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Neuronal activity is believed to be important for brain development; however, it remains unclear as to how spatiotemporal distributions of synaptic excitation contribute to neural network formation. Bifurcated axons of cerebellar granule cells, parallel fibers (PFs), are made in an orderly inside-out manner during postnatal development. In this study, we induced a blockade of neurotransmitter release from specific bundles of developing PFs and tested the effects of biased PF inputs on cerebellar development. The blockade of different layers of PFs at different developmental times results in varying degrees of abnormal cerebellar development. Furthermore, cerebellar network abnormalities are not restored when PF inputs are restored in adulthood and, hence, result in motor dysfunction. We thus conclude that spatiotemporally unbiased synaptic transmission from sequentially developed PFs is crucial for cerebellar network formation and motor function, supporting the idea that unbiased excitatory synaptic transmission is crucial for network formation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber-forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1-/- animals showed a defective PF-Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1-/- Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1-/- Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1-/- Cgcs. In line with our in vitro data, Pkn1-/- mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.

An axon is thought to faithfully conduct action potentials to its terminals. However, many features of the axon and axon terminals, especially at inhibitory synapses, remain unknown. By directly recording from the axon and terminal of a cultured cerebellar Purkinje cell (PC), we demonstrate that low membrane excitability of axon terminals shapes synaptic output. Simultaneous measurements of presynaptic capacitance and evoked IPSCs revealed PC axon terminals contained large readily releasable synaptic vesicles that exhibited a low release probability. Nevertheless, IPSCs evoked by stimulating a PC soma underwent frequency-dependent depression. Direct axonal recordings showed that high-frequency action potentials were faithfully conducted over axonal bifurcations but were attenuated around terminals. Sparse Na(+) channels relative to enriched voltage-gated K(+) channels in terminals caused short-term depression of IPSCs by reducing Ca(2+) influx. Together with confirmation in slice recordings, our findings reveal a presynaptic mechanism that shapes short-term synaptic depression without depleting releasable vesicles., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Based on extrapolation from excitatory synapses, it is often assumed that depletion of the releasable pool of synaptic vesicles is the main factor underlying depression at inhibitory synapses. In this issue of Neuron, using subcellular patch-clamp recording from inhibitory presynaptic terminals, Kawaguchi and Sakaba (2015) show that at Purkinje cell-deep cerebellar nuclei neuron synapses, changes in presynaptic action potential waveform substantially contribute to synaptic depression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The spatiotemporal pattern of synaptic inputs to the dendritic tree is crucial for synaptic integration and plasticity. However, it is not known if input patterns driven by sensory stimuli are structured or random. Here we investigate the spatial patterning of synaptic inputs by directly monitoring presynaptic activity in the intact mouse brain on the micron scale. Using in vivo calcium imaging of multiple neighbouring cerebellar parallel fibre axons, we find evidence for clustered patterns of axonal activity during sensory processing. The clustered parallel fibre input we observe is ideally suited for driving dendritic spikes, postsynaptic calcium signalling, and synaptic plasticity in downstream Purkinje cells, and is thus likely to be a major feature of cerebellar function during sensory processing.

Paranodal axo-glial junctions (PNJs) play an essential role in the organization and maintenance of molecular domains in myelinated axons. To understand the importance of PNJs better, we investigated cerebroside sulfotransferase (CST; a sulfatide synthetic enzyme)-deficient mice, which partially lack PNJs in both the central nervous system (CNS) and the peripheral nervous system (PNS). Previously, we reported that axonal mitochondria at the nodes of Ranvier in the PNS were large and swollen in CST-deficient mice. Although we did not observed significant defects in the nodal regions in several areas of the CNS, myelinated internodal regions showed many focal swellings in Purkinje cell axons in the cerebellum, and the number and the size of swellings increased with age. In the present analysis of various stages of the swellings in 4-12-week-old mutant mice, calbindin-positive axoplasm swellings started to appear at an early stage. After that, accumulation of neurofilament and mitochondria gradually increased, whereas deposition of amyloid precursor protein became prominent later. Ultrastructural analysis showed accumulations of tubular structures closely resembling smooth endoplasmic reticulum (ER). Staining of cerebellar sections of the mutant mice for type I inositol 1,4,5-trisphosphate receptor (IP3 R1) revealed high immunoreactivity within the swellings. This IP3 R1 deposition was the initial change and was not observed in development prior to the onset of myelination. This suggests that local calcium regulation through ER was involved in these axonal swellings. Therefore, in addition to the biochemical composition of the internodal myelin sheath, PNJs might also affect maintenance of axonal homeostasis in Purkinje cells., (© 2014 Wiley Periodicals, Inc.)

The establishment of precise neural circuits during development involves a variety of contact-mediated and secreted guidance molecules that are expressed in a complementary fashion by different cell types. To build a functional circuit, each cell type must first trigger an intrinsic genetic program that is led by their environment at a key time point. It is therefore essential to identify the different cell-specific and stage-specific transcriptional profiles expressed by neurons. However, very few studies have been done to address this issue thus far. Herein, we have carried out a large-scale quantitative real-time PCR analysis of all classical axon guidance molecules (i.e., Semaphorins, Netrins, Ephrins, and Slits) and their receptors expressed by Purkinje cells (PCs) at specific stages of postnatal cerebellar development in vivo. Most cerebellar connections are setup in a well-characterized sequential manner during postnatal development and lead to the fine regulation of the PC, the sole output of the structure. Our analysis of the relative expression of these guidance cues has uncovered a dynamic expression pattern corresponding to specific stages of cerebellar development, thus providing a starting point for studying the role of these axon guidance molecules in cerebellar wiring.

