Purkinje Cell Axon Terminals Research Articles

Activity-dependent distribution of protein kinase C-delta within rat cerebellar Purkinje cells following unilateral labyrinthectomy.

Protein kinase C isoforms PKC-delta and PKC-gamma are expressed in Purkinje cells in the uvula-nodulus of the cerebellum. We examined the effect of Purkinje cell activity on the transcription, expression and intracellular distribution of PKC-delta and PKC-gamma. Relative changes in activity were induced by unilateral labyrinthectomy (UL), decreasing the activity of Purkinje cells on the side of the labyrinthectomy relative to the contralateral side with intact vestibular input. After a UL, there was decreased immunolabeling of Purkinje cell axon terminals in the ipsilateral caudal vestibular complex by antisera to PKC-delta, but not PKC-gamma. Western blots prepared from the uvula-nodulus and caudal vestibular complex showed an increase in the cytosolic PKC-delta and a decrease in membrane-associated PKC-delta in the ipsilateral uvula-nodulus 12-48 h after the UL. Hybridization histochemistry and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) demonstrated no change in transcription of PKC-delta mRNA in the uvula-nodulus 1-240 h after unilateral labyrinthectomy. We conclude that both PKC-delta and PKC-gamma are constitutively expressed in Purkinje cells. The targeting of PKC-delta, but not PKC-gamma, to Purkinje neuron synaptic terminals is influenced by activity.

Phenytoin alters Purkinje cell axon morphology and targeting in vitro.

In adult mice, administration of the anticonvulsive drug phenytoin caused focal swellings along the Purkinje cell axon correlated with ataxia and incoordination of movements. In our model, we used murine cerebellar slice cultures to study the influence of phenytoin on postnatal Purkinje cell axon differentiation. Almost all of our untreated cultures developed to mature-like cerebellar tissue. Immunohistochemistry with anti-calbindin-D28k or UCHTI (anti-CD3) antibodies revealed numerous Purkinje cell axons in the white matter. In the area of the deep cerebellar nuclei, immunolabelled axons formed a large axonal plexus. The few neurofilament-positive neurons in this area were densely covered with Purkinje cell axon terminals. The synaptophysin immunoreactivity revealed connections between the terminals and the neurons of the deep cerebellar nuclei. Treatment of cerebellar slice cultures with phenytoin (10-80 microM) for 10-16 days resulted in focal swellings of different size along the axon. The number of swellings increased with an increasing dosage. At concentrations of 40 microM phenytoin, Purkinje cell axons seemed to be unable to invade the deep cerebellar nuclei, but numerous aberrant, recurrent collaterals could be detected immunohistochemically with the two specific Purkinje cell antibodies. Possible cytotoxic effects after treatment, such as dendritic degeneration and a decrease in the number of immunolabelled Purkinje cells, were observed above 40 microM phenytoin. These data suggest that the response of juvenile Purkinje cells is dependent upon the dosage of the antiepileptic drug because of morphological alterations as well as a misrouting of previously established connections.

Loss of the dentate nucleus neurons is associated with torpedo formation: a morphometric study in progressive supranuclear palsy and dentatorubro-pallidoluysian atrophy.

Cerebellar torpedoes can be induced by direct damage to Purkinje cell axons. This raises the possibility that the loss of dentate nucleus neurons (DNNs) may also cause torpedo formation through synaptic detachment between DNNs and Purkinje cell axon terminals. To investigate this possibility, we conducted a morphometric study with an image analyzer in progressive supranuclear palsy (PSP) and dentatorubro-pallidoluysian atrophy (DRPLA). Using horizontal and sagittal sections of the cerebellar hemispheres containing the greatest proportion of the dentate nucleus, we determined the line densities of torpedoes, Purkinje cells, and DNNs. In PSP and DRPLA, the densities of DNNs were significantly lower, and the densities of torpedoes much greater than in controls, while those of Purkinje cells were normal in both diseases. In addition, the torpedo densities in PSP and DRPLA showed a strong negative correlation with the DNN densities. Thus, this study clearly demonstrated that torpedoes are formed in association with the loss of DNNs, suggesting that they may occur in consequence of synaptic disconnection of Purkinje cells from DNNs.

