Polarised Localisation of the Voltage-Gated Sodium Channel Nav1.2 in Cerebellar Granule Cells

Voltage-gated sodium channels are responsible for action potential initiation and propagation in electrically excitable cells. In this study, we used biochemical, immunohistochemical and quantitative immunoelectron microscopy techniques to reveal the temporal and spatial expression of the Na_v1.2 channel subunit in granule cells of cerebellum. Using histoblot, we detected Na_v1.2 widely distributed in the adult brain, but prominently expressed in the cerebellum. During postnatal development, Na_v1.2 mRNA and protein were detected low during the first and second postnatal week, increased to P15 and then continue to decrease until adult levels. At the light microscopic level, Na_v1.2 immunoreactivity concentrated in the molecular layer of the cerebellar cortex. Using immunofluorescence, Na_v1.2 colocalised with VGluT1, but not with VGluT2, demonstrating that the subunit was preferentially present in parallel fibre axons and axon terminals. At the electron microscopic level, Na_v1.2 immunoparticles were exclusively detected at presynaptic sites in granule cell axons and axon terminals of granule cells, with occasional clustering in their axon initial segment. This was demonstrated using quantitative immunogold analysis. In the axon terminals, the distribution of Na_v1.2 was relatively uniform along the extrasynaptic plasma membrane and never detected in the active zone. We could not find detectable levels of Na_v1.2 at postsynaptic elements of granule cells or other cerebellar cell types. The present findings show a polarised distribution of Na_v1.2 along the neuronal surface of granule cells and suggest its primary involvement in the transmission of information from granule cells to Purkinje cells.     

Origin of Aspartate-induced Responses in Rat Cerebellar Purkinje Cells

The effects of glutamate, aspartate and N-methyl-d -aspartate (NMDA) on Purkinje cells and interneurons were investigated in cerebellar slice cultures using the whole-cell configuration of the patch-clamp technique. l -Glutamate and l -aspartate induced inward currents in Purkinje cells voltage-clamped at -60 mV. In standard external solution, the amplitude of the responses induced by these two amino-acids was a linear function of the membrane potential. l -Aspartate-induced currents were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA receptors. NMDA, a selective agonist of NMDA receptors, had no effect of its own on the excitability of Purkinje cells, but was effective in blocking the responses induced by aspartate in Purkinje cells in a voltage-independent manner. In contrast, d -(–)-2-amino-5-phosphonovaleric acid (d -APV), a selective antagonist of NMDA receptors, had no effect on aspartate-induced responses. d -Aspartate also induced responses in Purkinje cells, and the amplitude of these responses was a linear function of the membrane potential. Currents induced by l - and d -aspartate were inhibited by dihydrokainate, a glutamate uptake blocker. In sodium-free external solution, glutamate still induced outward currents in Purkinje cells, whereas l - and d -aspartate no longer evoked any current. When sodium was replaced by lithium in the external medium, no change in the holding current could be detected in Purkinje cells maintained at -60 mV; moreover, in this bathing medium l -aspartate no longer evoked any current whereas glutamate-induced responses were still present. In contrast, interneurons were sensitive to both NMDA and aspartate applications, and these responses were antagonized by d -APV. In addition, aspartate still induced an outward current in sodium-free external solution. This study presents rather direct evidence in favour of l -aspartate as being a very selective NMDA receptor agonist in the cerebellum. l -Aspartate-induced currents in Purkinje cells are not due to activation of mixed NMDA/non-NMDA receptors, but are probably due to the release of l -glutamate induced by aspartate through glutamate uptake.     

Contributions of T-Type Voltage-Gated Calcium Channels to Postsynaptic Calcium Signaling within Purkinje Neurons

Low threshold voltage-gated T-type calcium channels have long been implicated in the electrical excitability and calcium signaling of cerebellar Purkinje neurons although the molecular composition, localization, and modulation of T-type channels within Purkinje cells have only recently been addressed. The specific functional roles that T-type channels play in local synaptic integration within Purkinje spines are also currently being unraveled. Overall, Purkinje neurons represent a powerful model system to explore the potential roles of postsynaptic T-type channels throughout the nervous system. In this review, we present an overview of T-type calcium channel biophysical, pharmacological, and physiological characteristics that provides a foundation for understanding T-type channels within Purkinje neurons. We also describe the biophysical properties of T-type channels in context of other voltage-gated calcium channel currents found within Purkinje cells. The data thus far suggest that one specific T-type isoform, Ca_v3.1, is highly expressed within Purkinje spines and both physically and functionally couples to mGluR1 and other effectors within putative signaling microdomains. Finally, we discuss how the selective potentiation of Ca_v3.1 channels via activation of mGluR1 by parallel fiber inputs affects local synaptic integration and how this interaction may relate to the overall excitability of Purkinje neuron dendrites.     