The spike propagation on nerve axons, like synaptic transmission, is essential to ensure neuronal communication. The secure propagation of sequential spikes toward axonal terminals has been challenged in the neurons with a high firing rate, such as cerebellar Purkinje cells. The shortfall of spike propagation makes some digital spikes disappearing at axonal terminals, such that the elucidation of the mechanisms underlying spike propagation reliability is crucial to find the strategy of preventing loss of neuronal codes. As the spike propagation failure is influenced by the membrane potentials, this process is likely caused by altering the functional status of voltage-gated sodium channels (VGSC). We examined this hypothesis in Purkinje cells by using pair-recordings at their somata and axonal blebs in cerebellar slices. The reliability of spike propagation was deteriorated by elevating spike frequency. The frequency-dependent reliability of spike propagation was attenuated by inactivating VGSCs and improved by removing their inactivation. Thus, the functional status of axonal VGSCs influences the reliability of spike propagation.

Axon degeneration is a critical pathological feature of many neurodegenerative diseases. Misregulation of local axonal ion homeostasis has been recognized as an important yet understudied mechanism for axon degeneration. Here we report a chemically induced, recessive mouse mutation, vacillator (vac), which causes ataxia and concomitant axon degeneration of cerebellar Purkinje cells. By positional cloning, we identified vac as a point mutation in the calcineurin-like EF hand protein 1 (Chp1) gene that resulted in the production of mutant CHP1 isoforms with an amino acid substitution in a functional EF-hand domain or a truncation of this motif by aberrant splicing and significantly reduced protein levels. CHP1 has been previously shown to interact with the sodium hydrogen exchanger 1 (NHE1), a major regulator of intracellular pH. We demonstrated that CHP1 assists in the full glycosylation of NHE1 that is necessary for the membrane localization of this transporter and that truncated isoforms of CHP1 were defective in stimulating NHE1 biosynthetic maturation. Consistent with this, membrane localization of NHE1 at axon terminals was greatly reduced in Chp1-deficient Purkinje cells before axon degeneration. Furthermore, genetic ablation of Nhe1 also resulted in Purkinje cell axon degeneration, pinpointing the functional convergence of the two proteins. Our findings clearly demonstrate that the polarized presynaptic localization of NHE/CHP1 is an important feature of neuronal axons and that selective disruption of NHE1-mediated proton homeostasis in axons can lead to degeneration, suggesting that local regulation of pH is pivotal for axon survival.

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABAA receptors (GABAARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice ("γ2 knockdown mice") with an engineered allele of the GABAAR γ2 subunit gene which produced a mosaic expression of synaptic GABAARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABAARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABAARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABAARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input, while depressing synaptic gain for sustained parallel fiber inputs.

Background: Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated., Results: We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step., Conclusions: Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.

Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By using a recent improvement in voltage-sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than approximately 250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission.

Background: During development, neurons modify their axon growth mode switching from an elongating phase, in which the main axon stem reaches the target territory through growth cone-driven extension, to an arborising phase, when the terminal arbour is formed to establish synaptic connections. To investigate the relative contribution of cell-autonomous factors and environmental signals in the control of these distinct axon growth patterns, we examined the neuritogenesis of Purkinje neurons in cerebellar cultures prepared at elongating (embryonic day 17) or arborising (postnatal day zero) stages of Purkinje axon maturation., Methodology/principal Findings: When placed in vitro, Purkinje cells of both ages undergo an initial phase of neurite elongation followed by the development of terminal ramifications. Nevertheless, elongation of the main axon stem prevails in embryonic Purkinje axons, and many of these neurons are totally unable to form terminal branches. On the contrary, all postnatal neurites switch to arbour growth within a few days in culture and spread extensive terminal trees. Regardless of their elongating or arborising pattern, defined growth features (e.g. growth rate and tree extension) of embryonic Purkinje axons remain distinct from those of postnatal neurites. Thus, Purkinje neurons of different ages are endowed with intrinsic stage-specific competence for neuritic growth. Such competence, however, can be modified by environmental cues. Indeed, while exposure to the postnatal environment stimulates the growth of embryonic axons without modifying their phenotype, contact-mediated signals derived from granule cells specifically induce arborising growth and modulate the dynamics of neuritic elongation., Conclusions/significance: Cultured Purkinje cells recapitulate an intrinsically coded neuritogenic program, involving initial navigation of the axon towards the target field and subsequent expansion of the terminal arborisation. The execution of this program is regulated by environmental signals that modify the growth competence of Purkinje cells, so to adapt their endogenous properties to the different phases of neuritic morphogenesis.

The shapes of dendritic arbors are fascinating and important, yet the principles underlying these complex and diverse structures remain unclear. Here, we analyzed basal dendritic arbors of 2,171 pyramidal neurons sampled from mammalian brains and discovered 3 statistical properties: the dendritic arbor size scales with the total dendritic length, the spatial correlation of dendritic branches within an arbor has a universal functional form, and small parts of an arbor are self-similar. We proposed that these properties result from maximizing the repertoire of possible connectivity patterns between dendrites and surrounding axons while keeping the cost of dendrites low. We solved this optimization problem by drawing an analogy with maximization of the entropy for a given energy in statistical physics. The solution is consistent with the above observations and predicts scaling relations that can be tested experimentally. In addition, our theory explains why dendritic branches of pyramidal cells are distributed more sparsely than those of Purkinje cells. Our results represent a step toward a unifying view of the relationship between neuronal morphology and function.

