Purkinje Cell Membrane Research Articles

Immunohistochemical expression of P 400 protein in purkinje cells of sphingomyelinosis mouse

Immunohistochemical expression of P400 protein, a glycoprotein localized to the Purkinje cell membrane, has been studied in the cerebellum of spm mouse using anti-P400 monoclonal antibody. The initial change observed in the Purkinje cells was a swelling of the cell body with distortion of the neurites; this occurred as early as 5 weeks of age. A significant, patchy loss of Purkinje cells started at 6 weeks before cerebellar signs became manifest. With progression of the disease the dendritic processes in the molecular layer showed a marked swelling, followed by irregular arborization and finally by disintegration. A few, heterotopic Purkinje cells were found in the subcortical white matter; this was interpreted as an indication that a disturbance in neuronal migration could be superimposed on the sphingolipid metabolic disorder. Additionally, P400-immunoreactive nerve cells were occasionally encountered in areas of the deep cerebellar nuclei and in the lateral vestibular nuclei of the pontine tegmentum. The number of P400-immunoreactive Purkinje cells correlated well with the percentages of the remaining Purkinje cells during the ages of 4 to 7 wks. At the late stage of 10 to 12 weeks almost all Purkinje cells had lost their P400-immunoreactivity. It is suggested that Purkinje cells that fail to express P400 protein may undergo an immunohistochemical degeneration of the plasma membrane.

A developmentally regulated axonal glycoprotein (7-8D2 antigen) with a restricted distribution in mature rat brain

7-8D2 is a mouse monoclonal IgGl antibody which recognizes a neuronal cell surface antigen in rat brain. Immunohistochemical techniques reveal the antigen to be present most abundantly in the cerebellum of the adult brain, where it is expressed by the cell bodies and fibres of the granule neurons. Lower levels of staining were found in the neuropil of the hippocampus, in some fibres of the corpus callosum and in the most superficial layer of the cerebral cortex. Immunoelectron microscopy confirmed that the antigen was present on the surface of the parallel fibres in the cerebellum and showed that it was absent from glial, Purkinje or stellate cell membranes. The antigen had a more widespread distribution early in developmentand the restricted adult distribution was achieved by the end of the second postnatal week. Immunoblotting of samples of adult rat brain shows that the antigen appears as a close doublet of bands at 211,000 mol. wt. This result was confirmed by immunoprecipitating the antigen from metabolically labelled glycoproteins prepared from cultured cerebellar interneurons. Immunoblotting and immunohistochemical experiments were in agreement that the cerebellum contained high levels of the antigenand that lower but significant amounts could be found in other brain regions, notably the hippocampus and the cerebral cortex. The localization data and the changes in the distribution of the antigen may suggest some role for this molecule during early brain development.

Effects of electric fields on transmembrane potential and excitability of turtle cerebellar Purkinje cells in vitro.

1. Transmembrane potential (TMP) responses of Purkinje cells (PCs) in isolated turtle cerebellum to externally applied quasi-steady-state electric fields aligned with the dendritic axis were continuously measured using simultaneous intracellular and extracellular recording. TMP was obtained by subtraction of extracellular voltage fields from intracellular potential recorded at the same depth in the cerebellum. 2. The applied field changed the TMP with the polarity and amplitude dependent on the location on the PC membrane. This response at a given location increased linearly with external field up to a threshold level, beyond which active responses appeared. 3. The basic effect on TMP consisted of depolarization in the half of the dendrite towards which the fields were directed, and hyperpolarization in the other half. A pooled TMP depth-profile shows a steady increase in polarization from the middle of the molecular layer towards each end. This profile correlates with that predicted from previously proposed cable models, giving them empirical support for the first time. 4. Active responses were triggered by the field-induced depolarization. Tetrodotoxin (TTX)-sensitive action potentials arose with the primary depolarization in the somatic region. Notched, Ca2+-dependent action potentials arose with primary depolarization in the distal and mid-dendritic regions. 5. A TTX-sensitive voltage plateau was triggered by TMP-depolarization in the proximal region. It in turn activated Na+-spike trains. The frequency of spiking was proportional to the external field. At around 160 spikes/s, the Na+ spikes inactivated, and the TMP level rose to a more depolarized plateau. This latter plateau was also TTX-sensitive. 6. During depolarization of the distal dendritic region, sometimes a Ca2+-dependent plateau was observed. It appears to be associated with a small conductance increase. 7. Field-induced hyperpolarization suppressed local spiking and voltage plateaux, but remote Ca2+ spikes with reduced amplitude appeared in recordings from the proximal region. Similarly, in the distal region, low-amplitude, remote Na+ spikes and a Na+ plateau were observed superimposed on the hyperpolarizing baseline. The Na+ plateau apparently did not contribute to shunting of membrane currents in the distal dendrite. 8. The phase characteristics of the action potentials correlate with the modulation pattern noted in our extracellular study (Chan & Nicholson, 1986). Thus, the extracellular units ("giant spikes") were probably Na+ spikes activated in the soma and spread distally. Occasionally Ca2+ spikes, with a higher threshold, might also be activated to give dual-phase response.(ABSTRACT TRUNCATED AT 400 WORDS)

