Ca/calmodulin-dependent protein kinase II in the rat cerebellum: An immunohistochemical study with monoclonal antibodies specific to either α or β subunit

Monoclonal antibodies specific to either α or β subunit of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for α subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these α subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. β Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of α and β subunits of CaM kinase II in the cerebellum were demonstrated.     

Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system.

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat central nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase alpha-expressing cells throughout the telencephalon and diencephalon. CaM II kinase alpha mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase alpha gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the central nervous system. In several regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase alpha genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase alpha gene expression only. Several regions contained both glutamic acid decarboxylase and CaM II kinase alpha expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase alpha and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissural nucleus of the stria terminalis. The results imply that CaM II kinase alpha is primarily expressed in non-GABAergic neurons. In several regions CaM II kinase alpha mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.     

Anatomy of cerebellar Purkinje cells in the rat determined by a specific immunohistochemical marker

In the present study we have used guanosine 3′: 5′-phosphate-dependent protein kinase antiserum, a specific immunohistochemical marker for cerebellar Purkinje cells, [Lohmann, Walter, Miller, Greengard and De Camilli (1981)Proc. natn. Acad. Sci. U.S.A.78, 653–657], to carry out a detailed analysis of the architecture and projections of Purkinje cells in the adult rat. We have obtained a novel view of aspects of Purkinje cell morphology that were already known and, in addition, we have provided some new information, in particular on the targets of Purkinje cell axons and their pattern of innervation, and on the morphology and course of Purkinje cell axons. Furthermore, we have found a few cells positive for guanosine 3′: 5′ phosphate-dependent protein kinase which are very similar morphologically to Purkinje cells but are located outside of the cerebellar cortex.A unique feature of Purkinje cells is their peculiar monoplanar shape. Not only do their dendritic arbors lie in planes perpendicular to the major axis of the folia, but their axons, including the collaterals, also travel roughly in the same planes. Thus, Purkinje cells can be imagined as lying in longitudinal sheets radiating from the deep cerebellar nuclei. In these sheets, Purkinje cell axons originating from cells located at different rostrocaudal levels of the cortex converge towards the deep cerebellar nuclei without intersecting each other. It is as a result of this precise organization that Purkinje cell axons reach the deep cerebellar nuclei with a mediolateral and rostrocaudal topology that closely reflects the position of their parent cells in the cerebellar cortex. In the subcortical rays of white matter, Purkinje cell axons are interspersed with other axons, being excluded only from longitudinal strips which correspond to the cerebellar raphes. Upon converging towards the deep cerebellar nuclei they segregate into tracts of white matter that alternate with tracts of white matter from which they are excluded. The great majority of Purkinje cell axons terminate in the deep cerebellar nuclei. Recurrent collaterals terminate in close proximity to the Purkinje cell layer. Dense innervation by these axons is found around large interneurons (Lugaro and Golgi cells) and around the Purkinje cell pinceaux. No direct input of recurrent collaterals to Purkinje cell somata is evident in immunostained material.A substantial number of Purkinje cell axons continue beyond the cerebellar nuclei to innervate nearby regions in the brain stem. The most prominently innervated extracerebellar target region is the dorsal part of the lateral vestibular nucleus, which is as heavily innervated by Purkinje axons as the deep cerebellar nuclei are. All the other major parts of the vestibular formation and some adjacent nuclei (including the parabrachial nuclei, the prepositus hypoglossal nucleus and the nucleus of the solitary tract) are innervated to various degrees by Purkinje cells. In these regions heavily innervated cells are interspersed with cells which receive only a moderate degree of innervation and with cells which apparently lack Purkinje cell inputs. Upon reaching the deep cerebellar nuclei, axons destined to extracerebellar targets deviate from the planes of dendritic arborization of their parent cells. They converge into tight bundles which follow an irregular course and intersect each other to reach their targets. Axons travelling in the same bundle often appear to terminate on the same cell.At all target sites Purkinje axons end as varicose terminals which synapse primarily with the perikarya and proximal dendrites of target cells. On the surface of these cells Purkinje cell terminals are often tightly apposed to form a compact mosaic. Both the course of Purkinje cell axons and their pattern of innervation of target cells are consistent with the possibility that contacts between Purkinje cells and their target neurons in the deep cerebellar nuclei and the brain stem are established early in ontogenesis.Purkinje cell-like cells positive for guanosine 3′:5′-phosphate-dependent protein kinase not located in the cerebellar cortex were found predominantly at the dorsal surface of the brain stem and, in particular, in the cortex of the dorsal cochlear nucleus. Only on exception were they found in the cerebellar medulla or in nearby noncortical regions of the brain stem. While some of these cells might be ectopies, the significance of Purkinje cell-like cells in the dorsal cochlear nucleus, a region strikingly similar in architecture to the cerebellar cortex, remains to be established.     