Most axons in the mammalian brain are unmyelinated and thin with pre-synaptic specializations (boutons) along their entire paths. The parallel fibers in the cerebellum are examples of such axons. Unlike most thin axons they have only one branch point. The granule cell soma, where they originate, can fire bursts of action potentials with spike intervals of about 2 ms. An important question is whether the axons are able to propagate spikes with similarly short intervals. By using extracellular single-unit and population-recording methods we showed that parallel fibers faithfully conduct spikes at high frequencies over long distances. However, when adding 20 microm ZD7288 or 1 mm Cs(+), or reducing the temperature from 35 to 24 degrees C, the action potentials often failed even when successfully initiated. Ba(2+)(1 mm), which blocks Kir channels, did not reproduce these effects. The conduction velocity was reduced by ZD7288 but not by Ba(2+). This suggests that the parallel fibers have an H-current that is active at rest and that is important for their frequency-following properties. Interestingly, failures occurred only when the action potential had to traverse the axonal branch point, suggesting that the branch point is the weakest point in these axons.

Very fast oscillations (VFO; > 75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (approximately 40 Hz, evoked by nicotine) or in isolation [evoked by nicotine in combination with gamma-aminobutyric acid (GABA)(A) receptor blockade]. The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1000 multicompartment Purkinje cells) replicates the experimental data when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by g(Na) inactivation, and experiments prolonging this inactivation time constant, with beta-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons that are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model) and axonal firing rates are much lower in the uncoupled state than in the coupled state.

Mossy and climbing fibers convey a broad array of signals from vestibular end organs to Purkinje cells in the vestibulo-cerebellum. We have shown previously that Purkinje cell simple spikes (SSs) and climbing fiber-evoked complex spikes (CSs) in the mouse uvula-nodulus are arrayed in 400 microm wide sagittal climbing fiber zones corresponding to the rotational axes of the vertical semicircular canals. It is often assumed that mossy fibers modulate a higher frequency of SSs through the intermediary action of granule cells whose parallel fibers course through the Purkinje cell dendritic tree. This assumption is complicated by the diffuse topography of vestibular primary afferent mossy fiber projections to the uvula-nodulus and the dispersion of mossy fiber signals along folial axes by parallel fibers. Here we measure this parallel fiber dispersion. We made microinjections of neurobiotin into the molecular layers of different folia within the mouse vestibulo-cerebellum and measured the distribution of granule cells retrogradely labeled by the injected neurobiotin. Sixty-two percent of labeled granule cells were located outside a 400 microm sagittal zone flanking the injection site. The dispersion of labeled granule cells was approximately 2.5 mm along folial axes that were 2.7-2.9 mm wide. Our data suggest that topographic specificity of SSs could not be attributed to the topography of vestibular primary afferent mossy fiber-granule cell projections. Rather the response specificity of SSs must be attributed to other mechanisms related to climbing fiber-evoked Purkinje cell and interneuronal activity.

In a previous study, it was shown that populations of climbing fibers, derived from the inferior olivary complex (IOC) contain the peptide corticotropin releasing factor (CRF) and that the expression of this peptide in climbing fibers could be modulated by the level of activity in olivary afferents. The intent of this study was to determine if there was comparable plasticity in the distribution of the type 1 CRF receptor (CRF-R1) in the cerebellum of the rat. Our results indicate that CRF-R1 was localized primarily to Purkinje cell somata and their primary dendrites and granule cells. In addition, scattered immunolabeling was present over the somata of Golgi cells, basket cells and stellate cells, as well as Bergmann glial cells and their processes. IOC stimulation for 30 min at 1 Hz increased CRF-R1 expression in molecular layer interneurons and processes of Bergmann glial cells. Little to no effect on CRF receptor distribution was observed in Purkinje cells, granule cells, or Golgi cells. IOC stimulation at 5 Hz however, increased CRF-R1 expression in the processes of Bergmann glial cells while decreasing its expression in basket, stellate and, to some extent, in Purkinje cells. The present results suggest that there is activity-dependent plasticity in CRF-R1 expression that must be considered in defining the mechanism by which the CRF family of peptides modulates activity in cerebellar circuits. The present results also suggest that the primary targets of CRF released from climbing fibers are Bergmann glial cells and interneurons in the molecular layer. Further, interneurons responded with a decrease in receptor expression following more intense levels of stimulation suggesting the possibility of internalization of the receptor. In contrast, Bergmann glial cells showed an increased expression in receptor expression. These data suggest that CRF released from climbing fibers may modulate the physiological properties of basket and stellate cells as well as having a heretofore unidentified and potentially unique effect on Bergmann glia.

Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes (Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5(flox/flox) with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell-specific promoter, pcp2. In Atg5(flox/flox); pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons.

Autophagy is a regulated lysosomal degradation process that involves autophagosome formation and transport. Although recent evidence indicates that basal levels of autophagy protect against neurodegeneration, the exact mechanism whereby this occurs is not known. By using conditional knockout mutant mice, we report that neuronal autophagy is particularly important for the maintenance of local homeostasis of axon terminals and protection against axonal degeneration. We show that specific ablation of an essential autophagy gene, Atg7, in Purkinje cells initially causes cell-autonomous, progressive dystrophy (manifested by axonal swellings) and degeneration of the axon terminals. Consistent with suppression of autophagy, no autophagosomes are observed in these dystrophic swellings, which is in contrast to accumulation of autophagosomes in the axonal dystrophic swellings under pathological conditions. Axonal dystrophy of mutant Purkinje cells proceeds with little sign of dendritic or spine atrophy, indicating that axon terminals are much more vulnerable to autophagy impairment than dendrites. This early pathological event in the axons is followed by cell-autonomous Purkinje cell death and mouse behavioral deficits. Furthermore, ultrastructural analyses of mutant Purkinje cells reveal an accumulation of aberrant membrane structures in the axonal dystrophic swellings. Finally, we observe double-membrane vacuole-like structures in wild-type Purkinje cell axons, whereas these structures are abolished in mutant Purkinje cell axons. Thus, we conclude that the autophagy protein Atg7 is required for membrane trafficking and turnover in the axons. Our study implicates impairment of axonal autophagy as a possible mechanism for axonopathy associated with neurodegeneration.