L-Aspartate and l-glutamate binding sites in developing normal and ‘nervous’ mutant mouse cerebellum

This study concerns the ontogeny and the cellular localization of l-aspartate and l-glutamate binding sites in normal and ‘nervous’ mutant mouse cerebellar membranes. The binding kinetics revealed for l-aspartate a single binding system (Kd = 750 nM) and for l-glutamate also a single binding component of higher affinity (Kd = 344 nM). The pharmacological study, using various amino acid analogues, revealed a differential specificity for the binding sites of the two amino acids.The developmental study showed that the binding sites of both amino acids appear mainly during the second and third week of life, a period when parallel and climbing fiber synaptogenesis occurs, but they follow a slightly different developmental pattern. The study using ‘nervous’, mutant mouse cerebellum showed an age-dependent decrease of l-aspartate and l-glutamate binding, which coincides in time with the Purkinje cell degeneration in this mutant, indicating a cellular localization of these binding sites on the Purkinje cell membranes. These results suggest that l-aspartate and l-glutamate binding sites may be respectively associated with the postsynaptic target of climbing and parallel fibers on the Purkinje cell dendrites. However, the decrease of specific binding in ‘nervous’ mutant mouse cerebellum was about 50% for l-aspartate and 60% for l-glutamate, implying that a significant number of l-aspartate and l-glutamate binding sites are located on cerebellar elements other than the Purkinje cell membranes.

Cardiac dysrhythmias in the acute setting: Pathophysiology or anyone can understand cardiac dysrhythmias

Cardiac dysrhythmias are easy. Unlike the lung (which has formidable neuroendocrine, metabolic, and respiratory responsibilities), the heart is simple. It is an innervated muscular pump. A resting Purkinje or ventricular muscle cell membrane maintains a charge of about 90 millivolts. The five phases of a cardiac action potential are similar to the action potential in skeletal muscle, however, the cardiac action potential lasts a hundred times longer. When sodium specific “fast” channels and calcium specific “slow” channels open, positive ions rush into the myocardial cell, thus causing rapid membrane depolarization. In order to produce an action potential, some stimulus must decrease the membrane potential from − 90 millivolts to “threshold” or − 60 millivolts. Purkinje fibers do not have a stable phase for diastolic potential. These fibers continuously depolarize during diastole. Hypoxemia or hypokalemia may exacerbate this diastolic depolarization, thus promoting “hyperexcitability” or “automatic” ectopy. When myocardium is damaged, characteristically with myocardial ischemia, rapid conduction of cardiac impulses may be slowed dramatically. Very slow impulses may course through muscle such that by the time the activation wave front returns to the initiating site, this origin has had a chance to repolarize. This is the basis for re-entrant dysrhythmias. All cardiac dysrhythmias are automatic, re-entrant or both.

Cellular distribution of gangliosides in the developing mouse cerebellum: analysis using the staggerer mutant.

