Immunohistochemical localization of cannabinoid CB<Subscript>1</Subscript> receptor in inhibitory interneurons in the cerebellum

Inhibitory interneurons terminating on Purkinje cell dendrites contribute to cannabinoid-mediated cerebellar plasticity, consistent with the intense expression of cannabinoid CB1 receptor protein in the cerebellar molecular layer. CB1 labelling in the molecular layer has been attributed to parallel fibers originating from granule cells, climbing fibers originating in the inferior olive, and inhibitory interneurons in the deep molecular layer (basket cells). However, the cellular distribution of CB1 in the cerebellar molecular layer has remained poorly understood. We used double fluorescence labelling to test for co-localization of nuclei with CB1 receptor protein. Labelling was intense surrounding nuclei in the deep and superficial molecular layer; consistent with basket cell and stellate cell inhibitory interneurons that regulate depolarization-induced suppression of inhibition (DSI) of Purkinje cells.     

Decrease of [C]2-deoxyglucose uptake at the intracerebellar nuclei during cerebellar cortex stimulation

The effect of electrical stimulation of the cerebellar cortex at 10–30 c/s on metabolism in the intracerebellar nuclei has been studied using the [¹⁴C]2-deoxyglucose method. The experiments, performed on anesthetized or immobilized animals did not produce any detectable changes in the radioautographic labeling of the intracerebellar nuclei compared with controls.Experiments were also performed in animals pretreated with 3-acetylpyridine neurotoxin which selectively destroys the inferior olive and produces an intense labeling of the intracerebellar nuclei. Less marking was observed in restricted regions of the intracerebellar nuclei receiving the axon terminals of the stimulated Purkinje cells if the experiments were done within the first few hours. Following 3-acetylpyridine intoxication, in this early phase, destruction of the inferior olivary cell bodies occurs, but the climbing fibers remain intact. At 2 days or more following 3-acetylpyridine, changes in marking with stimulation could no longer be obtained.The finding is interpreted as being due to an actual reduction of the Purkinje cell activity upon stimulation of the cerebellar cortex, leading to a reduction of the metabolic activity at their presynaptic terminals.     

Cerebellar control of the inferior olive

A subpopulation of neurones in the cerebellar nuclei projects to the inferior olive, the source of the climbing fibre input to the cerebellum. This nucleo-olivary projection follows the zonal and, probably also, the microzonal arrangement of the cerebellum so that closed loops are formed between the neurones in the olive, the cerebellar cortex and the nuclei. The nucleo-olivary pathway is GABAergic, but several investigators argue that its main effect is to regulate electrotonic coupling between cells in the inferior olive rather than inhibit the olive. However, there is now strong evidence that the nucleo-olivary fibres do inhibit the olive. Three functions have been suggested for this inhibition: (i) feedback control of background activity in Purkinje cells, (ii) feedback control of learning, and (iii) gating of olivary input in general. Evidence is consistent with (i) and (ii). Activity in the nucleo-olivary pathway suppresses both synaptic transmission and background activity in the olive. When learned blink responses develop, the blink related part of the olive is inhibited while blinks are produced. When the nucleo-olivary pathway is interrupted, there is a corresponding increase in complex spike discharge in Purkinje cells followed by a strong suppression of simple spike firing. Stimulation of the pathway has the opposite results. It is concluded that the nucleo-olivary fibres are inhibitory and that they form a number of independent feedback loops, each one specific for a microcomplex, that regulate cerebellar learning as well as spontaneous activity in the olivo-cerebellar circuit.     

Spatiotemporal distribution of Connexin45 in the olivocerebellar system

The olivocerebellar system is involved in the transmission of information to maintain sensory motor coordination. Gap junctions have been described in various types of neurons in this system, including the neurons in the inferior olive that provide the climbing fibers to Purkinje cells. While it is well established that Connexin36 is necessary for the formation of these neuronal gap junctions, it is not clear whether these electrical synapses can develop without Connexin45. Here we describe the development and spatiotemporal distribution of Connexin45 in relation to that of Connexin36 in the olivocerebellar system. During development Connexin45 is expressed in virtually all neurons of the inferior olive and cerebellar nuclei. During later postnatal development and adulthood there is a considerable overlap of expression of both connexins in subpopulations of all main olivary nuclei and cerebellar nuclei as well as in the stellate cells in the cerebellar cortex. Despite this prominent expression of Connexin45, ultrastructural analysis of neuronal gap junctions in null-mutants of Connexin45 showed that their formation appears normal in contrast to that in knockouts of Connexin36. These morphological data suggest that Connexin45 may play a modifying role in widely distributed, coupled neurons of the olivocerebellar system, but that it is not essential for the creation of its neuronal gap junctions.     

Thyrotropin-releasing hormone and its receptor in the cerebellum of inferior olive destroyed rat brain

Abstract

To study the pathophyhsiology of olivopontocerebellar atrophy (OPCA), we destroyed inferior olive nuclei of male Wistar rats using 3-acetyl pyridine (3-APJ+ harmaline+ niacinamide. These rats showed a sluggish and ataxic gait. To elucidate the relationship between thyrotropin releasing hormone (TRH) in the Purkinje cell of cerebellum and the inferior olive nucleus, we investigated the concentrations of TRH in the cerebellar cortex, nuclei, and medulla oblongata including the inferior olive nuclei using radioimmunoassay method as well as TRH receptor in the Purkinje cells of cerebellum using immunohisto-chemical method. All statistical comparisons were done using non-parametric tests (Mann-Whitney U-test). We found that two weeks after the treatment, TRH concentrations in the cerebellar cortex as well as nuclei were significantly lower than in the controls but no significant difference in the medulla oblongata was observed between 3-AP treated rats and controls. Moreover, four weeks after the treatment, TRH-receptor positive Purkinje cell counts were significantly fewer than in the controls. These results suggest that TRH in the Purkinje cell of cerebellum may play a role in the ataxic gait observed in the rats whose inferior olive were destroyed.[Neurol Res 2000; 22: 401^403]     