In the last few years Purkinje cells have become a most interesting model to investigate cellular/molecular mechanisms of axon regeneration and plasticity. Adult Purkinje cells are most peculiar for their weak cell body response to axotomy, which is accompanied by a strong resistance to injury and a virtually absolute inability to regenerate severed neurites, even in the presence of favourable environmental conditions. The same neurons show a vigorous intrinsic inclination toward axonal sprouting and structural plasticity, which can be elicited by removing extrinsic growth-inhibitory cues. These features gradually develop during early postnatal life, but the underlying mechanisms and biological significance remain unclear. This article reviews recent studies aimed at addressing these questions with respect to the general issue of brain repair. Indeed, understanding the reasons for the extremely poor regenerative capacity of Purkinje cells will be most important to elucidate basic biological mechanisms of axon regeneration and plasticity, and to promote circuit rewiring in the adult CNS.

Cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses in rodents provides a good model to study mechanisms underlying postnatal development of synaptic functions and elimination of redundant synapses in the central nervous system. At birth, each PC is innervated by multiple CFs. Then, single CF input is selected, matured and strengthened, while surplus CFs are eliminated. By the end of the third postnatal week, most PCs become innervated by single CFs. This up-date article aims to provide an overview of recent studies on the mechanisms of this process.

The molecular layer of the cerebellar cortex is populated by glial progenitors that express ionotropic glutamate receptors and extend numerous processes among Purkinje cell dendrites. Here, we show that release of glutamate from climbing fiber (CF) axons produces AMPA receptor currents with rapid kinetics in these NG2-immunoreactive glial cells (NG2+ cells) in cerebellar slices. NG2+ cells may receive up to 70 discrete inputs from one CF and, unlike mature Purkinje cells, are often innervated by multiple CFs. Paired Purkinje cell-NG2+ cell recordings show that one CF can innervate both cell types. CF boutons make direct synaptic junctions with NG2+ cell processes, indicating that this rapid neuron-glia signaling occurs at discrete sites rather than through ectopic release at CF-Purkinje cell synapses. This robust activation of Ca2+-permeable AMPA receptors in NG2+ cells expands the influence of the olivocerebellar projection to this abundant class of glial progenitors.

By blocking electrical activity in the cerebellar cortex the Purkinje cell dendrites become a uniform territory with a high density of spines all bearing the glutamate receptor delta2 subunit (GluRdelta2) and being mainly innervated by parallel fibers. Such a subunit, which is constitutively targeted specifically to the parallel fiber synapses, appears in the spines contacted by the climbing fibers before they disconnect from the target. A similar pattern of hyperspiny transformation and innervation occurs a few days after a subtotal lesion of the inferior olive, the source of climbing fibers. During the climbing fiber reinnervation process which follows the removal of the electrical block or by collateral sprouting of surviving inferior olive neurons, the new active climbing fibers establish synaptic contacts with proximal dendritic spines that bear GluRdelta2s. After, they repress these subunits and displace the parallel fibers to the distal dendritic territory. These findings suggest the following operational principle in the axonal competition for a common target. The Purkinje cells have an intrinsic phenotypic profile which is compatible with the parallel fiber innervation, this mode being operational in targets innervated by a single neuronal population, like the neuromuscular system. An additional input, the climbing fibers, in order to achieve its own territory on the proximal dendrite needs the ability to displace the competitor. Such an inhibition is activity-dependent and the activity needs to be present in order to allow the climbing fiber to maintain its territory, even when the developmental period is over.

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

Distinct classes of GABAergic synapses are segregated into subcellular domains (i.e., dendrite, soma, and axon initial segment-AIS), thereby differentially regulating the input, integration, and output of principal neurons. In cerebellum, for example, basket interneurons make exquisitely precise "pinceau synapses" on AIS of Purkinje neurons, but the underlying mechanism is unknown. Using BAC transgenic reporter mice, we found that basket axons always contacted Purkinje soma before innervating AIS. This synapse targeting process followed the establishment of a subcellular gradient of neurofascin186 (NF186), an L1 family immunoglobulin cell adhesion molecule (L1CAM), along the Purkinje AIS-soma axis. This gradient was dependent on ankyrinG, an AIS-restricted membrane adaptor protein that recruits NF186. In the absence of neurofascin gradient, basket axons lost directional growth along Purkinje neurons and precisely followed NF186 to ectopic locations. Disruption of NF186-ankyrinG interactions at AIS reduced pinceau synapse formation. These results implicate ankyrin-based localization of L1CAMs in subcellular organization of GABAergic synapses.