Differential expression of protein kinase C isozymes in rat cerebellum

Protein kinase C (PKC) is a family of enzymes found throughout the body, with the greatest diversity and concentration in brain tissue. To understand further the differential role of PKC isozymes in brain, we have raised antibodies against four PKC isoforms--alpha, beta I, beta II and gamma--using specific amino acid sequences predicted from cDNA sequences. On Western blot of rat cerebellar homogenate, these antibodies stained a band of Mr 80,000, a known molecular mass for PKC, except for anti-PKC(beta II), which stained an additional minor Mr 90,000 band. On rat cerebellar sections, specific sets of cells were stained by each antibody. In the molecular layer, stellate and basket cells were stained with anti-PKC(alpha) but not with anti-PKC(beta I), -(beta II), or -(gamma). Anti-PKC (alpha) and anti-PKC(beta II) seemed to stain the Bergmann glial fibre. In the Purkinje cell layer, Purkinje cells were stained with anti-PKC[beta I) and -(gamma). Anti-PKC (alpha) and -(beta II) were negative. Interestingly, a few Purkinje cells were not stained with anti-PKC(gamma). In the granule cell layer, both granule and Golgi cells were stained with anti-PKC(beta I) and -(beta II) but not with anti-PKC(alpha) or -(gamma). Glial cells were stained with anti-PKC(beta II) but not with anti-PKC(alpha), -(beta I), or -(gamma). This study suggests a differential distribution of PKC isoforms in different cells, irrespective of their neurotransmitters. The presence of both positive and negative staining of Purkinje cells with anti-PKC(gamma) raised the possibility of their heterogeneity.     

The cyclin-dependent kinase 5 activator,p39, is expressed in stripes in the mouse cerebellum

Cyclin-dependent kinase 5 (Cdk5) activity is required for CNS development. The Cdk5 activator, p35, is well characterized but its isoform, p39, has been less studied. Previously, p39 mRNA expression in rat brain was shown to peak at 3 weeks postnatal, and the level remains high in the adult cerebellum [Neurosci Res 28 (1997) 355]. However, p39 protein expression and specific localization in the cerebellum, where p39 mRNA level significantly exceeds that of p35, have not been examined. Here, we explored the specific cerebellar localization of the p39 protein in the developing and adult mice. Adult cerebellar Purkinje cell somata and dendritic arbors were strongly positive for p39 but only rare and barely detectable p39 was observed in Purkinje cell axons. Cdk5 also localized in Purkinje cell somata and dendrites of the adult cerebellum, but p35 localized only in Purkinje cell somata, further suggesting a functional difference between p35 and p39. During development, cerebellar p39 was first noted at P10. Primary cultures of a developing cerebellum also showed strong p39 immunoreactivity in Purkinje cell somata and dendrites, but weak p39 immunoreactivity in Purkinje cell axons. Starting from P10, p39 was observed in a subset of Purkinje cells that form parasagittal bands throughout the vermis and hemispheres. These bands were bilaterally symmetrical and continuous from one lobule to another. Conversely, Cdk5 and p35 showed a uniform staining pattern. The pattern of p39 closely resembled that of zebrin II/aldolase C, suggesting that p39 may play a role in the adult cerebellum rather than in pattern development. This premise is consistent with the normal pattern of zebrin II/aldolase C zones and stripes in mutant p39-/- mice. The alternating p39 parasagittal band pattern may reflect a role for p39 or Cdk5/p39 in the functional compartmentation of the cerebellum.     