Distribution Pattern and Ultrastructural Localization of Rxt1, an Orphan Na +/Cl −-Dependent Transporter, in the Central Nervous System of Rats and Mice

The cellular and subcellular localization of Rxt1 protein, an orphan Na +/Cl −-dependent transporter, was investigated in the central nervous system of rats and mice, with rabbit polyclonal antibodies specifically directed against its C-terminal region. At the light microscope level, the distribution of Rxt1, visualized by the immunoperoxidase method, was found to be similar in rats and mice. Labelled elements were present in numerous gray matter regions of the central nervous system, from the olfactory bulb to the spinal cord. In all labelled regions, immunoreactivity was confined to the neuropil where both a diffuse labelling of low intensity and an intense punctate staining were noted. To further identify the nature of the cellular elements bearing the punctate staining, possible changes in this labelling pattern were investigated: (i) in deep cerebellar nuclei and lateral vestibular nucleus of the Lurcher mutant mouse, in which all cerebellar Purkinje cells are missing and (ii) in the rat cervical spinal cord, 10 days after multiple resections of dorsal roots. The vast majority of the punctate structures, delineating the neuronal perikaryal and stem dendritic contours, had disappeared in the mutant mouse, providing evidence that they belong to Purkinje cell axon terminals. In rhizotomized rats, the intense labelling in laminae I and III had disappeared, demonstrating that it occurred in subclasses of axonal projections of primary afferent fibres. These results strongly suggest that Rxt1 is present in presynaptic axon terminals. The electron microscopic study was carried out in the hippocampus, cerebellum and lateral vestibular nucleus of control mice, where Rxt1-labelled punctate structures were found to be abundant. Immunostaining was confined to axon terminals, particularly in hippocampal and cerebellar mossy fibres and in Purkinje cell axonal terminations of the cerebellar deep nuclei and lateral vestibular nucleus. In the cerebellar cortex, axon terminals belonging to inhibitory local circuit neurons (basket and Golgi cells), were free of labelling. The observations reported in this study have shown that: (1) The Rxt1 transporter is neuron-specific, and is expressed by only some classes or even subclasses of neuronal systems. (2) This transporter can be encountered in excitatory axons using glutamate as neurotransmitter (hippocampal and cerebellar mossy fibres; primary afferent fibres), as well as in inhibitory axons known by their GABAergic nature (Purkinje cell axon terminals) where it might be involved in the re-uptake process of one or several molecules released from corresponding terminals.

Anti-Zebrin II immunopositivity in the cerebellum and octavolateral nuclei in two species of stingrays.

Anti-Zebrin II is an antibody directed against a 36kDa aldolase epitope expressed by Purkinje cells. Two patterns of Zebrin II immunolabeling have been described in the cerebellar corpus. In mammalian cerebella, the anti-Zebrin II labels longitudinal zones of Purkinje cells, whereas in teleosts, all Purkinje cells of the cerebellar corpus are Zebrin II immunopositive. An outgroup analysis is required to determine which of these distribution patterns represents the primitive condition for jawed vertebrates. The sister group of the Osteichthyans (rayfinned fishes, amphibians, and amniotes) is the Chondrichthyans (sharks, skates, and rays). In the present study the distribution of Zebrin II immunopositivity was examined in the Atlantic stingray and the Southern stingray. Western-blot analysis demonstrates that the Zebrin II antibody recognizes an antigen of the same molecular weight in stingrays, teleosts, and mammals. In stingrays, anti-Zebrin II immunohistochemistry reveals a staining pattern in which all Purkinje cells are immunopositive, no banding pattern or zonal compartmentation is observed. Purkinje cell axon projections to the cerebellar nucleus and the octaval nuclei are also revealed. Within the octaval nuclei, immunopositive Purkinje cell axon terminals and boutons en passant were found in the anterior, descending, and posterior nuclei. These immunopositive profiles are found throughout these nuclei, but they are most dense in the lateral and ventral portions. Except for the dorsolateralmost portion, the magnocellular nucleus does not appear to receive Purkinje cell inputs. Based on these results it is concluded that the Zebrin II distribution pattern in which all Purkinje cells of the cerebellar corpus are immunopositive is the primitive condition for jawed vertebrates.

Astrocytic processes compensate for the apparent lack of GABA transporters in the axon terminals of cerebellar Purkinje cells.

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT-1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.