Dendritic Self-Avoidance and Morphological Development of Cerebellar Purkinje Cells

Cerebellar Purkinje cells arborize unique dendrites that exhibit a planar, fan shape. The dendritic branches fill the space of their receptive field with little overlap. This dendritic arrangement is well-suited to form numerous synapses with the afferent parallel fibers of the cerebellar granule cells in a non-redundant manner. Purkinje cell dendritic arbor morphology is achieved by a combination of dynamic local branch growth behaviors, including elongation, branching, and retraction. Impacting these behaviors, the self-avoidance of each branch terminal is essential to form the non-overlapping dendritic configuration. This review outlines recent advances in our understanding of the cellular and molecular mechanisms of dendrite formation during cerebellar Purkinje cell development.     

Transynaptic Changes Evident in Peripheral Axonal Function After Acute Cerebellar Infarct

The cerebellum has a vital role in fine motor control of the limbs. Consequently, downstream changes in peripheral axonal function may develop following a cerebellar infarct, in part, to adapt to the resultant impairment. The present study investigated changes in excitability in ipsilateral and contralateral upper limb peripheral motor axons in patients with acute cerebellar infarct to determine whether plastic changes may have functional relevance. Peripheral nerve excitability studies and detailed clinical assessments of functional impairment were undertaken in 13 patients with acute unilateral cerebellar infarct within 1 week of ictus. Changes were followed longitudinally over 1 year at 3, 6 and 12 months with results compared to 15 age-matched control subjects. Immediately following stroke, there were significant alterations in peripheral nerve excitability parameters in the upper limbs of patients compared to controls that were most evident in the more severely impaired group. There were significant correlations between excitability indices and functional scores in the entire cohort that demonstrated greater changes in axonal function associated with more impairment. Peripheral excitability trended towards normal over the study period in the context of clinical improvement. Following an acute cerebellar infarct, changes were observed in peripheral motor axons bilaterally that were more pronounced in patients with severe functional impairment. The peripheral changes may represent a functionally relevant plastic process reflecting altered activity to adapt to the disability of the stroke.     

Defining the Role of Cerebellar Purkinje Cells in Autism Spectrum Disorders

Understanding the contribution of cerebellar dysfunction to complex neurological diseases such as autism spectrum disorders (ASD) is an ongoing topic of investigation. In a recent paper, Tsai et al. (Nature 488:647–651, 2012) used a powerful combination of conditional mouse genetics, electrophysiology, behavioral tests, and pharmacological manipulations to address the role of Tuberous sclerosis complex 1 (Tsc1) in Purkinje cells and cerebellar function. The authors make the staggering discovery that morphological and electrophysiological defects in Purkinje cells are linked to system-wide ASD-like behavioral deficits. In this journal club, I discuss the major findings of this paper and critically assess the implications of this seminal work.     

Simple and Complex Spike Firing Patterns in Purkinje Cells During Classical Conditioning

Classical blink conditioning is known to depend critically on the cerebellum and the relevant circuitry is gradually being unravelled. Several lines of evidence support the theory that the conditioned stimulus is transmitted by mossy fibers to the cerebellar cortex whereas the unconditioned stimulus is transmitted by climbing fibers. This view has been dramatically confirmed by recent Purkinje cell recordings during training with a classical conditioning paradigm. We have tracked the activity of single Purkinje cells with microelectrodes for several hours in decerebrate ferrets during learning, extinction, and relearning. Paired peripheral forelimb and periocular stimulation, as well as paired direct stimulation of cerebellar afferent pathways (mossy and climbing fibers) causes acquisition of a pause response in Purkinje cell simple spike firing. This conditioned Purkinje cell response has temporal properties that match those of the behavioral response. Its latency varies with the interstimulus interval and it responds to manipulations of the conditioned stimulus in the same way that the blink does. Complex spike firing largely mirrors the simple spike behavior. We have previously suggested that cerebellar learning is subject to a negative feedback control via the inhibitory nucleo-olivary pathway. As the Purkinje cell learns to respond to the conditioned stimulus with a suppression of simple spikes, disinhibition of anterior interpositus neurons would be expected to cause inhibition of the inferior olive. Observations of complex spike firing in the Purkinje cells during conditioning and extinction confirm this prediction. Before training, complex spikes are unaffected or facilitated by the conditioned stimulus, but as the simple spike pause response develops, spontaneous and stimulus-evoked complex spikes are also strongly suppressed by the conditioned stimulus. After extinction of the simple spike pause response, the complex spikes reappear.     

