Moderate alcohol consumption and loss of cerebellar Purkinje cells

OBJECTIVE: To examine the dose-response effect of alcohol consumption on the number of cerebellar Purkinje cells. DESIGN: A prospective necropsy study combined with detailed reports on use of alcohol from a relative or friend. The number of Purkinje cells was counted in the anterior midsagittal section of the cerebellar vermis, the area of which was measured by computer assisted morphometry. SETTING: Department of forensic medicine, University of Helsinki. SUBJECTS: 66 men, aged 35 to 69 years, subjected to medicolegal necropsy because of sudden or violent death. The average all year daily alcohol consumption over the year was 0 to 10 g in 17 men, 11 to 80 g in 24 men, and more than 80 g in 25 men. MAIN OUTCOME MEASURES: Number of Purkinje cells, alcohol consumption. RESULTS: The numbers and density of Purkinje cells in the cross section of vermis showed a consistent but weak decrease with increasing daily alcohol intake but not with age. A wide variation in the cell counts was observed, especially in men drinking more than 80 g, suggesting differences in the susceptibility to effects of alcohol. Compared with men drinking 40 g or less, a long term moderate consumption of an average of 41 to 80 g daily was associated with a significant average loss of 242 (95% confidence interval 45 to 439) Purkinje cells (15.2%) from a mean of 1583 to 1341 cells. In those drinking 81 to 180 g the average loss was 535 (259 to 811) cells (33.4%) to a mean of 1048 cells. The density of cells in the cross section of vermis also fell significantly by 0.9 cell/mm (0.1 to 1.7) when the daily consumption exceeded 40 g and by 1.4 cell/mm (0.3 to 2.5) when the intake was 81 to 180 g. Only three cases (4.5%) in the series showed macroscopical cerebellar atrophy. CONCLUSION: Long term intake of moderate doses of alcohol daily for 20-30 years may damage the cerebellum before the onset of macroscopical atrophy. Despite distinct individual differences an all year average daily alcohol intake of 41-80 g results in a risk of significant loss of Purkinje cells.     

Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI

Intradendritic recordings in Purkinje cells from a defined area in parasaggital slices of cerebellar lobule HVI, obtained after rabbits were given either paired (classical conditioning) or explicitly unpaired (control) presentations of tone and periorbital electrical stimulation, were used to assess the nature and duration of conditioning-specific changes in Purkinje cell dendritic membrane excitability. We found a strong relationship between the level of conditioning and Purkinje cell dendritic membrane excitability after initial acquisition of the conditioned response. Moreover, conditioning-specific increases in Purkinje cell excitability were still present 1 month after classical conditioning. Although dendritically recorded membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in cells from paired or control animals, the size of a potassium channel-mediated transient hyperpolarization was significantly smaller in cells from animals that received classical conditioning. In slices of lobule HVI obtained from naive rabbits, the conditioning-related increases in membrane excitability could be mimicked by application of potassium channel antagonist tetraethylammonium chloride, iberiotoxin, or 4-aminopyridine. However, only 4-aminopyridine was able to reduce the transient hyperpolarization. The pharmacological data suggest a role for potassium channels and, possibly, channels mediating an IA-like current, in learning-specific changes in membrane excitability. The conditioning-specific increase in Purkinje cell dendritic excitability produces an afterhyperpolarization, which is hypothesized to release the cerebellar deep nuclei from inhibition, allowing conditioned responses to be elicited via the red nucleus and accessory abducens motorneurons.     

Differential expression of glutamate decarboxylase messenger RNA in cerebellar Purkinje cells and deep cerebellar nuclei of the genetically dystonic rat