Development of Purkinje cell bodies and processes with basic fibroblast growth factor-like immunoreactivity in the rat cerebellum

The development of basic fibroblast growth factor-like immunoreactivity was investigated in the nuclei, cell bodies and processes of Purkinje cells with attention to basic fibroblast growth factor-containing neuronal input to the deep cerebellar nuclei. Immunoblot analysis with the use of the antisera against basic fibroblast growth factor revealed that crude homogenate of the developing rat cerebellum exhibits a main band with the same molecular weight (18,000 mol. wt) as basic fibroblast growth factor in all the postnatal stages examined. Cerebellar cells were not labeled with the antisera during embryonic life. Under light microscopy, basic fibroblast growth factor-like immunoreactivity was detected initially in cortical cells located close to deep cerebellar fissures of the newborn rat but not in superficial cortical regions. It was difficult to determine whether or not they are Purkinje cells at the fusiform stage. On postnatal day 7, immunoreactive Purkinje cells were identified throughout the cerebellar cortex, and they expressed basic fibroblast growth factor-like immunoreactivity mainly in the apical cytoplasm and proximal dendrites. From postnatal day 14 to postnatal day 28, basic fibroblast growth factor-like immunoreactivity was noted not only throughout the cytoplasm of Purkinje cells but also in the nuclei of the immunopositive cells. Our statistical anatysis showed that Purkinje cells with nuclear immunoreaction peaked on postnatal day 21. At these stages, nerve fibers immunoreactive for basic fibroblast growth factor were numerous in the cerebellar medulla and deep cerebellar nuclei. After postnatal day 42, Purkinje cells with intense immunoreactivity in the nuclei showed a marked decrease in number, and immunoreactive structures were distributed in the cerebellum in a fashion similar to that in adult rats. Electron microscopy demonstrated that immunoreactivity was located mainly in the apical cytoplasm of Purkinje cells on postnatal day 7 and throughout the cytoplasm and in the nuclear euchromatin from postnatal day 14 to postnatal day 28, as was expected from light-microscopic observations. Immunoreactivity, even though distributed diffusely in the cytoplasm, was absent from the lumen of endoplasmic reticulum and mitochondria. A small population of Purkinje cell axon terminals forming synapses with the soma and dendrites of deep cerebellar nucleus neurons began to express basic fibroblast growth factor on postnatal day 21. This is much later than the starting age for synaptogenesis between Purkinje cells and deep cerebellar nucleus neurons. The age-dependent changes in the localization of basic fibroblast growth factor within Purkinje cell nucleus, soma and processes suggest a complex transport system of this factor within Purkinje cells during postnatal development.

Cerebellar benzodiazepine receptors: cellular localization and consequences of neurological mutations in mice

The distribution of cerebellar [ 3H]flunitrazepam binding sites was studied autoradiographically in Purkinje cell degeneration ( pcd/pcd), weaver ( wv/wv), staggerer ( sg/sg) and reeler ( rl/rl) mutant mice. In the normal 78-day-old C57BL/6J mouse cerebellum, the highest concentration of [ 3H]flunitrazepam binding sites was observed over the molecular layer. Intermediate grain density was present over the Purkinje cell layer and intermediate to high density over the deep cerebellar nuclei. Low labeling was observed over the granule cell layer. Negligible concentrations of binding sites were seen in the white matter. In 45–49-day-old Purkinje cell degeneration mutants, where essentially all Purkinje cells have disappeared by day 45, there was a small decrease in grain density over the cerebellar cortex. Concomitantly, a substantial increase in grain density was observed over the deep cerebellar nuclei of the pcd/pcd mutants when compared to littermate controls. A significant increase in [ 3H]flunitrazepam labeling was observed over the cerebellar cortex of 81–86-day-old wv/wv mutants; this was most pronounced in the vermis where the granule cell loss was greatest. Over the hemispheres, where fewer granule cells degenerate, a lower density of binding sites was seen. Grain density over the wv/wv deep cerebellar nuclei was comparable to that of littermates. Substantially lower [ 3H]flunitrazepam labeling was detected over the cerebellar cortex of 25–27-day-old sg/sg mutants in which the number of granule. Purkinje and Golgi cells is greatly reduced; the labeling over the deep nuclei, however, was significantly increased. In 27–29-day-old rl/rl mutant cerebella, where all classes of cells are malpositioned, labeling density over all areas of the cerebellar cortex, including the Purkinje cell masses. was increased. Our autoradiographic data suggest that a proportion of cerebellar cortical benzodiazepine receptors are associated with Purkinje cells; we propose that the remainder of the receptors are localized on Golgi cells, while granule cells are devoid of receptors. In the deep cerebellar nuclei, the observed increase in benzodiazepine receptors in the pcd/pcd and sg/sg mutants may be a manifestation of denervation supersensitivity subsequent to the loss of innervation by Purkinje cell axon terminals. The finding of a high receptor density in the Purkinje cell masses of the rl/rl mutant, where Purkinje cells are devoid of afferent basket cell input, suggests that benzodiazepine receptors are expressed and maintained in the absence of a full complement of GABAergic afferents.