Previous studies have described the embryonic and postnatal development of CRF, as well as the type 1 CRF receptor in the mouse cerebellum. The present immunohistochemical study localizes the cellular distribution of the type 2 CRF receptor (CRF-R2) during postnatal development of the mouse cerebellum. Western blot analysis indicates that the antibody used in this analysis recognizes both a full-length and a truncated isoform of the type 2 receptor. We propose that each isoform has a unique cellular distribution. In the present study, the postnatal (P) development (P0-P14) and cellular localization of CRF-R2 in different cell types was analyzed using PAP and double-label fluorescent immunohistochemistry; cell-specific antibodies were used to identify cells expressing CRF-R2 at different stages of postnatal development. At P0, CRF-R2 immunoreactivity was localized within the somata of Purkinje cells and migrating GABAergic interneurons. CRF-R2 was first observed in the initial axonal segments of some Purkinje cells at P5, and was evident in many Purkinje cell axon hillocks at P8. Punctate immunoreactivity is present in the molecular layer by P5 and is interpreted to be immunolabeled parallel fibers. Between P8 and P14, CRF-R2 immunostaining is present in the initial axonal segments of Golgi cells, within the internal granule cell layer. Finally, CRF-R2 is present in both radial glia in the molecular layer as well as in astrocytes in the white matter and internal granule cell layer from P5 to P14. The present results suggest that CRF-R2, both the truncated and the full-length isoforms, are present in the developing cerebellum, each with a unique cellular distribution. The immunohistochemical evidence indicates that the truncated isoform of the type 2 CRF receptor is in the axons of several different types of cerebellar cortical neurons, and suggests that CRF could play a role in cerebellar development by modulating the release of transmitters from excitatory and/or inhibitory interneurons, which in turn could directly alter the maturation of cerebellar circuits. In contrast, the binding of a ligand to the full-length isoform of CRF-R2 or to CRF-R1, both in a postsynaptic location, may have a more direct effect on regulating the responsiveness of these cells to growth factors or neurotransmitters released from afferent axons by regulating permeability of ion channels or altering second messenger systems.

Axonal regeneration in the mammalian CNS is a property of immature neurons that is lost during development. Using organotypic culture of cerebellum, we have shown that in vitro Purkinje cells lose their regenerative capacity in parallel with the process of myelination. We have investigated whether myelination is involved in the age-dependent loss of regeneration of these neurons. By applying a high dose of bromodeoxyuridine in the culture medium of newborn cerebellar slices during the first 3 d in vitro, we have succeeded in obtaining cultures with oligodendrocyte depletion, together with a lack of ameboid microglia and enhancement of Purkinje cell survival. These cultures, after 14 d in vitro, are completely devoid of myelin. We have compared the ability of Purkinje cells to regenerate their axons in the presence or absence of myelin. Purkinje cells in cerebellar explants taken at birth, treated with bromodeoxyuridine and axotomized after 7 d in vitro, survive better than similar neurons in untreated cultures. However, despite the lack of myelin and the enhanced survival, Purkinje cells do not regenerate, whereas they do regenerate when the axotomy is done at postnatal day 0. Thus, the Purkinje cell developmental switch from axonal regeneration to lack of regeneration does not appear to be regulated by myelin.

The highly specific connection patterns of the mature CNS are shaped through finely regulated processes of axon growth and retraction. To investigate the relative contribution of cell-autonomous mechanisms and extrinsic cues in these events, we examined the development of Purkinje axon intracortical plexus in the rat cerebellum. During the first postnatal week, several new processes sprout from focal swellings along the initial portion of the Purkinje neurite and spread in the granular layer. Intense structural plasticity occurs during the following week, with pruning of collateral branches and remodeling of terminal arbors. The mature distribution of the Purkinje infraganglionic plexus, confined within the most superficial portion of the granular layer, is attained at approximately postnatal day 15. A similar neuritic branching pattern is also developed by Purkinje cells grown in cultures of dissociated cerebellar cells or transplanted to extracerebellar CNS regions, suggesting that cell-autonomous mechanisms contribute to determining the Purkinje axon phenotype. The structural remodeling of Purkinje intracortical plexus is concomitant with the development of cerebellar myelin. To ask whether myelin-associated factors contribute to the morphological maturation of Purkinje neurites, we prevented normal myelinogenesis by killing oligodendrocyte precursors with 5'-azacytidine or by applying neutralizing antibodies against the myelin-associated neurite growth inhibitor Nogo-A. In both conditions, Purkinje axons retained exuberant branches, and the terminal plexus spanned the entire extent of the granular layer. Thus, the formation of Purkinje axon collaterals is, in part, controlled by intrinsic determinants, but their growth and distribution are regulated by environmental signals, among which are myelin-derived cues.

Purkinje cells aligned on the medio-lateral axis share a large proportion of their approximately 175,000 parallel fiber inputs. This arrangement has led to the hypothesis that movement timing is coded in the cerebellum by beams of synchronously active parallel fibers. In computer simulations I show that such synchronous activation leads to a narrow spike cross-correlation between pairs of Purkinje cells. This peak was completely absent when shared parallel fiber input was active in an asynchronous mode. To determine the presence of synchronous parallel fiber beams in vivo I recorded from pairs of Purkinje cells in crus IIa of anesthetized rats. I found a complete absence of precise spike synchronization, even when both cells were strongly modulated in their spike rate by trains of air-puff stimuli to the face. These results indicate that Purkinje cell spiking is not controlled by volleys of synchronous parallel fiber inputs in the conditions examined. Instead, the data support a model by which granule cells primarily control Purkinje cell spiking via dynamic population rate changes.

Long-distance axon regeneration requires the activation of a specific set of neuronal growth-associated genes. Adult Purkinje cells fail to upregulate these molecules in response to axotomy and show extremely weak regenerative properties. Nevertheless, starting from several months after injury, transected Purkinje axons undergo spontaneous sprouting. Here, we asked whether long-term injured Purkinje cells acquire novel intrinsic growth properties that enable them to upregulate growth-associated genes and sustain axon regeneration. To test this hypothesis, we examined axon growth and cell body changes in adult rat Purkinje neurons following axotomy and implantation of embryonic neocortical tissue or Schwann cells into the injury track. Purkinje cells that survived over 6 months after injury/transplantation displayed profuse sprouting in the injured cerebellum and developed extensive networks of terminal branches into embryonic neocortical grafts. In addition, severed Purkinje axons exposed to these transplants 6 months after injury grew faster than their counterparts confronted with the same environment immediately after axotomy. Nevertheless, long-term injured Purkinje cells failed to regenerate stem neurites into Schwann cell grafts, and, under all experimental conditions, they did not upregulate growth-associated molecules, including c-Jun, GAP-43, SNAP-25, and NADPH-diaphorase. These results indicate that the long-term injured Purkinje cells remain unable to activate the gene program required to sustain axon regeneration and their plasticity is restricted to terminal arbor remodeling. We propose that the delayed growth of injured Purkinje cells reflects an adaptive phenomenon by which the severed axon stump develops a new terminal arbor searching for alternative connections with local partners.

