The intermediate filament protein peripherin is a marker for cerebellar climbing fibres

Immunocytochemical staining with antibodies to the class III intermediate filament protein peripherin reveals discrete subpopulations of neurons and nerve fibres throughout the rat central nervous system. Some of these fibres enter the cerebellar granular and molecular layers. Here we use light and electron microscopic immunocytochemistry and confocal fluorescence microscopy to identify the peripherin positive fibres in the molecular layer of the cerebella of various mammals. 1) The peripherin positive fibres in the molecular layer have morphological attributes of climbing fibres, and peripherin positive fibres are also detected in the olivo-cerebellar tract. Furthermore peripherin positive neurons can be seen in the inferior olive, from which climbing fibres originate. (2 ) The peripherin positive molecular layer fibres rapidly degenerate in rats treated with 3-acetylpyridine (3-AP), a reagent which destroys neurons in the inferior olive, and the time course of degeneration of these mirrors that previously described for 3-AP induced destruction of climbing fibres. (3) Cerebella of other mammal species tested (mouse, rabbit, pig, cow and human) revealed a similar peripherin staining pattern in the cerebellum, including fibres in the molecular layer with the morphology of climbing fibres. (4) We also noted peripherin positive spinocerebellar and vestibulocerebellar mossy fibres in the cerebellar granular layer of folia known to receive these inputs. (5) A subset of perivascular nerve fibres are also peripherin positive. These results show that peripherin is a useful marker for mammalian cerebellar climbing fibres, and that a subset of morphologically distinct cerebellar mossy fibres are also peripherin positive.     

Cytospectrophotometric study of GABA-transaminase in the rat cerebellar cortex

Modification of the histochemical method for detection of GABA-transaminase activity is suggested. Optimal concentrations of the substrates and cofactors are chosen on the basis of kinetic study of the enzymatic reaction in the cryostat sections of the rat cerebellar cortex by the quantitative microspectrophotometric method. The method is intended for the quantitative histochemical analysis of GABA-transaminase activity in the brain sections.     

Age-related changes of monoaminooxidases in rat cerebellar cortex

Age-related changes of the monoaminoxidases, evaluated by enzymatic staining, quantitative analysis of images, biochemical assay and statistical analysis of data were studied in cerebellar cortex of young (3-month-old) and aged (26-month-old) male Sprague-Dawley rats. The enzymatic staining shows the presence of monoamino-oxidases within the molecular and granular layers as well as within the Purkinje neurons of the cerebellum of young and aged animals. In molecular layer, and in Purkinje neurons the levels of monoaminooxidases were strongly increased in old rats. The granular layer showed, on the contrary, an age-dependent loss of enzymatic staining. These morphological findings were confirmed by biochemical results. The possibility that age-related changes in monoaminooxidase levels may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs is discussed.     

Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43

The adult mammalian central nervous system has a limited ability to establish new connections and to recover from traumatic or degenerative events. The olivo-cerebellar network represents an excellent model to investigate neuroprotection and repair in the brain during adulthood, due to its high plasticity and ordered synaptic organization. To shed light on the molecular mechanisms involved in these events, we focused on the growth-associated protein GAP-43 (also known as B-50 or neuromodulin). During development, this protein plays a crucial role in growth and in branch formation of neurites, while in the adult it is only expressed in a few brain regions, including the inferior olive (IO) where climbing fibres (CFs) originate. Following axotomy GAP-43 is usually up-regulated in association with regeneration. Here we describe an in vivo lentiviral-mediated gene silencing approach, used for the first time in the olivo-cerebellar system, to efficiently and specifically downregulate GAP-43 in rodents CFs. We show that lack of GAP-43 causes an atrophy of the CF in non-traumatic conditions, consisting in a decrease of its length, branching and number of synaptic boutons. We also investigated CF regenerative ability by inducing a subtotal lesion of the IO. Noteworthy, surviving CFs lacking GAP-43 were largely unable to sprout on surrounding Purkinje cells. Collectively, our results demonstrate that GAP-43 is essential both to maintain CFs structure in non-traumatic condition and to promote sprouting after partial lesion of the IO.     

Locomotor performance and lesions of the posterior cerebellar cortex in the rat

Rats with posterior cerebellar cortex lesions were compared with sham operated controls on a parallel rod locomotor performance task. Lesioned animals showed marked deficits in negotiating the task even though gross signs of locomotor disturbance were no longer obvious.     

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.     

Heroin-induced changes of catecholamine-containing particles in male rat cerebellar cortex.

The content and distribution of catecholamine-containing formations in the cerebellum of untreated and heroin-treated male rats, was visualized by glyoxylic acid-induced histofluorescence, in an attempt to define the adaptive mechanisms leading to heroin dependent tolerance as well as identify a biological role for these formations. Repeated heroin administration increased the number of specifically organized intracellular catecholamine containing particles, including grain (diameter less than 0.8 microm) and aggregate (diameter greater than 1 microm) forms, in all cerebellar cortical layers examined one hour after the last injection of the drug, relative to controls. The number of grains in all cerebellar cortical layers examined and aggregates in the granular layer, returned to normal or near normal baseline levels within twenty four hours after the last injection of the drug. The analogous baseline of the aggregates in the Purkinje cell layer primarily and the Molecular layer secondarily remained significantly elevated by 86% and 50% respectively, relative to controls. Catecholamine-heroin interactions most likely mediated this elevation that was related directly to the heroin-dependent state of tolerance. These findings indicate that heroin administration to heroin-tolerant rats leads to the formation of unusually large intracellular aggregates with catecholamines in the Purkinje cells of the cerebellum primarily and support a direct role for these formations in the modulation of biogenic amine bioavailability. We conclude that adaptation to drug exposure involves multiple homeostatic interactions, with sympathetic activation at the level of catecholamine reorganization and redistribution playing a major role in rat cerebellar cortex.     

Electron cytochemical localization of gamma-aminobutyric acid catabolism in rat cerebellar cortex

Summary An electron cytochemical technique is described for the localization of GABA-T, the enzyme which degrades the neurotransmitter GABA, in rat cerebellar cortex. The technique allows ultrastructural demonstration of GABA-T activity by the final deposition of an electron dense formazan precipitate at reaction sites, whilst maintaining adequate ultrastructural preservation for recognition of cellular and subcellular structures. Numerous electron dense precipitates are evident as discrete punctate deposits situated mainly in mitochondria of stellate cells, basket cells and astrocytic glial cells; they are also seen in axonal or dendritic profiles at some synaptic junctions. The technique enables the first cytochemical demonstration of the mitochondrial localization of GABA-T activity in nervous tissue to be presented. It establishes that GABA-T is present in supposed GABA neurones, in pre- or post-synaptic endings, or both, of presumed inhibitory synapses and in glial cells which may be associated with these synapses. From this seemingly ubiquitous distribution, functional aspects of GABA-T in these cells is considered.     

Localization of the mGlu4a metabotropic glutamate receptor in rat cerebellar cortex

 The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses. Accepted: 1 July 1997