The genetically dystonic rat exhibits a motor syndrome that closely resembles the human disease, generalized idiopathic dystonia. Although in humans dystonia is often the result of pathology in the basal ganglia, previous studies have revealed electrophysiological abnormalities and alterations in glutamate decarboxylase, the synthetic enzyme for GABA, in the cerebellum of dystonic rats. In this study, we further characterized the alterations in cerebellar GABAergic transmission in these mutants by examining the expression of the messenger RNA encoding glutamate decarboxylase (67000 mol. wt) with in situ hybridization histochemistry at the single cell level in Purkinje cells and neurons of the deep cerebellar nuclei. Glutamate decarboxylase (67000 mol. wt) messenger RNA levels were increased in the Purkinje cells and decreased in the deep cerebellar nuclei of dystonic rats compared to control littermates, suggesting opposite changes in GABAergic transmission in Purkinje cells and in their target neurons in the deep cerebellar nuclei. In contrast, levels of glutamate decarboxylase (67000 mol. wt) messenger RNA in the pallidum, and of enkephalin messenger RNA in the striatum, were unaffected in dystonic rats. The data indicate that both the Purkinje cells and GABAergic neurons of the deep cerebellar nuclei are the site of significant functional abnormality in the dystonic rat.     

Expression pattern of integrin beta 1 subunit in Purkinje cells of rat and cerebellar mutant mice

We found that integrin beta 1 subunit (INT beta 1)-immunoreactive Purkinje cells first appeared caudally at postnatal day (PD) 6 of rat and most Purkinje cells gradually became positive by PD 12. The expression of INT beta 1 was then suppressed in some of these cells, so that the positive Purkinje cells in the adult were organized into parasagittal bands interposed by negative cells throughout the vermis and hemispheres. When Purkinje cells were deprived of their climbing fiber innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the subsequent patterning of INT beta 1-positive Purkinje cells was not changed. In both reeler and weaver mice, the INT beta 1-positive parasagittal bands were observed, however, the Purkinje cells in the staggerer mice did not express INT beta 1 at any stage. These data suggest that the expression of INT beta 1 in Purkinje cells is genetically programmed in the developing cerebellum, and that the afferent synaptic inputs by climbing and parallel fibers are not prerequisites for INT beta 1 expression in Purkinje cells. Therefore, the unique distribution patterns of INT beta 1-positive Purkinje cells provides a new marker for postnatal development of rodent cerebella.     

Kinetic and stochastic properties of a persistent sodium current in mature guinea pig cerebellar Purkinje cells

Whole cell voltage-clamp techniques were employed to characterize the sodium (Na) conductances in acutely dissociated, mature guinea-pig cerebellar Purkinje cells. Three phenomenological components were noted: two inactivating and a persistent component (I(P)(Na). All exhibited similar sensitivities to tetrodotoxin (TTX; IC50 approximately 3 nM). The inactivating Na current demonstrates two components with different rates of inactivation. The persistent component activates at a more negative membrane potential than the inactivating components and shows little inactivation during a 5-s pulse. The amplitude of the persistent Na conductance had a higher Q10 than the inactivating Na conductance (2.7 vs. 1.3). (I(P)(Na) rapidly activates (approximately 1 ms) and deactivates (< 0.2 ms) and like the fast component appears to be exclusively Na permeable. (I(P)(Na) is not a "window" current because its range of activation exceeds the small overlap between the steady-state activation and inactivation characteristics of the inactivating current. Anomalous tail currents were observed during voltage pulses above -40 mV after a prepulse above -30 mV. The tails rose to a maximum inward current with a time constant of 1.5 ms and decayed to a persistent inward current with a time constant of 20 ms. The tails probably arose as a result of recovery from inactivation through the open state. The noise characteristics of (I(P)(Na) were anomalous in that the measured variance was lower at threshold voltages than would be predicted by a binomial model. The form of the variance could be partially accounted for by postulating that the maximum probability of activation of the persistent current was less than unity. The noise characteristics of (I(P)(Na) are such as to minimize noise near spike activation threshold and sharpen the threshold.     

Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase

BACKGROUND: The pathophysiological mechanisms in non-ulcer dyspepsia are incompletely understood. AIMS: To compare gastric motor and sensory functions in Helicobacter pylori positive or negative patients with non-ulcer dyspepsia. PATIENTS: Seventeen patients with non-ulcer dyspepsia and 16 asymptomatic controls. METHODS: The following were evaluated: gastrointestinal symptoms; gastric emptying and orocaecal transit of solids; abdominal vagal function; gastric compliance; fasting and postprandial gastric tone and phasic contractions; symptoms during ingestion of cold water and during the distension of an intragastric bag; and somatic sensitivity and personality profile (Minnesota Multiphasic Personality Inventory, MMPI). RESULTS: Gastric accommodation was reduced in H pylori negative dyspeptics relative to controls; the degree of accommodation was unrelated to H pylori status in dyspeptics. Increased postprandial gastric sensation was more frequent among H pylori positive patients (4/5 H pylori positive versus 4/12 H pylori negative patients). Intragastric meal distribution and orocaecal transit were normal; gastric emptying at four hours was abnormal in 4/17 patients. Vagal dysfunction was rare. Eight of 17 patients had somatisation or depression on MMPI. CONCLUSION: Impaired gastric accommodation is frequent in non-ulcer dyspepsia and seems to be unrelated to vagal efferent dysfunction. H pylori infection does not seem to influence gastric accommodation, but is associated with heightened sensitivity in dyspeptics. Therapeutic approaches that restore normal postprandial accommodation and gastric sensitivity should be tested in non-ulcer dyspepsia.     

Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Galphaq

Mice lacking the alpha-subunit of the heterotrimeric guanine nucleotide binding protein Gq (Galphaq) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Galphaq mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Galphaq is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.     

Synchronization of golgi and granule cell firing in a detailed network model of the cerebellar granule cell layer

The granular layer of the cerebellum has a disproportionately large number of excitatory (granule cells) versus inhibitory neurons (Golgi cells). Its synaptic organization is also unique with a dense reciprocal innervation between granule and Golgi cells but without synaptic contacts among the neurons of either population. Physiological recordings of granule or Golgi cell activity are scarce, and our current thinking about the way the granular layer functions is based almost exclusively on theoretical considerations. We computed the steady-state activity of a large-scale model of the granular layer of the rat cerebellum. Within a few tens of milliseconds after the start of random mossy fiber input, the populations of Golgi and granule cells became entrained in a single synchronous oscillation, the basic frequency of which ranged from 10 to 40 Hz depending on the average rate of firing in the mossy fiber population. The long parallel fibers ensured, through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-mediated synapses, a coherent excitation of Golgi cells, while the regular firing of each Golgi cell synchronized all granule cells within its axonal radius through transient activation of their gamma-aminobutyric acid-A (GABAA) receptor synapses. Individual granule cells often remained silent during a few successive oscillation cycles so that their average firing rates, which could be quite variable, reflected the average activities of their mossy fiber afferents. The synchronous, rhythmic firing pattern was robust over a broad range of biologically realistic parameter values and to parameter randomization. Three conditions, however, made the oscillations more transient and could desynchronize the entire network in the end: a very low mossy fiber activity, a very dominant excitation of Golgi cells through mossy fiber synapses (rather than through parallel fiber synapses), and a tonic activation of granule cell GABAA receptors (with an almost complete absence of synaptically induced inhibitory postsynaptic currents). These three conditions were associated with a reduction in the parallel fiber activity, and synchrony could be restored by increasing the mossy fiber firing rate. The model predicts that, under conditions of strong mossy fiber input to the cerebellum, Golgi cells do not only control the strength of parallel fiber activity but also the timing of the individual spikes. Provided that their parallel fiber synapses constitute an important source of excitation, Golgi cells fire rhythmically and synchronized with granule cells over large distances along the parallel fiber axis. According to the model, the granular layer of the cerebellum is desynchronized when the mossy fiber firing rate is low.     

Monoclonal antibodies reveal expression of the CGRP receptor in Purkinje cells, interneurons and astrocytes of rat cerebellar cortex

The molecular layer of adult rat cerebellum displays high levels of calcitonin gene-related peptide (CGRP) receptors, but the cellular location of the receptor remains unidentified. In an attempt to reveal the expression sites of these receptors, monoclonal antibodies raised against purified CGRP receptors from porcine cerebellar membranes were used in double-immunofluorescence experiments combined with confocal microscopy. PEP-19, a marker that is highly enriched in Purkinje cells (Pc), revealed that CGRP receptors are located in Pc cytoplasm and dendrites, where they label small puncta sometimes arranged in a row along the course of the dendrite itself. CGRP receptors were also located in inhibitory interneurons. Furthermore, as shown by double-labeling experiments with GFAP, CGRP receptor-IR labeled Golgi epithelial cells and their radial fibers (Bergmann fibers), as well as astrocytic processes encircling Pc somata. The simultaneous presence of CGRP receptors in Purkinje cells and in the glial cells that heavily enshroud Purkinje cells allows us to hypothesize that these receptors may be involved in neuron-glia interactions influencing neuronal activity.     

