrine and dopamine were also determined chemically in the same mutants. Green fluorescent fibers in cerebella of normal mice are similar in size and distribution to those described in the rat. Weanling and adult weaver, reeler and staggerer mice all manifest greatly increased specific catecholamine fluorescence per unit area in cerebellar cortex, but the patterns of fluorescent fibers are distinctive. In weaver, the fibers are of normal diameter, surround Purkinje cell bodies and appear to climb along major dendrites. In reeler, similar fibers form a plexus around cortical and non-cortical Purkinje cells; relatively normal fluorescent fiber patterns are present in well-organized cortical regions, while stouter disoriented fibers course through the shallow molecular layer in disorganized regions. Staggerer cerebellar cortex exhibits the greatest fluorescence with most terminals appearing as matted tangles adjacent cell bodies. Clearly defined fibers, however, can be distinguished in the molecular layer running perpendicular to the pia or for long distances in the coronal plane parallel to the pia. The major catecholamine determined chemically is norepinephrine. Reeler cerebella contain normal absolute levels and a doubled concentration of norepinephrine. In contrast, and despite the fluorescence findings, the total norepinephrine content of weaver and staggerer cerebella is significantly reduced and concentrations are not significantly different from normal.     

Saxitoxin-sensitive Na channels: presynaptic localization in cerebellum and hippocampus of neurological mutant mice

The autoradiographic distribution of saxitoxin (STX) binding sites associated with voltage-sensitive Na⁺ channel was studied in the cerebellum of neurological weaver (wv/wv), Purkinje cell degeneration (pcd/pcd), nervous (nr/nr) and reeler (rl/rl) mutant mice. The Purkinje cell layer contains the highest density of STX binding sites in normal mice. High densities were observed in the molecular layer. Intermediate and very low densities were present in the granular layer and the white matter respectively. There was an important decrease of grain density in the molecular layer and Purkinje cell layer ofwv/wv cerebellum, where a large majority of granular cells had disappeared. Inpcd/pcd andnr/nr mutants, a small decrease was observed in Purkinje cell layer where the Purkinje cells had almost all degenerated. Inrl/rl mutants where all neuronal cells were malpositioned, the compacted molecular layer contained an increased STX binding sites density. Conversely the labelling of Purkinje cells areas was decreased. The hippocampal formation ofrl/rl mutants presents an homogeneous repartition of the Na⁺ channel protein in contrast with the laminated distribution observed in normal mice. Our autoradiographic data suggest that a major proportion of STX-sensitive Na⁺ channels are localized in parallel fibers of granular cells and in axons of basket cells in a presynaptic position. In Purkinje cells, the dendritic arborization seems to be devoid of STX binding sites conversely to somata.     

Differential ontogenesis of type I and II benzodiazepine receptors in mouse cerebellum

The ontogeny of type I and type II benzodiazepine binding sites was studied in mouse cerebellum by displacement of [3H]flunitrazepam binding by zolpidem, a ligand specific for the type I sites. Type I binding sites predominate throughout development and in the adult while type II sites account for 25% of total cerebellar benzodiazepine binding sites at birth and, during development, decrease to 10% or less in the adult. On a per cerebellum basis type II sites increase during the first postnatal week and then remain at a steady level while type I sites increase until adulthood. These results may indicate a specific localization of the type II sites (and of the corresponding alpha-protein subunits in the GABA/benzodiazepine receptor complex) in structures already present at birth and developing during a short early postnatal period. The affinity of zolpidem for its high affinity (type I) binding sites increases during cerebellar ontogeny, this increase possibly indicates an epigenetic (post-translational) 'maturation' process of the corresponding receptor molecule. Hill numbers indicate the existence of an additional binding site heterogeneity greater during development but still present in the adult; probably this is to be related to the simultaneous presence of different 'maturation' stages during development and with a certain variety of the final products.     