Purkinje Cell-Specific Ablation of CaV2.1 Channels is Sufficient to Cause Cerebellar Ataxia in Mice

The Cacna1a gene encodes the α(1A) subunit of voltage-gated Ca(V)2.1 Ca(2+) channels that are involved in neurotransmission at central synapses. Ca(V)2.1-α(1)-knockout (α1KO) mice, which lack Ca(V)2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of Ca(V)2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the Ca(V)2.1-α(1A) subunit and thereby Ca(V)2.1 channels in Purkinje cells. Purkinje cell Ca(V)2.1-α(1A)-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of Ca(V)2.1 channels, we show that ablation of Ca(V)2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of Ca(V)2.1 channels may help in unraveling mechanisms of human disease.     

The Fine Structure of the Axonal Torpedo in Purkinje Cells of the Human Cerebellum

Axonal torpedoes on Purkinje cells of the cerebellum have been observed at electron microscopic level in the case of a 5-year-old boy suffering from a juvenile astrocytoma present in the roof of the fourth ventricle and cerebellar hemisphere.

The axon torpedo is characterised by a central accumulation of disoriented neurofilaments, which displace the mitrochondria and endoplasmic reticular elements to the periphery. The mitochondira are small and densely staining with longitudinally arranged cristae and are intimately associated with the smooth endoplasmic reticulum that occurs as stacked complexes.

Multivesicular and lamellar bodies, typical of degenerating axons, are not consistently seen and this indicates that axon torpedoes are more likely to represent a regenerating state within the nerve fibre than a degenerative condition.     

Changes in Spontaneous Firing Patterns of Cerebellar Purkinje Cells in p75 Knockout Mice

The p75 neurotrophin receptor is highly expressed in the developing nervous system and is required for neuronal survival, growth, and synaptic transmission. In young mice, p75 is present in both granular cells and Purkinje cells of the cerebellum. Although p75 has been implicated in modulation of neuronal excitability in several neuronal types, whether and how it affects the excitability of cerebellar Purkinje neurons remained unclear. Using extracellular recordings of spontaneous firing of Purkinje neurons in cerebellar slices prepared from wild type and p75 knockout mice, we measured intrinsic firing properties in the presence of fast synaptic blockers of more than 200 Purkinje cells, each for a period of 5 min, for each genotype. We detected a significant increase in the mean firing frequency in p75^?/? neurons comparing to the wild type littermates. Upon separating tonically firing from phasically firing cells, i.e., cells with firing pauses of longer than 300 ms, we observed that the change mainly arose from phasic firing cells and can be explained by an increase in the firing/silence ratio and a decrease in the number of long pauses during the 5-min recording period. We conclude that p75 plays an important role in regulating the firing-to-silence transition during the phasic firing period of the spontaneous firing of Purkinje cells. Thus, p75 exerts a modulatory function on Purkinje cell firing patterns, through which it may act as a key player in motor coordination and other cerebellum-regulated activities since Purkinje cells represent the sole neuronal output of the cerebellar cortex.     

Survival of Purkinje Cells in Cerebellar Cultures is Increased by Insulin-like Growth Factor I

Insulin-like growth factor I (IGF-I) is a trophic factor for both neurons and glia. Its presence in the developing and adult cerebellum suggests a role for this growth factor in this area of the brain. Recently, we have described the existence of an IGF-I-containing pathway in afferents of Purkinje neurons arising from the inferior olive. In addition, IGF-I receptors are present in the molecular layer of the cerebellar cortex. These observations prompted us to investigate whether the Purkinje cell is a target for IGF-I. Addition of IGF-I to rat cerebellar cultures produced a 7-fold increase in the number of Purkinje cells (calbindin-positive) together with an increase in the calbindin content of the cultures. IGF-I also doubled the number of surviving neurons and produced a moderate, non-significant increase in [³H]thymidine incorporation by the cultures. On the other hand, basic fibroblast growth factor (bFGF), which is also present in the cerebellum, produced a dramatic increase in both the proportion of astrocytes and in the mitotic activity of the cultures, without affecting neuron survival. We conclude that IGF-I is a specific promoter of Purkinje cell survival and that its effects differ from those produced by bFGF in fetal cerebellar cultures. These findings reinforce our hypothesis that the Purkinje cell is a target neuron for IGF-I action in the developing cerebellum.     