The distribution of cerebellar gangliosides was studied in staggerer (sg/sg) mutant mice, where the majority of granule cells die after completing their migration across the molecular layer. In addition, the external granule cell layer in sg/sg mice persists longer than in normal mice. Moreover, in the sg/sg cerebellum, Purkinje cells are significantly reduced in number, and almost none have tertiary branchlet spines. The loss of Purkinje cells and granule cells in sg/sg mice is accompanied by an early-onset reactive gliosis that continues through adulthood. By correlating changes in ganglioside composition with the well-documented histological events of cerebellar development in normal and sg/sg mice, we obtained strong evidence for a nonrandom cellular distribution of gangliosides. The sharpest reduction in the GD1a content of sg/sg cerebellum occurred after 15 days of age, coincident with granule cell loss. GT1a, on the other hand, was significantly reduced from 15 through 150 days in the sg/sg mice. GD3 is a major ganglioside of the undifferentiated granule cell, but it becomes rapidly displaced by the more complex gangliosides with the onset of granule cell maturation. In the sg/sg mice, GD3 persisted at abnormally high levels from 15 to 28 days and then accumulated through adulthood. These findings, and those from other cerebellar mouse mutants, suggest that GD1a is enriched in granule cells and that GT1a is enriched in Purkinje cells. Our findings also suggest that GT1a is more concentrated in branchlet spines than in other regions of the Purkinje cell membrane. GT1b appears to be enriched in both granule cells and Purkinje cells, whereas GM1 appears to be enriched in myelin. Furthermore, the apparent persistence of the embryonic ganglioside GD3 in sg/sg mice results from an early-onset reactive gliosis, together with a partial retardation in granule cell maturation. The accumulation of GD3 beyond 28 days reflects the continued accretion of GD3 in reactive glia.

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro

A voltage clamp analysis of the effects of l-aspartate, l-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications of l-aspartate and l-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl- d, l-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl- d, l-aspartate much more severely depressed l-aspartate than l-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due to l-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due to l-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl- d-aspartate and quisqualate receptors.

Cultured cerebellar neurons: endogenous and exogenous components of purkinje cell activity and membrane response to putative transmitters

Modified explant cultures of fetal rat cerebellum were developed for electrophysiological and pharmacological studies, at the membrane level, of Purkinje neurons. The goals of the present series of experiments were to identify possible endogenous and exogenous components to the electrical activity of Purkinje neurons, to assess the sensitivity of these neurons to putative excitatory and inhibitory neurotransmitters, and to characterize the membrane response to the transmitters. Intracellular recordings were made from Purkinje neurons, identified on a morphological basis, using conventional electrophysiological techniques. Virtually all Purkinje neurons displayed spontaneous activity. A contribution of both endogenous and exogenous components to the spontaneous activity was indicated by alterations in the pattern and amount of activity when the membrane potential was varied and by the characteristics of the individual potentials themselves. Several types of activity were considered to be endogenous: the most common type consisted of pacemaker-like potentials which generated a pattern of firing similar to that characterized as simple spike activity in previous in vivo studies; another type of endogenous activity consisted of large membrane depolarizations that evoked one or two spikes. These depolarizing responses were similar to the membrane response generated by climbing fiber input to Purkinje cells in vivo. The exogenous components to the spontaneous activity consisted of synaptic potentials including excitatory (EPSPs) and inhibitory (IPSPs) synaptic potentials and biphasic EPSP/IPSPs. Several putative transmitters thought to mediate these synaptic potentials were tested by focal micropressure application to determine if they could mimic the action of the endogenous transmitters. The putative transmitter glutamate depolarized the cultured Purkinje neurons and evoked action potentials, characteristics which were displayed by the excitatory synaptic potentials. The putative inhibitory transmitter GABA hyperpolarized the cultured Purkinje neurons and depressed activity, characteristics which were displayed by the inhibitory synaptic potentials. The putative inhibitory transmitters glycine and taurine were ineffective. Norepinephrine, the transmitter mediating the inhibitory input from the locus coeruleus to Purkinje neurons, was also tested. When applied in the μM range, NE effects were variable. When applied in the mM range, NE depressed the spontaneous activity in a manner suggestive of a presynaptic action.

Effects of Taurine and GABA on Ca spikes and Na spikes in cerebellar Purkinje cells in vitro: Intrasomatic study

For the purpose of throwing some light on the inhibitory neuronal mechanisms in the cerebellum, particularly that by interneurons, the effects of taurine, a strong candidate for the transmitter of stellate interneurons, on spontaneous and evoked Ca spikes and Na spikes in Purkinje cells in guinea pig cerebellar slices were intrasomatically investigated in comparison with GABA, the transmitter of basket interneurons. In common, both amino acids hyperpolarized Purkinje cell membrane, inhibited somatic Na spikes and dendritic Ca spikes, and decreased somatic membrane resistance. Taurine applied focally onto Purkinje cell dendrites, however, was found to block Ca spikes and decrease the membrane resistance more potently than when it was applied onto the soma. GABA applied onto Purkinje cell somata was more potent in blocking somatic Na spikes and in decreasing the membrane resistance than taurine applied onto the somata. The dendritic sites of Purkinje cells, which were sensitive to iontophoretically applied taurine, responded also to focal electrical stimulation in an inhibitory manner. These results not only suggest that Purkinje cell dendrites are the primary site of action for taurine, but also support the previous concept that taurine acts as a transmitter in the dendritic interneuron, stellate cells.