The highly specific relationships between parallel fiber (PF) and climbing fiber (CF) receptive fields in Purkinje cells and interneurons suggest that normal PF receptive fields are established by CF-specific plasticity. To test this idea, we used PF stimulation that was either paired or unpaired with CF activity. Conspicuously, unpaired PF stimulation that induced long-lasting, very large increases in the receptive field sizes of Purkinje cells induced long-lasting decreases in receptive field sizes of their afferent interneurons. In contrast, PF stimulation paired with CF activity that induced long-lasting decreases in the receptive fields of Purkinje cells induced long-lasting, large increases in the receptive fields of interneurons. These properties, and the fact the mossy fiber receptive fields were unchanged, suggest that the receptive field changes were due to bidirectional PF synaptic plasticity in Purkinje cells and interneurons.

In organotypic cultures, mouse Purkinje cells regenerate their axons from embryonic day 18 (E18) to postnatal day 0 (P0), die of apoptosis between P1 and P7, and survive but do not regenerate at P10. This particular behavior of Purkinje cells did not allow us to find out when the developmental switch between regeneration and lack of regeneration occurs. This work was undertaken to suppress Purkinje cell apoptosis and to investigate whether the same molecules that prevent apoptosis could also influence axonal growth, regeneration, or both. We show that brain-derived neurotrophic factor, neurotrophin 3, and insulin-like growth factor I have marginal effects on P3 Purkinje cell death. The use of Gö6976 [a protein kinase C (PKC) inhibitor] or a transgenic mouse line, in which a pseudosubstrate PKC inhibitor has been specifically targeted to Purkinje cells, prevents the massive Purkinje cell death in P3 organotypic cultures. In addition, Gö6976 promotes axotomized Purkinje cell survival up to P7. Thus, the inhibition of PKC activity is able to prevent Purkinje cell apoptosis in organotypic cultures. Furthermore, Gö6976 increases the outgrowth of dendrites and axon collateralization, as shown after gene gun enhanced green fluorescent protein transfection. In contrast, PKC inhibitors do not influence the axonal regenerative capability of Purkinje cell during development; the latter decreases between E18 and P7 after the same time course in control and Gö6976-treated slices. Thus, because inhibition of PKC prevents Purkinje cell death but does not affect axonal regeneration, these two events (cell death and axonal regeneration) seem to be differentially regulated.

The reestablishment of topographically organized connections is a necessary prerequisite to obtain a full anatomical repair following brain injury. One system where such an issue can be addressed is the olivocerebellar system, where, normally, clusters of inferior olive neurons project to neurochemically heterogeneous Purkinje cell compartments defined by the expression of cell-specific markers, such as zebrin II. To assess whether adult injured olivocerebellar axons that regenerate into cerebellar transplants are able to establish target-specific innervation of grafted Purkinje cells, we made surgical transections in the white matter of adult rat cerebella and placed solid grafts from the embryonic cerebellar anlage into the lesion site. The transplanted tissue developed highly organized minicerebella, in which Purkinje cells were distributed into distinct clusters of zebrin II-immunopositive or -immunonegative neurons, mimicking the cortical compartments present in the normal adult cerebellum. Olivocerebellar axons, labeled by biotinylated dextran amine tracing, regenerated into the transplants where they formed discrete patches made of several terminal arbors impinging upon Purkinje cell dendrites. Among 401 such climbing fiber patches, 96% exclusively innervated Purkinje cells of either phenotype and stopped at the border of the zebrin II(+/-) Purkinje cell clusters, whereas only 4% were extended across this boundary and innervated both zebrin II-positive and -negative Purkinje cells. The results obtained support the view that the embryonic cerebellar tissue provides target-specific information that can be decoded by ingrowing adult olivocerebellar axons in order to establish appropriate innervation patterns with zebrin II-positive or -negative Purkinje cell compartments., (©2002 Elsevier Science (USA).)

We have analyzed the influence of the calcium-dependent cell adhesion molecule, N-cadherin, on events leading to CNS myelination. Interactions between axons and oligodendrocyte progenitor (OP) cells and the CG4 OP cell line were examined by video-microscopy. OPs cocultured with dorsal root ganglia explants migrated around the culture and formed numerous contacts with axons. The duration of these contacts depended on the morphology of the OP, with OPs containing four or more processes forming long-lasting contacts and OPs with three or fewer processes forming short-termed contacts. Treatment with N-cadherin function blocking peptides approximately halved the duration of contacts made by cells with four or more processes but contact times for cells with three or less processes were unaffected. The L7 cadherin-blocking antibody and calcium withdrawal had similar effects. Contacts with axons regenerating from explants of adult retina, which do not have N-cadherin on their surface was examined. The contact duration of OPs to adult retina axons was short and similar in length to those formed between OPs and dorsal root ganglion axons in the presence of cadherin blocking reagents. Oligodendrocyte myelination was examined in organotypic rat cerebellar slice cultures, taken before myelination at postnatal day 10 and then allowed to myelinate in vitro over 7 days. When incubated with an N-cadherin function-blocking peptide, myelination of Purkinje cell axons was reduced to about half of control levels, while control peptides were without effect. Cadherin-blockade did not prevent maturation of OPs, since oligodendrocytes showing myelin basic protein immunostaining were still found in these cultures. However, many of the cell processes did not colocalize with calbindin-positive axons. From these data we conclude that N-cadherin is important for the initial contact between a myelinating oligodendrocyte and axons and significantly contributes to the success of myelination., (Copyright 2001 Academic Press.)