Immunohistochemical Studies on Cerebellar Purkinje Cells of Patients with Menkes' Kinky Hair Disease

Immunohistochemical investigations were carried out on cerebellar Purkinje cells of eight patients with Menkes' kinky hair disease (MD) using an antibody against the inositol 1, 4, 5-triphosphate receptor which is identical to the cerebellar Purkinje cell glycoprotein P₄₀₀ (P₄₀₀/IP₃R). In normal cerebellar Purkinje cells, the cell bodies, axons and dendrites including spiny branchlets were specifically stained by the antibody. By comparison, the immunoreactivity of the MD Purkinje cells ranged from negative to positive. The most frequent staining pattern in the MD Purkinje cells was that of cell bodies and proximal portions of the dendrites expressing P₄₀₀/IP₃R in varying degrees, with the peripheral dendrites including spiny branchlets not being stained. The Purkinje cells of the eight patients were also analyzed for expression of phosphorylated neurofilament proteins (pNFP), αB-crystallin, stress-response protein (srp) 72, srp 27 and ubiquitin. Most MD Purkinje cell bodies reacted with the antibody against pNFP. Although the proportion of stained Purkinje cells was less, a positive reaction was also observed with the antibodies to αB-crystallin, srp 72 and srp 27. By contrast, no normal Purkinje cell was stained by these antibodies. Our results suggest that the function of P₄₀₀/IP₃R in the Purkinje cells of MD patients is impaired, that these cells have an aberrant phosphorylation of NFP and that some of the imparied Purkinje cells synthesize specific sets of stress-related proteins.     

Patch-clamp Analysis of Synaptic Transmission to Cerebellar Purkinje Cells of Prion Protein Knockout Mice

The prion protein (PrP) plays a pivotal role in transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. Previous experiments have suggested that the normal cellular prion protein (PrP^C) is involved in synaptic function in the hippocampus. Here, we utilized the controlled recording conditions of the patch-clamp technique to investigate the synaptic function of prion protein in cerebellar Purkinje cells. By performing whole-cell and outside-out patch-clamp experiments in thin slices, we investigated synaptic transmission in prion protein knockout mice (PrP-null) and control animals. In PrP-null mice, the kinetics of GABA- and glutamate receptor-mediated currents showed no significant deviation from those in control animals. In contrast to previous results in hippocampal neurons, our findings support the view that synaptic transmission is unimpaired in prion protein-deficient mice.     

Characteristics of Calcium Channels Responsible for Voltage-activated Calcium Entry in Rat Cerebellar Granule Cells

The properties and characteristics of calcium channel openings in cerebellar granule cells were analysed by the cell-attached patch-clamp technique. At depolarized potentials, with 110 mM Ba²+ as the divalent charge carrier, 36% of the patches displayed activity that consisted of elementary events whose amplitude ranged from – 0.3 to –1. 75 pA at 0 mV, giving rise to a high threshold current. In this population of events at least four different types of channel openings were identified by their distinct biophysical and pharmacological properties. Two types of channel openings, with conductances around 24 and 7 pS, had similar characteristics in that both opened following two modes of gating characterized by brief (˜ 2 ms) and longer openings (˜ 8 ms) and both were sensitive to dihydropyridines. A further type of channel opening, with a conductance around 11 pS gated mainly with brief openings (∼1 ms), was shown to be insensitive to dihydropyridines but was undetectable in recordings from the cells that had been treated with ω-conotoxin. The last type of event was revealed after treatment of the cell with nicardipine or nifedipine and ω-conotoxin. The corresponding channel had a conductance of 19 pS and opened in one dominant mode characterized by brief openings (∼1 ms). The data obtained on single-channel activity of cerebellar granule cells are compared with the properties of the total current recorded in whole-cell conditions.     

Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells

Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor–mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells.     