Block of P-type Ca2+ channels in freshly dissociated rat cerebellar Purkinje neurons by diltiazem and verapamil

Osteoporosis is a slowly progressing disease resulting from an imbalance between bone accretion and degradation. As interstitial collagenase is a key enzyme in the degradation of bone matrix, we investigated a possible relationship between the collagenase gene and osteoporosis. Analysis of an amplified genomic DNA fragment from -524 to +52 by denaturing gradient gel electrophoresis and sequencing allowed us to detect three dimorphic sites upstream of base -300, one of them leading to a BanI restriction site. None of the sites could be directly associated with osteoporosis. The allele frequencies of the three dimorphic sites were estimated. The interallelic ratios were high, thus providing new useful genetic markers for linkage analysis. When comparing these ratios in osteoporotic and nonosteoporotic subjects, no significant differences could be observed.     

CD40 ligand signals optimize T helper cell cytokine production: role in Th2 development and induction of germinal centers

The role of CD40 in the development of germinal centers (GC) is not simply to initiate the B cell response, as rudimentary GC can develop in CD40-/- mice that are injected with CD40-immunoglobulin (Ig) fusion protein. This indicates that CD40 ligand (CD40L) transduces a signal to T cells that is important in the process. In this study we have used an in vitro model of GC development to investigate the role of CD40L, cytokines and other co-stimuli. The model involves the specific induction of an H-2E transgene in GC B cells (in Sma58 mice). We find that Th2 cytokines together with Ig and CD40 cross-linking are the most efficient means of induction of the GC phenotype. Although IL-4 plays some inductive role, it is not the sole active ingredient in the mix of cytokines made by Th2 cells. Our studies on primary T cells and T cell clones activated in the absence of CD40 on antigen-presenting cells or CD40L on T cells indicate that the CD40L co-stimulus does not directly bias the response to Th2 cells, as previously reported, but that it augments terminal effector T cell differentiation or the level of secretory activity. However, both in vitro and in vivo, the CD40L co-stimulus is crucially important for Th2 development as in its absence IL-4 production is suboptimal and does not compete with a larger, more rapid IFN-gamma response.     

Ca2+ release from Ca2+ stores, particularly from ryanodine-sensitive Ca2+ stores, is required for the induction of LTD in cultured cerebellar Purkinje cells

1. Primary-cultured cerebellar Purkinje cells (PCs) from mouse embryos were whole cell voltage clamped, and L-glutamate (Glu) was applied iontophoretically to the dendrite. Long-term depression (LTD) of Glu-evoked currents was induced through the conjunction of repeated depolarizations and Glu applications. 2. Thapsigargin, a specific inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, and ryanodine and ruthenium red, inhibitors of the ryanodine receptor, blocked the induction of LTD. 3. Thapsigargin and ryanodine alone did not affect influx of Ca2+ through voltage-gated Ca2+ channels and inward currents evoked by Glu applications. 4. Our results suggest that Ca2+ release from internal stores, particularly from ryanodine-sensitive stores, is necessary for the induction of LTD in cultured PCs.     