To investigate the tissue distribution and subcellular localization of ST3GalV (CMP-NeuAc:lactosylceramide alpha2,3 sialyltransferase/GM3 synthase) in the adult mouse, we generated two antisera against mouse ST3GalV that were designated CS2 (directed against amino acids K227-I272) and CS14 (directed against amino acids D308-H359). We previously reported that CS2 antiserum stains medial and trans-Golgi cisternae in all cell types investigated. In neural tissue, however, CS14 antiserum reveals a subpopulation of ST3GalV with a subcellular distribution complementary to CS2 antiserum. CS14 antiserum strongly stains axons in cortical, cerebellar, brainstem, and spinal cord tissue sections. The subcellular localization of neuronal ST3GalV is maintained in primary cultures of rat hippocampal neurons and in PC12 cells. In PC12 cells, ST3GalV localization evolves during NGF-induced differentiation such that a pool of enzyme leaves the Golgi for a distal compartment in conjunction with neurite outgrowth. In PC12 cells transfected with an epitope-tagged form of ST3GalV, staining for the epitope tag coincides with expression of endogenous enzyme. The non-Golgi pool of ST3GalV does not colocalize with markers for the trans-Golgi network, endosome, or synaptic vesicles, nor is it detected on the cell surface. Distinct subpopulations of ST3GalV imply that ganglioside synthesis can occur outside of the Golgi or, alternatively, that a portion of the total ST3GalV pool subserves a nonenzymatic function. Significantly fewer transfected cells were found in PC12 cultures treated with plasmid encoding ST3GalV than in cultures treated with control plasmid, indicating that the expression of ST3GalV in excess of endogenous levels results in either cell death or a decreased rate of cell division.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

The morphology of olivocerebellar (OC) axons originating from the inferior olive (IO) was investigated in the rat by reconstructing the entire trajectories of single axons that had been labeled with biotinylated dextran amine. Virtually all of the OC axons entered the cerebellum through the inferior cerebellar peduncle (ICP) contralateral to the IO, with a few exceptions. Although most OC projection was contralateral, a few axons projected bilaterally by crossing the midline within the cerebellum. Collaterals of OC axons could be classified into thick branches and thin collaterals. Thick branches of each OC axon (6.1 +/- 3.7/OC axon, mean +/- SD for n = 16 axons) terminated as climbing fibers (CFs) on single Purkinje cells (PCs) in a one-to-one relationship. Besides terminal arborization around PC thick dendrites, CFs had terminals that surrounded a PC soma, fine branchlets that extended transversely in the molecular layer, and thin retrograde collaterals that re-entered the PC and granular layers. Innervation of a single PC by two CFs originating from the same axon was seen, although infrequently. Concerning thin collaterals, about half of the OC axons had one or only a few collaterals terminating in the white matter of the ICP, most had 1 to 6 collaterals terminating in a single cerebellar nucleus, and all had 3 to 16 collaterals that terminated mainly in the granular layer, but occasionally in the cerebellar white matter and the PC layer. Some swellings of thin collaterals touched somata of presumed Golgi cells and PCs. No OC axons terminated solely in the ICP, cerebellar nucleus or granular layer without giving rise to CFs., (Copyright 1999 Wiley-Liss, Inc.)

To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va-associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein-myosin Va tail construct. Myosin Va-associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.

The semaphorins constitute a large gene family of transmembrane and secreted molecules, many of which are expressed in the nervous system. Genetic studies in Drosophila have revealed a role for semaphorins in axon guidance and synapse formation, and several in vitro studies in mice have demonstrated a dramatic chemorepellent effect of semaphorin III (Sema III) on the axons of several populations of neurons. To investigate the function of Sema III during in vivo axon guidance in the mammalian CNS, we studied the development of axonal projections in mutant mice lacking Sema III. Projections were studied for which either the in vitro evidence suggests a role for Sema III in axon guidance (e.g., cerebellar mossy fibers, thalamocortical axons, or cranial motor neurons) or the in vivo expression suggests a role for Sema III in axon guidance (e.g., cerebellar Purkinje cells, neocortex). We find that many major axonal projections, including climbing fiber, mossy fiber, thalamocortical, and basal forebrain projections and cranial nerves, develop normally in the absence of Sema III. Despite its in vitro function and in vivo expression, it appears as if Sema III is not absolutely required for the formation of many major CNS tracts. Such data are consistent with recent models suggesting that axon guidance is controlled by a balance of forces resulting from multiple guidance cues. Our data lead us to suggest that if Sema III functions in part to guide the formation of major axonal projections, then it does so in combination with both other semaphorins and other families of guidance molecules., (Copyright 1998 Academic Press.)