The constitutive production of the endocannabinoid 2-arachidonoylglycerol participates in oligodendrocyte differentiation

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLβ), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and β abundantly, resulting in the stronger production of 2-AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB2 receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC-80267 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous 2-AG. Likewise, 2-AG synthesis disruption using specific siRNAs against DAGLα and DAGLβ significantly reduced myelin protein expression in vitro, whereas a pharmacological gain-of-function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC-80267 all diminished basal ERK1/2 phosphorylation, effects that were partially reversed by the addition of 2-AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of 2-AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.     

Thyrotropin-releasing hormone and its receptor in the cerebellum of inferior olive destroyed rat brain

To study the pathophysiology of olivopontocerebellar atrophy (OPCA), we destroyed inferior olive nuclei of male Wistar rats using 3-acetyl pyridine (3-AP) + harmaline + niacinamide. These rats showed a sluggish and ataxic gait. To elucidate the relationship between thyrotropin releasing hormone (TRH) in the Purkinje cell of cerebellum and the inferior olive nucleus, we investigated the concentrations of TRH in the cerebellar cortex, nuclei, and medulla oblongata including the inferior olive nuclei using radioimmunoassay method as well as TRH receptor in the Purkinje cells of cerebellum using immunohistochemical method. All statistical comparisons were done using non-parametric tests (Mann-Whitney U-test). We found that two weeks after the treatment, TRH concentrations in the cerebellar cortex as well as nuclei were significantly lower than in the controls but no significant difference in the medulla oblongata was observed between 3-AP treated rats and controls. Moreover, four weeks after the treatment, TRH-receptor positive Purkinje cell counts were significantly fewer than in the controls. These results suggest that TRH in the Purkinje cell of cerebellum may play a role in the ataxic gait observed in the rats whose inferior olive were destroyed.     

Feedback control of Purkinje cell activity by the cerebello-olivary pathway

The pathway from the deep cerebellar nuclei to the inferior olive, the source of the climbing fibre input to the cerebellum, inhibits olivary transmission. As climbing fibre activity can depress the background firing of the Purkinje cells, it was suggested that nucleo-olivary (N-O) inhibition is a negative feedback mechanism for regulating Purkinje cell excitability. This suggestion was investigated, in a set-up with decerebrate ferrets, both by blocking and by stimulating cerebellar output while recording Purkinje cell activity. Blocking the N-O pathway was followed by an increased climbing fibre activity and a dramatic reduction in simple spike firing. Stimulation of the N-O fibres depressed climbing fibre responses and caused an increase in simple spike firing. These results are taken as support for the feedback hypothesis.     

Orthograde transport and release of insulin-like growth factor I from the inferior olive to the cerebellum

Insulin-like growth factor I (IGF-I) and its receptor are expressed in functionally related areas of the rat brain such as the inferior olive and the cerebellar cortex. A marked decrease of IGF-I levels in cerebellum is found when inferior olive neurons are lesioned. In addition, Purkinje cells in the cerebellar cortex depend on this growth factor to survive and differentiate in vitro. Thus, we consider it possible that IGF-I forms part of a putative trophic circuitry encompassing the inferior olive and the cerebellar cortex and possibly other functionally connected areas. To test this hypothesis we have studied whether IGF-I may be taken up, transported, and released from the inferior olive to the cerebellum. We have found that ¹²⁵I-IGF-I is taken up by inferior olive neurons in a receptor-mediated process and orthogradely transported to the cerebellum. Thus, radioactivity found in the cerebellar lobe contralateral to the injection site in the inferior olive was immunoprecipitated by an anti-IGF-I antibody, co-eluted with ¹²⁵I-IGF-I in an HPLC column, and co-migrated with ¹²⁵I-IGF-I in an SDS-urea polyacrylamide gel electrophoresis. Time-course studies indicated that orthograde axonal transport is relatively rapid since 30 min after the injection, radiolabeled IGF-I was already detected in the contralateral cerebellum. Furthermore, transport of IGF-i from the inferior olive is specific since when ¹²⁵I-neurotensin was injected in the inferior olive or when ¹²⁵I-IGF-I was injected in the pontine nucleus, no radiactivity was found in the contralateral cerebellum. In addition, no specific transport of ¹²⁵I-IGF-I was found in climbing fiber-deafferented rats or when excess unlabeled IGF-I was co-injected with ¹²⁵I-IGF-I. We next studied whether IGF-I is released by inferior olive neurons. We found that the release of IGF-I from cerebellar slices of normal rats was significantly greater in response to depolarizing stimuli than that from slices obtained of climbing fiber-deafferented animals. Indeed, in vitro release of IGF-I in response to KCI or veratridine was almost completely abolished in the latter. These data suggest that IGF-I is taken up by inferior olive neurons through IGF-I receptors and transported to the cerebellum through their axons without any major modification. Moreover, the release of IGF-I from the cerebellum after depolarization depends on the presence of climbing fiber afferents. Altogether these results indicate that the olivo-cerebellar pathway is able to take up, orthogradely transport, and release IGF-I. Since a similar process has been described in the visual system for basic fibroblast growth factor (bFGF), we propose that IGF-I, bFGF, and possibly other growth factors may constitute afferent trophic signals involved in plastic mechanisms within specific neural circuitries. © 1993 Wiley-Liss, Inc.