Histochemical and immunohistochemical studies of the cerebellum from the reeler mutant mouse

The organization of the fiber connections and architecture of the cerebellum of reeler mutant mice was analyzed by an immunohistochemical and histochemical procedure. By immunohistochemical staining of the myelinated fiber arborization with antiserum against myelin basic protein, it was found in the reeler cerebellum that the fibers ran in all directions throughout the white matter. Some of the fibers surrounded Purkinje cells. The distribution of AChE-positive fibers was abnormal and some Purkinje cells surrounded by AChE-positive fibers were present. Molecular layers and glomeruli showed strong succinic dehydrogenase (SDH) activity. The white matter of the reeler cerebellum showed a mosaic of SDH-positive and -negative sites. The results indicate that changes in the distribution of myelinated fiber arborization and the change in the distribution of SDH and AChE activity are different in each part of the cerebellum as a result of the disorganization of the architecture of the reeler mutation.     

CB2 cannabinoid receptors promote mouse neural stem cell proliferation

Neurospheres are clonal cellular aggregates of neural stem/precursor cells that grow in culture as free-floating clusters. Activation of CB1 cannabinoid receptors, which are expressed by these cells, promotes proliferation. In the present study we investigated the expression of CB2 cannabinoid receptors and the effect of exogenous cannabinoids on neural stem/precursor cell proliferation. Neurospheres containing nestin-positive and sn-1 diacylglycerol lipase alpha-positive cells expressed both CB1 and CB2 receptors, which were maintained through several passages. Application of the non-selective cannabinoid agonist (HU-210, 0.5 microM) stimulated bromodeoxyuridine incorporation and neurosphere formation. This action involved both CB1 and CB2 receptors as neurosphere formation was stimulated by either selective CB1 [arachidonyl-2'chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA), 200 nM and 1 microM] or CB2 (JWH-056, 0.5 microM) agonists. In addition, CB1 or CB2 antagonists (1 microM SR-141716A and SR-144528, respectively) blocked basal proliferation, suggesting that endogenous cannabinoids are implicated in neurosphere proliferation. In addition, cannabinoid agonist-stimulated proliferation was reduced by the Akt translocation inhibitor BML-257 (12.5 microM), suggesting a role for phosphoinositide-3 kinase signalling. Together, our results suggest that cannabinoids stimulate proliferation of neural stem/precursor cells acting on both CB1 and CB2 cannabinoid receptors through a phosphoinositide-3 kinase/Akt pathway.     

Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum

Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.     

Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain

Cholesterol 24-hydroxylase is a cytochrome P450 (CYP46A1) that is selectively expressed in the brain and is responsible for the majority of cholesterol turnover in the central nervous system. Mice deficient in 24-hydroxylase exhibit impaired learning and defective hippocampal long-term potentiation, suggesting that the metabolism of cholesterol by this enzyme is required for learning and memory formation. To determine where in the neuron cholesterol turnover was taking place, monoclonal antibodies directed against 24-hydroxylase were generated by immunization of mice with recombinant protein and used to detect the enzyme in brain homogenates, cultured neurons, and histological sections. 24-Hydroxylase was localized to the endoplasmic reticulum and was distributed throughout the cell bodies and dendrites of multiple types of neurons; the enzyme was not detected in axon terminals or in the cells of 24-hydroxylase knockout mice. 24-Hydroxylase was highly expressed in pyramidal neurons of the hippocampus and cortex, in Purkinje cells of the cerebellum, and in hippocampal and cerebellar interneurons. Within the retina, 24-hydroxylase was detected in ganglion cells and some but not all cells of the inner nuclear layer. These findings reveal the microsomal localization of 24-hydroxylase and provide subcellular insight into cholesterol turnover in the brain.     

Alteration of benzodiazepine receptors in mouse cerebellum following methylazoxymethanol treatment during development

The specific binding of [3H]flunitrazepam was studied to biochemically specify the morphological alterations induced in mouse cerebellum by a single injection of an antimitotic agent, methylazoxymethanol (MAM) performed at the beginning of the postnatal life. The MAM injection causes a general reduction of the benzodiazepine receptors in the adult mice which is particularly severe in mice having been injected the 1st day of postnatal life (so-called MAM0 mice) as compared to animals injected the 5th day (MAM5 mice): in MAM0 mice the benzodiazepine receptor is reduced to half of the control value. The affinity of the benzodiazepine towards its receptor was not affected and the topographic and biochemical action of MAM in the central nervous system was ascertained. Correlations could be made between the biochemical modifications and the morphological alterations otherwise described.     

Ultrastructural localization of cannabinoid CB1 and mGluR5 receptors in the prefrontal cortex and amygdala

Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.     

Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study

A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.