The ability of Schwann cells to induce the regeneration of severed olivocerebellar and Purkinje cell axons across an injury up to their deafferented targets was tested by transplanting freshly dissociated cells from newborn rat sciatic nerves into surgically lesioned adult cerebella. The grafted glial cells consistently filled the lesion gap and migrated into the host parenchyma. Transected olivocerebellar axons vigorously regenerated into the graft, where their growth pattern and direction followed the arrangement of Schwann cell bundles. Although some of these axons terminated within the transplant, many of them rejoined the cerebellar parenchyma beyond the lesion. Here, their fate depended on the territory encountered. No growth occurred in the white matter. Numerous fibres penetrated into the granular layer and formed terminal branches that remained confined within this layer. A few of them, however, regenerated up to the molecular layer and formed climbing fibres on Purkinje cell dendrites. By contrast, the growth of transected Purkinje cell axons into the grafts was very poor. These results underscore the different intrinsic responsiveness of Purkinje cell and olivocerebellar axons to the growth-promoting action of Schwann cells, and show that the development and outcome of the regenerative phenomena is strongly conditioned by the spatial organization and specific features of the environmental cues encountered by the outgrowing axons along the course they follow. However, Schwann cells effectively bridge the lesion gap, induce the regeneration of olivocerebellar axons, and direct their growth up to the deafferented host cortex, where some of them succeed in reinnervating their natural targets.

Purkinje cells are among the most resistant neurons to axotomy and the most refractory to axonal regeneration. By using organotypic cultures, we have studied age- and environment-related factors implicated in Purkinje cell survival and axonal regeneration. Most Purkinje cells taken from 1- to 5-d-old rats, the period in which these neurons are engaged in intense synaptogenesis and dendritic remodeling, die 1 week after plating, whereas if cultured before or after this period, Purkinje cells survive, even in the absence of deep nuclear neurons, their postsynaptic targets. Cerebellar slices taken from 10-d-old rats and kept in vitro for 1 week acquire a cellular composition resembling mature cerebellum. Their Purkinje cells are resistant to axotomy, but even when confronted with permissive environments (sciatic nerves or fetal cerebellar slices), their axons do not regenerate. In contrast, fetal rat and mouse Purkinje cells are able to regenerate their axons on mature cerebellar slices. This regeneration is massive, and the regrowing axons invade all cerebellar regions of the apposed mature slices, including white matter. These results show that Purkinje cell survival and axonal regeneration are age-related and independent from environmental constraints. Moreover, our observations suggest strongly that the onset of synaptogenesis of Purkinje cell axons could provide a signal to turn off their growth program and that, thereafter, permissive microenvironment alone is unable to reestablish such a program.

The formation of the olivocerebellar projection is supposed to be regulated by positional information shared between pre- and postsynaptic neurons. However, experimental evidence to support this hypothesis is missing. In the chick, caudal neurons in the inferior olive project to the anterior cerebellum and rostral ones to the posterior cerebellum. We here report in vitro experiments that strongly support the existence of anteroposterior polarity cues in the embryonic cerebellum. We developed an in vitro system that was easily accessible to experimental manipulations. Large hindbrain explants of E7.5-E8 chick embryos, containing the cerebellum and its attached brainstem, were plated and studied using axonal tracing methods. In these cultures, we have shown that the normal anteroposterior topography of the olivocerebellar projection was acquired, even when the cerebellar lamella was detached from the brainstem and placed again in its original position. We also found that, following various experimental rotations of the anteroposterior axis of the cerebellum, the rostromedian olivary neurons still project to the posterior vermis and the caudolateral neurons to the anterior vermis, that now have inverted locations. Thus, the rotation of the target region results in the rotation of the projection. In addition, we have shown that the formation of the projection map could be due to the inability of rostromedian inferior olivary axons to grow in the anterior cerebellum. All these experiments strongly indicate that olivocerebellar fibers recognize within their target region polarity cues that organize their anteroposterior topography, and we suggest that Purkinje cells might carry these cues.

In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.

Monoclonal antibody mabQ113 has been used to study the distribution of Purkinje cell axon collaterals in the rat cerebellar cortex. MabQ113 recognizes a polypeptide antigen, zebrin I, that is confined to a subset of Purkinje cells. Antigenic Purkinje cells are arranged in parasagittal compartments running throughout the cortex. No other cerebellar cells are immunoreactive. Immunoreactive axon collaterals are confined principally to the infraganglionic plexus with only a few extending into the molecular layer. Three probable target cells for the axon collaterals have been identified: Golgi cells, Lugaro cells, and other Purkinje cells. About 90% of immunoreactive axon collaterals in the anterior lobe are located beneath the mabQ113+ Purkinje cell compartment in which they originate but some do invade the neighboring mabQ113- territory. In the anterior lobe vermis, the distribution of invading mabQ113+ collaterals is not symmetrical, such that the probability of an invading collateral from the P2+ compartment is greater at the medial boundary into P1- than at the lateral into P2-. The distribution of immunoreactive collaterals is consistent with their playing a role in synchronizing the firing of Purkinje cells within the same compartment.

The proximal portion of the Purkinje cell axon in normal cerebellum was investigated using the Golgi-Cox method. The axon emerging from the axon hillock tapered as it proceeded distally along the initial segment. The most distal portion of the initial segment was the narrowest (about 1 micron). Then the axon became thicker again in the probable myelinated portion. The length of the axon hillock plus the initial segment ranged from 21 micron to 52 micron, 35 +/- 6 micron on average +/- SD. The axon arose from any site of the soma and the primary dendrite of the Purkinje cell. Almost half of the axons emanated from a lateral surface of the soma. The dendritic arbores of the Purkinje cell with a torpedo were atrophic.

Focal axonal swellings are characteristic of a wide range of neuropathies. Three neuron-specific monoclonal antibodies have been used to identify focal axonal swellings in the normal developing rat cerebellar cortex. Between 7 and 15 days postnatal, swellings are a common feature of the granular layer and white matter tracts. Using a Purkinje cell-specific antibody, the majority of swellings were shown to occur in Purkinje cell axons. Focal axonal swellings therefore seem to be a normal adjunct of Purkinje cell maturation.

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.