Cholinergic mechanisms and neurotransmission in the cerebellum of the rat. An in vitro study

The effects of acetylcholine (ACh) and ACh antagonists on synaptic transmission, membrane resistance and resting potential of Purkinje cells in Larsell's lobules IX and X of the adult rat cerebellum were studied in sagittal slices maintained in vitro. Typical disynaptic excitatory responses mediated via the mossy fibre-granule cell pathway, as well as characteristic antidromic activation and climbing fibre responses were routinely evoked in Purkinje cell soma by monopolar or bipolar white matter stimulation. Marked IPSPs were also evoked in Purkinje cell soma and dendrites by white matter stimulation or by local stimulation of the molecular layer. In all Purkinje cells tested, d-tubocurarine, mecamylamine and gallamine triethiodide at concentrations up to 1 mM/l in the bath had no detectable effect on the excitatory and inhibitory synaptic responses. Atropine, at concentrations up to3 × 10 −6M/l in the bath did not affect climbing fibre and mossy fibre mediated EPSPs evoked in Purkinje cells by white matter stimulation, but abolished the fast spikes evoked in these neurones by synaptic or direct electrical stimulation. No synaptic responses recorded in Purkinje cell somata were affected by a bath concentration of2 × 10 −5M/l of eserine. Finally, bath or iontophoretic application of high doses of ACh led in most cases to a slow depolarization of Purkinje cells, which was accompanied by a noticeable increase in input resistance and which was no longer observed when atropine was also added to the bath. These results suggest that the presence of a massive contingent of mossy or climbing fibres using ACh as a neurotransmitter is unlikely in cerebellar lobules IX and X of the rat, and indicate that the action of ACh on Purkinje cell membranes is very similar to its muscarinic effect on other central neurones.

Climbing fiber destruction affects dendrite and spine membrane organization in purkinje cells

Destruction of climbing fibers, one of the presynaptic inputs to Purkinje cells, was achieved by intraperitoneal injection of 3-acetylpyridine in rats. Freeze-fracture morphology of the Purkinje cell membrane was studied under these conditions. Quantitative analysis reveals a decrease in the number of intramembrane particles (IMP) in the membrane E-face of dendrites and spines of large dendrites, both post-synaptic targets for climbing fibers. The membrane of the perikaryon and of the spines from the spiny branchlets were unaffected by climbing fiber destruction. These results suggest that under the conditions studied, the membrane organization in well-defined areas of the Purkinje cell may be influenced by presynaptic factors.

Structure of the Purkinje cell membrane in staggerer and weaver mutant mice.

The structure of the plasma membrane of Purkinje cell dendrites was examined in weaver and staggerer mutant mice. Purkinje spines in weaver mice have clusters of intramembrane particles which resemble those at normal synapses with parallel fibers, even though no parallel fibers are formed in this mutant. There are very few spines in the staggerer, and these manifest normal intramembrane structure at contacts with climbing fibers. The spines which would normally be involved in synapses with parallel fibers are never formed in the staggerer, and the intramembrane structures which would have been associated with these spine synapses are also lacking. Thus, during postnatal cerebellar development in the mutants, acquisition of intramembrane specializations requires Purkinje spine formation but can occur independently of the development of parallel fibers.

Physiological and pharmacological properties of Purkinje cells in rat cerebellum degranulated by postnatal x-irradiation.

AbstractElimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x‐irradiation applied during the first two weeks of postnatal life. Purkinje neurons in these rats, studied when adults, exhibited sustained spiking activity in Halothane anesthetized preparations. Mean firing rates were 35‐‐40/sec, no different from normal. Spontaneous bursts presumed to be generated by climbing fiber synaptic activity differed from normal by often consisting of full sized spikes rather than characteristic inactivation responses. Intracellularly observed correlates of bursts consisted of epsp's of several discretely different amplitudes appearing independently in time. Stimulation of white matter revealed evidence for, (a) graded synaptic excitation of Purkinje cells indicating more than one converging excitatory synapse, and (b) inhibitory actions on Purkinje cells either through a few remaining inhibitory interneurons or through Purkinje cell recurrent collaterals. Iontophoretic drug application studies showed normal chemosensitivity of the Purkinje cell membrane, i.e., excitation by glutamate and inhibition by gamma‐amino butyric acid, serotonin, norepinephrine, and 3′5′ cyclic AMP.These studies indicate considerable autonomy of Purkinje cell ontogenesis in the absence of normal interneuronal input. A unique synaptic relation only rarely found in normal cerebellum is the innervation of single Purkinje cells by more than one climbing fiber.

Electrogenesis of cerebellar Purkinje cell responses in cats.

Electrogenesis of cerebellar Purkinje cell responses in cats.F E Martinez, W E Crill, and T T KennedyF E Martinez, W E Crill, and T T KennedyPublished Online:01 May 1971https://doi.org/10.1152/jn.1971.34.3.348MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByPhysiology of the cerebellumSomatosensory cortical neurons with an identifiable electrophysiological signatureBrain Research, Vol. 441, No. 1-2Membrane resistivity estimated for the purkinje neuron by means of a passive computer modelNeuroscience, Vol. 14, No. 1Plateau potentials evoked by climbing-fibre stimulation are restricted to the Purkinje cell dendrites of the catNeuroscience Letters, Vol. 45, No. 2Cultured cerebellar neurons: endogenous and exogenous components of purkinje cell activity and membrane response to putative transmittersBrain Research, Vol. 263, No. 2The changes in Purkinje cell simple spike activity following spontaneous climbing fiber inputsBrain Research, Vol. 237, No. 2The olivocerebellar system. I. Delayed and slow inhibitory effects: An overlooked salient feature of cerebellar climbing fibersBrain Research, Vol. 187, No. 1A computer model of cerebellar purkinje cellsNeuroscience, Vol. 2, No. 1Climbing fiber responses of cerebellar Purkinje cells to passive movement of the cat forepawBrain Research, Vol. 106, No. 1Anatomical, physiological and biochemical studies of the cerebellum from mutant mice. I. Electrophysiological analysis of cerebellar cortical neurons in the staggerer mouseBrain Research, Vol. 98, No. 1Cerebellar afferent systems: A reviewProgress in Neurobiology, Vol. 2Cerebellar output: Properties, synthesis and usesBrain Research, Vol. 40, No. 1The role of dendritic events in the initiation of monosynaptic spikes in frog motoneuronsBrain Research, Vol. 39, No. 2Synaptic potential summation and repetitive firing mechanisms: Input-output theory for the recruitment of neurons into epileptic bursting firing patternsBrain Research, Vol. 39, No. 1 More from this issue > Volume 34Issue 3May 1971Pages 348-56 https://doi.org/10.1152/jn.1971.34.3.348PubMed5560036History Published online 1 May 1971 Published in print 1 May 1971 Metrics

Origin of the negative potential shift of all-or-nothing character in the cerebellar cortex of the rabbit.

1. The negative potential shift, having variable durations (from 250 msec to several sec) and occurring in all-or-nothing fashion by white matter stimulation, was studied with the microelectrode in the cerebellum of the rabbit anaesthetized with Nembutal. The shift ranged from several to 60 mV in magnitude. The shift was recorded from the molecular layer as well as from the layer of Purkinje cell bodies, but never observed in the white matter.2. The shift was always preceded by the sequential spike discharge of the Purkinje cell dendrites, but did not influence the excitability of the dendrites. Furthermore, no intracellular potentials were ever recorded from the Purkinje cell dendrites and cell bodies, which could account for the long-lasting potential shif t. These results show that the shift is recorded when the microelectrode tip is positioned close to the Purkinje cell membrane, but it does not originate in the Purkinje cell. All other nerve cells in the cerebellar cortex are also excluded as candidates for possible site of origin of the shift because they are physically not in a close relationship with the Purkinje cell dendrites. Possible site of origin of the shift was discussed.3. During the shift amplitude of the spike of the Purkinje cell dendrites increased and positive-negative spikes of its cell body were converted into monophasic-positive spikes. Possible mechanism for this phenomenon was discussed.