Dendritic localization of type II calcium calmodulin-dependent protein kinase mRNA in normal and reinnervated rat hippocampus.

In situ hybridization histochemistry has revealed a diffuse distribution of the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) mRNA in the neuropil of regions containing CaM II kinase alpha-expressing cells and has led some to propose that it may be expressed in dendrites. In order to determine if CaM II kinase alpha mRNA is expressed in dendrites and if the gene encoding CaM II kinase alpha is regulated in response to synaptic reinnervation, we examined its expression in the hippocampus of normal rats, of rats that had received a unilateral injection of kainic acid and of rats with a unilateral entorhinal cortex lesion. The relatively specific elimination of the CA3 pyramidal cells by kainate lesions precisely correlated with the loss of CaM II kinase alpha cRNA hybridization in the stratum radiatum as well as the stratum pyramidale. Following entorhinal cortex lesions, during the period of new synapse formation in the dentate gyrus, there was no detectable change in the level of CaM II kinase alpha gene expression. These data suggest that CaM II kinase alpha mRNA is expressed in the dendrites of hippocampal pyramidal cells and, therefore, is likely to be expressed in dendrites in other regions of the central nervous system exhibiting CaM II kinase alpha cRNA labeling in the neuropil. However, changes in expression were not found to accompany new synapse formation.     

Immunohistochemical localization of secretogranin II in the rat cerebellum

Secretogranin II (chromogranin C) is a peptide related to chromogranin A and secretogranin I (chromogranin B) which is secreted by a regulated pathway from both neurons and endocrine cells. In the present study we have determined by light microscopic immunocytochemistry its distribution in the cerebellum and in adjacent brain stem regions. Secretogranin II was found to be widely distributed throughout the gray matter of these regions. Highly immunoreactive structures in the cerebellar cortex included the majority of climbing fibers, a large number of mossy fibers, sparse varicose fibers in the molecular layer and a subpopulation of neuronal perikarya in the granule cell layer. The location and shape of these neurons are very similar to those of a novel type of cerebellar neurons which has been recently described. A moderate level of immunoreactivity was observed on fibers travelling among Purkinje cells and parallel to the pial surface in the Purkinje cell layer. A variable, but in general low, degree of immunoreactivity was also detectable in the perikarya of Purkinje cells. In the deep cerebellar nuclei a loose network of secretogranin II-positive fibers was visible. Neurons of the nuclei, however, were non-immunoreactive. A dense network of highly immunoreactive fibers was found throughout the brain stem regions adjacent to the cerebellum. Our results indicate that secretogranin II has in the cerebellum and adjacent regions a distribution more widespread than that of known regulatory peptides and suggest that the peptide-mediated signaling in the cerebellum plays a role more important that has been acknowledged so far.     

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.     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400.

P400 protein, which is identical to the inositol 1,4,5-trisphosphate receptor protein, is a glycoprotein closely associated with the membranes of Purkinje cells. Three types of monoclonal antibodies against P400 protein were employed for the immunohistochemical detection of Purkinje cells in the cerebellum and brainstem of the normal and reeler mouse. Purkinje cells in both types of mice were immunoreactive against anti-P400 antibodies, and the soma, dendrites, axon and even terminal boutons in the cerebellar and vestibular nuclei could be clearly visualized. In the cerebellum of the reeler mutant, the heterotopic Purkinje cells both within and below the granule cell layer were also immunopositive and could be clearly differentiated from the deep cerebellar nuclei, in which neurons were immunonegative. The molecular layer of the reeler cerebellum varied in thickness and certain parts were completely defective. The dendrites within the molecular layer extended from Purkinje cells whose cell bodies were located in the normal position, abnormally in the granule cell layer, or at the surface of the central mass. Outside the cortex of the cerebellum, ectopic Purkinje cells were demonstrated in 3 cerebellar nuclei, the cerebellar medulla and peduncle, and brainstem of the normal and reeler mouse.     