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: dendritic differentiation, synaptic maturation, and formation of cell-class specific features

Cerebellar Purkinje cells (PC) display a highly distinctive form of polarity. We have cultured murine PCs from dissociated E16 cerebellar anlagen for 1 week to investigate the early stages of neuronal compartmentalization and synaptic interactions, features which are important for the establishment of neuronal polarity. To unequivocally identify the PCs we utilized light and electron microscopic immunocytochemistry with an anti-serum to the cell class-specific marker L7/pcp2 gene product. The PCs typically show a single, long axon, numerous short appendages classified as filopodia and protospines, and a small number of protodendrites. The nucleus is positioned asymmetrically in both the horizontal and vertical axes of the soma. The Golgi apparatus, coated and uncoated vesicles, and mitochondria are prominent ultrastructural features, while the endoplasmic reticulum is highly fragmented. The cell body receives rudimentary synapses on its smooth surfaces and appendages and no consistent morphological differences were detected between these elementary contacts. The axon is clearly identifiable; it emanates from either the cell body or a protodendrite, bifurcates at predominantly right angles, forms beaded collaterals, and terminates with relatively large growth cones. The varicosities of the PC axon contain pleomorphic synaptic vesicles and form rudimentary synapses primarily with the dendritic shafts of immunonegative neurons. The protodendrites are short, quickly tapering and sparsely branched; they emit numerous filopodia and immature spines and terminate with small growth cones. Rudimentary synapses are received on the proximal dendritic shafts and filopodia, and more mature synapses occur frequently on protospines. With few exceptions, PCs lie atop an astrocytic bed layer and glial processes are apposed to the various aspects of the PC body left free by the afferent axons. By contrast, PC processes are largely free of glial sheaths. We conclude that the "stellate stage" of PC development in situ is replicated rather faithfully in culture and that PCs have established polarity and have begun to form intercellular contacts by 1 week in vitro. Moreover, the PCs are already morphologically distinct from other cell types in the 1 week cultures, although they have yet to develop the differentiated features that distinguish mature PCs.     

Different functional effect of Ro 15-4513 and Ro 19-4603 on synaptic responses of Purkinje cells in the rat cerebellar slice

The Sugiura operation has been reported to have low operative mortality, rebleeding, and encephalopathy rates when carried out in a predominantly nonalcoholic Japanese population with good liver function. A literature review of reports of the Sugiura procedure outside Japan reveals a high complication and mortality rate when it is used as an emergency procedure in patients with advanced liver disease, especially in those with alcoholic cirrhosis. Uncontrolled studies report results that differ little from the Japanese series when the operation is confined to good-risk patients in the elective situation. Our experience with the Sugiura operation supports its role in these circumstances, especially in patients with portal vein thrombosis and normal liver function. The only good prospective controlled trial has been carried out in patients with schistosomiasis and suggests that the Sugiura operation is far superior to total shunt and may have a slight advantage over the Warren shunt because of its low incidence of postoperative encephalopathy. More controlled trials are required to establish its role in good- to moderate-risk patients with alcoholic cirrhosis.     

Lipofuscin pigment in cerebellar Purkinje neurones and choroid plexus epithelial cells of macaque monkeys with Plasmodium knowlesi cerebral malaria: an electron microscopical observation

Experimental infection of rhesus monkeys (Macaca mulatta) with a virulent (W1) strain of Plasmodium knowlesi resulted in cerebral malaria. Electron microscopical examination of the brain revealed large numbers of intracytoplasmic lipofuscin pigment deposits in cerebellar Purkinje neurones and choroid plexus epithelium of the lateral ventricle. This lesion may be part of the nervous system response to ischaemic hypoxia.     

Postnatal expression of Hu-bcl-2 gene in Lurcher mutant mice fails to rescue Purkinje cells but protects inferior olivary neurons from target-related cell death

Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ions partition into the hydrophobic phase and then diffuse across (the solubility-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theoretical predictions made by both models were tested for halide anions by measuring the permeability coefficients for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepared from monounsaturated phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms. The fluorescent dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) served as an indicator for halide concentration inside the liposomes and was used to follow the kinetics of halide flux across the bilayer membranes. The observed permeability coefficients ranged from 10(-9) to 10(-7) cm/s and increased as the bilayer thickness was reduced. Bromide was found to permeate approximately six times faster than chloride through bilayers of identical thickness, and iodide permeated three to four times faster than bromide. The dependence of the halide permeability coefficients on bilayer thickness and on ionic size were consistent with permeation of hydrated ions by a solubility-diffusion mechanism rather than through transient pores. Halide permeation therefore differs from that of a monovalent cation such as potassium, which has been accounted for by a combination of the two mechanisms depending on bilayer thickness.