Responses to Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells: Induction of an Inward Current

The responses to activation of metabotropic glutamate receptors (mGluRs) of Purkinje cells in rat cerebellar slice cultures were investigated using intracellular recordings in single-electrode voltage-clamp mode combined with microfluorometric measurements of cytosolic free calcium using fura-2. Purkinje cells were perfused with saline containing 0.5 μM tetrodotoxin and 10 μM bicuculline and voltage-clamped at –60 mV. Bath-applied trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid ( t -ACPD, 50–100 μM), a selective agonist of mGluRs, induced a transient inward current that was followed by an outward current. The response induced by t -ACPD was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, up to 40 μM). In contrast, inward currents caused by (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1–2 μM) were completely abolished, while inward currents caused by quisqualate (0.25 μM) were only partially depressed by CNQX (5–40μM). The inward current induced by t -ACPD was unaffected by external Ba²⁺ (1 mM), tetraethylammonium (10 mM) and Cs⁺ (1 mM), and was associated with an increase in apparent input conductance of the cell membrane. The extrapolated reversal potential of inward currents induced by t -ACPD was +18 mV while Cl⁻ currents induced by muscimol reversed at –66 mV. Inward currents induced by t -ACPD, but not those induced by AMPA, were associated with a rise in cytosolic Ca²⁺ concentration and suppressed by intracellular injection of a calcium chelator. Replacement of external Na⁺ by choline or Li⁺ depressed the inward current and resulted in a slower decay of the Ca²⁺ signal.     

Postsynaptic Currents and Short-term Synaptic Plasticity in Purkinje Cells Grafted onto an Uninjured Adult Cerebellar Cortex

It has been shown recently that embryonic Purkinje cells grafted extraparenchymally into an intact cerebellum, in the absence of any sign of damage, are able to migrate into the host molecular layer where they receive a climbing fibre innervation. Using the same technique, we investigated the development of the electrophysiological properties of the synapses between the grafted cells and their main afferents. Purkinje cells either in the graft or having migrated into the molecular layer of the host were recorded using the whole-cell patch-clamp method in acutely prepared slices 17–112 days after grafting. Spontaneous postsynaptic currents with a single-exponential decay and mediated by GABA_A receptors were very similar to those described in normal Purkinje cells. Excitatory postsynaptic currents (EPSCs) evoked by climbing fibre and by parallel fibre stimulation were blocked by an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate antagonist, and displayed the linear current-voltage relation typical of postnatal Purkinje cells. The attainment of normal functional properties by the adult axons at the newly formed synaptic sites was shown by the expression of short-term facilitation of parallel fibre EPSCs and of short-term depression of climbing fibre EPSCs. The grafted Purkinje cells showed climbing fibre polyinnervation 17-20 days after grafting which evolved to monoinnervation at 23-45 days, confirming the completion of the developmental programme up to maturation. Our experiments support the view that the adult intact brain is able to accept and integrate an additional number of neurons which show fully mature electrophysiological properties which are electrophysiologically indistinguishable from those of the host neurons.     

Exposure to Kainic Acid Mimics the Effects of Axotomy in Cerebellar Purkinje Cells of the Adult Rat

We have investigated the long-term structural changes which affect Purkinje cells exposed to a single dose of kainic acid. Following intraparenchymal injection of the excitotoxin in the cerebellar cortex (1 μ1 of a 1 mg/ml solution), Purkinje cells which survived within the lesioned area or close to its edges showed remarkable axonal abnormalities, involving the formation of torpedoes, hypertrophy of recurrent collaterals and atrophy of the corticofugal portion of the axon. In addition, their dendritic trees were often affected by conspicuous regressive alterations. The climbing fibres contacting these Purkinje cells were characterized by thick perisomatic plexuses, whereas their peridendritic branches were atrophic. The dendrites innervated by such atrophic olivary arbours were studded with huge numbers of newly formed spines. These alterations were already present a few days after kainic acid administration and persisted for the total period of observation of 6 months after the lesion. The remarkable similarity between the abnormalities of Purkinje cells exposed to kainic acid and those observed after axotomy indicates that in these two conditions common mechanisms determine analogous long-lasting modifications in the affected neurons. It is proposed that kainic acid-induced intracellular calcium overload disrupts cytoskeletal components and impairs axonal transport, thus depriving the affected Purkinje cells of retrograde trophic influences from their target neurons. As a consequence the affected neurons undergo long-lasting regressive modifications and compensatory remodelling phenomena.