Morphology of Purkinje cell axon terminals in intracerebellar nuclei following inferior olive lesion

We have examined the ultrastructural changes of axons and synaptic boutons in the intracerebellar nuclei of the rat at 3 days to one year after inferior olive lesion performed by means of electrocoagulation or 3-acetylpyridine injection. A large number of preterminal segments and axon terminals undergoes remarkable ultrastructural changes after total or subtotal olivary lesion. Large membrane bound vacuoles and clusters of small synaptic vesicles characterize a good number of these terminals at 3 days up to one month after the lesion. Tightly packed tubules and cisternae of smooth endoplasmic reticulum appear during the first week in an increasing number of axon terminals. Boutons with large whorled bodies formed by smooth membranes increase in number during the second half of the first month and further increase in density until the sixth month. They are still present in large amounts at one year. Immunoreactivity for 3′,5′-guanosine-phosphate-dependent protein kinase, which is specific for Purkinje neurons, can be detected in the axons and synaptic terminals displaying the ultrastructural changes described above. These results are discussed in relation to a possible trophic action of the climbing fibers on the Purkinje cells. We suggest that, at least in part, these alterations may be the consequence of the intense Purkinje cell hyperactivity which is present for up to one month from inferior olive lesion.

Long-term effects of 3-acetylpyridine-induced destruction of cerebellar climbing fibers on Purkinje cell inhibition of vestibulospinal tract cells of the rat

The inhibitory action of Purkinje cells on vestibulospinal tract (VST) cells was examined in rats deprived of climbing fibers with 3-acetylpyridine (3-AP) intoxication. In order to resolve discrepancies raised in previous studies with various means, special efforts were devoted to directly estimate Purkinje cell inhibition at synaptic levels by using intracellular recording, to avoid sampling bias by using a systematic survey of VST cells in each rat, and to evaluate the time-dependence of the effects of climbing fiber deafferentation by regular testing at 10 day intervals until 160 days after 3-AP intoxication. As compared with 661 VST cells impaled in 15 control rats, 1771 VST neurons impaled in 29 3-AP-treated rats revealed four basic changes in the monosynaptic inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons in the white matter of the cerebellar anterior lobe. First, the rate of IPSP occurrence among VST cells was 0.64 in control rats; at more than 10 days after 3-AP intoxication it decreased gradually, down to 0.37-0.38 at the 70th-81st days, and thereafter increased up to 0.53 by the 160th day. The rate of IPSP occurrence varied considerably between the rostral and caudal regions, and also between the dorsal and ventral divisions of the VST cell population, but its reduction after 3-AP intoxication occurred approximately in parallel in all divisions. Second, IPSPs evoked with standard 500 microA pulse stimuli were smaller in size on and after day 10. The reduction of IPSP size was by as much as 53% of control values at the 70th-101st days in the dorsal division, but no significant change occurred in the ventral division of the VST cell population. Third, the latency of the IPSPs was prolonged by about 0.25 ms on and after day 10. Analysis of the relationship between the IPSP latency and the dorsoventral location of VST cells in the medulla suggests that the major cause for the prolongation of IPSP latency is an increased synaptic delay at Purkinje cell axon terminals. Fourth, the cerebellar stimulation threshold for evoking IPSPs was almost always below 100 microA in control rats, but values of 100-250 microA were common after the 40th day. Thus, climbing fiber deafferentation exerts long-term influences on excitability of Purkinje cell axons, and on the connectivity and synaptic transmission from Purkinje cell axons to VST cells.

GABA-immunoreactive neurons in the rat cerebellum: a light and electron microscope study.