Region-specific expression of messenger RNAs encoding GABAA receptor subunits in the developing rat brain.

The distribution and levels of messenger RNAs encoding the alpha 1, beta 1, beta 2, beta 3, and gamma 2 subunits of the GABAA receptor in the developing and adult rat brain were investigated using quantitative in situ hybridization histochemistry and subunit-specific probes. Regional localization of the subunit messenger RNAs was determined with film autoradiography and expression in identified neuronal cell populations was examined using higher resolution techniques. Each of the GABAA receptor subunit messenger RNAs exhibits a distinct pattern of localization in the developing and adult brain. Of the subunits examined, the alpha 1, beta 2, and gamma 2 are the most abundant and are found in many brain regions, including the olfactory bulb, cortex, hippocampus, thalamic nuclei, and inferior colliculus. In addition, these subunit messenger RNAs are prominent in the cerebellum where virtually all cells of the deep cerebellar nuclei and Purkinje cell layer are labeled. The levels of most of the subunit messenger RNAs, with the exception of that encoding the beta 1 subunit, increase during postnatal development. While the alpha 1, beta 2, and gamma 2 subunit messenger RNAs rise in parallel in many regions and identified cell populations, different subsets of receptor subunit messenger RNAs are co-ordinately expressed at other sites. The greatest increases in subunit messenger RNA levels occur in the cerebellar cortex during the second postnatal week, a period coincident with cerebellar maturation. The co-distribution of different GABAA receptor subunit messenger RNAs in various regions of the developing and adult nervous systems supports the hypothesis that multiple receptor compositions exist. Moreover, that different subunit messenger RNAs exhibit coordinate changes in expression in different regions and cell populations suggests that receptor gene expression is modulated by cell type-specific signals. The temporal changes in subunit messenger RNA levels in the cerebellum raise the possibility that synaptogenesis may play a role in receptor gene regulation in this brain region.     

Formation of presynaptic dendrites in the rat cerebellum following neonatal X-irradiation

The ultrastructure of the cerebellum was studied after X-irradiation of the whole head of newborn rats. The neuronal elements forming the cerebellar circuitry in rats 30–60 days after irradiation were limited to climbing fibers, mossy fibers, and Purkinje and Golgi cells. Under these conditions the perikarya and dendrites of the Golgi neurons develop presynaptic vesicular grids. These unconventional presynaptic elements establish numerous synaptic contacts with spines and occasionally with shafts of Purkinje cell dendrites.The results indicate that interference with normal cerebellar development, such that granule, basket and stellate cells are absent, generates new types of cellular interactions during synaptogenesis which allow Golgi cells to express their latent potentiality to form presynaptic perikarya and dendrites. It is concluded that this latent potentiality is repressed during development of the normal cerebellum by the presence of the other interneurons.     

NMDAR1和GABA_A受体的α_1及α_3亚单位在成年猫小脑的定位分布

用免疫组织化学反应及 Nissl染色探讨了 NMDAR1及 GABAA受体α1 和α3亚单位在成年猫小脑皮质及小脑核的定位分布。结果表明 ,NMDAR1免疫反应产物主要分布在 Purkinje细胞胞质和分子层的树突 ,分子层的星形细胞和篮细胞以及颗粒细胞层的颗粒细胞和胶质细胞呈中等强度的阳性反应 ,小脑核的神经元胞质和部分突起着色明显。 GABAA受体的α1 亚单位免疫反应产物主要分布在 Purkinje细胞胞质和树突 ,分子层的星形细胞和胶质细胞呈弱阳性 ,小脑核的神经元阳性反应明显。GABAA 受体的α3亚单位免疫反应产物主要分布在 Purkinje细胞胞质和树突 ,分子层的星形细胞和篮细胞着色明显 ,胶质细胞呈免疫反应弱阳性 ,小脑核神经元及纤维着色明显。在 Purkinje细胞层 NMDAR1、GABAA受体α1 及α3亚单位免疫阳性神经元分别占 Purkinje细胞总数的 80 % ,61% ,88%。结论 :NMDAR1、GABAA受体的α1 及α3亚单位在成年猫小脑具有广泛的分布。这些受体在介导小脑的复杂功能中可能发挥重要的作用。     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum

Previous physiological and pharmacological studies have shown that the serotonin_2A (5-HT_2A) receptor is involved in cerebellar functions. However, the expression of 5-HT_2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT_2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT_2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT_2A receptors during the cerebellar development.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.     

Anosmin-1 stimulates outgrowth and branching of developing Purkinje axons

During development, Purkinje axons elongate along precise trajectories and acquire stereotypic branching patterns to innervate targets in the deep nuclei and cerebellar cortex. These processes are accomplished through cell-intrinsic mechanisms, whose operation is regulated by environmental signaling cues. Here, we show that Anosmin-1, the protein defective in the X-linked form of Kallmann syndrome, is one among such cues. Anosmin-1, that stimulates axon elongation and branching in the olfactory system, is expressed by Purkinje cells and deep nuclear neurons of the rat cerebellum during the ontogenetic period when Purkinje axons acquire their mature pattern. These neurons also express the putative Anosmin-1 receptor, fibroblast growth factor receptor 1. Application of Anosmin-1 to dissociated cultures of embryonic (embryonic day 17, E17) or postnatal (postnatal day 0, P0) rat cerebellar cells enhances neuritic elongation and exerts a strong promoting action on the budding of collateral branches and on the extension of terminal arbors. Opposite effects are observed when neutralizing anti-Anosmin-1 antibodies are applied to the same cultures. Comparable results are obtained by administering the protein or the blocking antibodies to organotypic cultures of postnatal (P0) rat cerebellum. In P10 cerebellar slices, Anosmin-1 does not enhance the spontaneous regenerative capabilities of severed Purkinje axons, but promotes the terminal outgrowth of injured neurites into embryonic neocortical explants apposed to the axotomy site. Although Anosmin-1 is unable to change the overall intrinsic growth competence of Purkinje cells, it exerts a powerful stimulatory action on the budding and extension of collateral branches and terminal plexus, contributing to the patterning of Purkinje axons.     

Calretinin-immunoreactive systems in the cerebellum and cerebellum-related lateral-line medullary nuclei of an elasmobranch, Scyliorhinus canicula

Calretinin immunohistochemistry was used to study the organization of some cerebellar structures and lateral line medullary nuclei of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula. In the cerebellar molecular layer, stellate cells are strongly calretinin-immunoreactive (CR-ir). Perikarya and dendrites of Purkinje cells are contacted by numerous stellate cell small CR-ir boutons. Some Purkinje cell perikarya are contacted by CR-ir climbing fibers forming complex axo-somatic contacts. In the granular layer, numerous CR-ir mossy fibers exhibited large swellings. Notable differences in density and diameter of mossy fibers are observed between the auricles and cerebellar body. Thin beaded CR-ir fibers are also present in the granular layer of the body. The lateral line nuclei of the octavolateralis region are comprised of a molecular-like cerebellar crest that covers the dorsal (electroreceptive) and the medial octavolateralis nuclei (mechanoreceptive). The cerebellar crest exhibited numerous CR-ir stellate cells. In the dorsal octavolateralis nucleus, the presence of conspicuous CR-ir cells and neuropil closely associated to the region of primary fiber terminals distinguishes it clearly from the medial nucleus, revealing major differences between the electroreceptive and mechanoreceptive primary nuclei of elasmobranchs. Moreover, CR distribution in the dogfish cerebellum showed interesting differences with those reported in cerebella of other vertebrates, indicating a high variability of cerebellar CR expression in phylogeny.