An antibody raised against gamma - amino-butyric acid (GABA) coupled to bovine serum albumin with glutaraldehyde (Hodgson et al: J. Histochem. Cytochem. 33:229-239, '85) was used to localise immunocytochemically the presumptive GABAergic neuronal elements in the cerebellar cortex of the adult rat. employing the unlabelled antibody enzyme method with pre- and post-embedding immunocytochemical procedures, the following cellular structures were observed to be GABA-immunopositive in both the light and electron microscopes: the somata, dendrites, and axonal processes (including axon terminals) of stellate, basket, and Golgi neurons. In immunopositive neuronal somata and dendrites, the reaction product was found to be associated with all intracellular organelles and with the postsynaptic densities of synaptic junctions. Specific GABA-like immunoreactivity was also seen around outer mitochondrial membranes, microtubules, and neurofilaments, and coating synaptic vesicles in presynaptic axon terminals. In the pre-embedding procedure with dilutions of the antiserum between 1:1,000 and 1:2,000, the perikarya and dendrites of Purkinje cells were GABA-immunonegative, whereas at an antiserum dilution of 1:500 the somata of Purkinje cells were mildly GABA-immunoreactive. Purkinje cell axon terminals in the infra- and supraganglionic plexuses and in the deep cerebellar nuclei were always strongly immunopositive. Neuroglia were invariably GABA-immunonegative, as were the dendrites, axons (parallel fibres), and somata of granule cells. Mossy fibre and climbing fibre afferents were also immunonegative. The pattern of immunoreactivity obtained with this antiserum directed against the inhibitory neurotransmitter GABA was found to resemble closely the immunocytochemical distribution of GABA and of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) as reported previously in other immunocytochemical investigations (Oertel et al. and Wu et al: Cytochemical Methods in Neuroanatomy. New York: A. R. Liss, '82; Seguela et al: Neuroscience 16:865-874, '85; Mugnaini and Oertel: GABA and Neuropeptides in the CNS. Handbook of Chemical Neuroanatomy, Vol. 4, Part I. Amsterdam: Elsevier, '85.

Immunocytochemical demonstration of topographic ordering of Purkinje cell axon terminals in the fastigial nuclei of the rat.

We have used a monoclonal antibody, mabQ113, which selectively stains a subset of cerebellar Purkinje cells, to study the topography of the corticonuclear projection of the median vermis to the fastigial nuclei in the rat. The fastigial projection zone contains both mabQ113+ and mabQ113- Purkinje cells grouped into parasagittal bands. The immunoreactivity extends throughout the Purkinje cell including the axon terminals and thus it is possible to investigate the topographic distribution of mabQ113+ terminals. In the fastigial nuclei the target cells receiving mabQ113+ axon terminals are concentrated in the caudal pole. In the rostral pole, cells receive anti-GAD+ terminals but not mabQ113+ terminals. There is no gradient in anti-GAD staining. Double-labelling experiments with mabQ113, anti-GAD, and an antisynaptic antibody mabQ155 suggest that there is little or no mixing of mabQ113+ and mabQ113- Purkinje cell terminals on the same target neuron.

Neurochemical and morphological consequences of axon terminal degeneration in cerebellar deep nuclei of mice with inherited purkinje cell degeneration

The concentrations of free amino acids and the activities of transmitter-related enzymes, glutamic acid decarboxylase (GAD), choline acetylase (ChAC) and GABA-transaminase (GABA-t) were measured in cerebellar cortex and deep cerebellar nuclei from the mouse mutant Purkinje cell degeneration (pcd) at various times before and after Purkinje cell loss. Axosomatic synapses on target cells in pcd deep nuclei were quantified by electron microscopy during and after degeneration. The concentration of GABA (nmol/mg wet weight), the Purkinje cell transmitter, was normal in pcd cerebellar cortex and deep nuclei before onset of Purkinje cell degeneration on postnatal day 15. Just after the major period of Purkinje cell loss in cerebellar cortex, GABA concentration was unchanged in the cortical layers but fell to 50% of normal values in the deep nuclei of pcd animals killed either by decapitation or by microwave irradiation. No other measured free amino acid decreased. There were no long-term increases following Purkinje cell degeneration in the concentration of any transmitter amino acids or related enzymes, GAD, ChAC or GABA-t, and thus no indication of axonal sprouting reactions. Progressive losses occurred in wet weight and protein and in activity of GABA-t in both the cerebellar cortex and the deep nuclei of pcd animals. Electron microscopic analysis indicated that Purkinje cell axon terminals contact 30% or more of the somatic surface of principal neurons of the lateral nucleus of the normal cerebellum, but only about 2% of the corresponding sites in the pcd cerebellum. Glial leaflets, rather than other synaptic terminals take their place. Axon terminals may degenerate earlier than Purkinje somata in the pcd disease.