Effects of serotonin on cerebellar Purkinje cells are dependent on the baseline firing rate

Summary Serotonin applied iontophoretically to cerebellar Purkinje cells elicited one of three effects: inhibition (62% of the cells), biphasic response (27%), and excitation (11%). This study describes a correlation between the spontaneous discharge rate of Purkinje cells and the action of serotonin. Purkinje neurons that responded to serotonin with an increase in firing rate had a significantly slower predrug firing frequency (40 Hz) than those cells that were suppressed by serotonin (51 Hz). Furthermore, it was shown that with increasing firing rates the proportion of excitations decreased, and the proportion of depressions increased. A quantitative comparison revealed a statistically significant correlation between the spontaneous discharge rate of cells displaying excitation and the magnitude of the excitatory response. On several occasions, the direction of the Purkinje cell response to serotonin reversed following a decrease or increase in the baseline spontaneous rate. Glutamate- or asparate-induced excitations elicited an augmentation of serotonin-mediated inhibition and in some cases a reversal of excitation to inhibition. Likewise, the lowering of neuronal activity by the continuous application of glycine augmented excitation and reduced and reversed serotonin inhibitions. Preliminary results from experiments in which various receptor antagonists were tested against serotonin actions suggest that the effects of serotonin occur, at least in part, at postsynaptic sites on Purkinje cells. These results strongly suggest that the overall qualitative effects of serotonin is to set Purkinje cells at a preferred firing rate. In this sense, the term biaser or modulator may best describe the role of serotonin in the cerebellum.     

Potassium currents modulation of calcium spike firing in dendrites of cerebellar Purkinje cells

The pattern of sustained Ca²⁺ spike firing was investigated, using macropatch clamp and intracellular recordings, in guinea pig cerebellar Purkinje cells. Under our standard experimental conditions (30°C, 5 mM [K⁺]_o, 2 mM [Ca²⁺]_o, 1 μM tetrodotoxin), each firing period started with uniform firing and gradually turned into a doublet pattern with a large spike afterhyperpolarization (AHP) between the doublets. Macropatch clamp recordings from localized dendritic regions revealed that each doublet is composed of two similar inward current deflections. This result indicated, for both peaks, an active process in the recording site and contradicted the possibility that they reflect firing in two completely separated dendritic regions. When [K⁺]_o was increased the transition to a doublet pattern occurred earlier and the doublets became more pronounced. A similar but more prominent effect occurred following application of 1–10 μM 4-aminopyridine, which also reduced the threshold, increased the spike amplitude, and shortened the initial delay of evoked Ca²⁺ spike firing. In contrast, membrane depolarization, increased [Ca²⁺ ]_o, and application of quinidine (but not apamine) markedly suppressed the generation of doublet pattern. During uniform initial firing, a short hyperpolarizing pulse that mimicked a large AHP induced a subsequent doublet. A short depolarizing pulse following a single spike induced an artificial doublet followed by a large AHP. These results indicate that the pattern of Ca²⁺ spike firing in the dendrites of Purkinje cells is dynamically modulated by a highly aminopyridine-sensitive K⁺ current, and probably also by a Ca²⁺ -activated potassium current. Received: 1 December 1997 / Accepted: 28 April 1998     

Effect of simple spike firing mode on complex spike firing rate and waveform in cerebellar Purkinje cells in non-anesthetized mice

Cerebellar Purkinje cells receive two different excitatory inputs from parallel and climbing fibers, causing simple and complex spikes, respectively. Purkinje cells present three modes of simple spike firing, namely tonic, silent and bursting. The influence of complex spike firing on simple spike firing has been extensively studied. However, it is unknown whether and how the simple spike firing mode may influence complex spike waveform and firing rate in vivo. We studied complex spike firing during tonic and silent mode periods in non-anesthetized mice. We found that complex spike firing rate is not influenced by simple spike firing modes, but that the complex spike waveform is altered following high frequency simple spike firing. This alteration is a specific decrement of the second depolarizing component of the complex spike. We demonstrate that the amplitude of the second depolarizing component is inversely proportional to the simple spike firing rate preceding the complex spike and that this amplitude is independent of previous complex spike firing. This waveform modulation is different from previously reported modulation in paired-pulse depression and refractoriness.     

4-Aminopyridine (4-AP) augments Ca(2+)-dependent action potential and changes oscillatory firing patterns in rat cerebellar Purkinje cells.

Intracellular recordings in cerebellar slice preparation showed that applications of 4-AP altered the pattern of oscillatory firing activity in Purkinje cells (PCs), especially yielding pronounced changes in action potential shape. 4-AP increased the amplitude and duration of action potential significantly and decreased the spike frequency. After 4-AP application, the duration of bursting was prolonged and the duration of after-burst hyperpolarization was progressively shortened. In all PCs tested, the rhythmicity of oscillatory firing activity was abolished completely at the steady state. These results suggest that 4-AP-sensitive currents determine the shape and frequency of individual Ca(2+)-dependent action potentials as well as maintaining oscillatory firing activity in PCs.     

Altered dendritic development of cerebellar Purkinje cells in slice cultures from protein kinase Cgamma-deficient mice

Protein kinase C (PKC) is a key molecule for the expression of long-term depression at the parallel fiber–Purkinje cell synapse in the cerebellum, a well known model for synaptic plasticity. We have recently shown that activity of PKC also profoundly affects the dendritic morphology of Purkinje cells in rat cerebellar slice cultures suggesting that synaptic efficacy and dendritic development may be controlled by similar intracellular signalling pathways. Here we have analyzed the role of the γ-isoform of protein kinase C (PKCγ), which is strongly and specifically expressed in Purkinje cells, during dendritic development. After pharmacological treatment with PKC modulators, phosphorylation of PKCγ at serine 660 was altered in cerebellar slices suggesting that a change of PKCγ activity by these treatments was taking place within the Purkinje cells. In PKCγ-deficient mice, Purkinje cell dendritic trees were enlarged and had an increased number of branching points compared to wild-type mice indicating a role for the PKCγ isoform as a negative regulator of dendritic growth and branching. Furthermore, the branching-stimulating effects of the PKC inhibitors 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide and Gö6976 found in wild-type cultures were abolished in the absence of PKCγ. In contrast, the strong inhibitory effect on dendritic growth by the PKC activator phorbol-12-myristate-13-acetate (PMA) did not require the presence of the PKCγ isoform since it was still present in the cultures of PKCγ-deficient mice.

Our results clearly demonstrate an involvement of PKCγ in Purkinje cell dendritic differentiation in cerebellar slice cultures.     

Maturation of the cerebellar Purkinje cells

Summary The postnatal evolution of the Purkinje cell spontaneous firing (simple spikes) has been studied in the white Rat from 3 days after birth until adulthood using the following parameters: modal interspike interval, mean interspike interval, and ratio of the modal over the mean interspike interval of the discharge. Furthermore, two experimental conditions have been used: rats anaesthetized with Nembutal and rats only locally analgesied, the two preparations being curarized. It is shown that:1) Between 3 and 5 days after birth, the spontaneous activity of many Purkinje cells is only formed by brief bursts of several spike potentials occuring more or less frequently. Evidence is given that some of these bursts are climbing fiber responses, the others being presumably elicited in Purkinje cells by parallel fibers activation. 2) After 5 days, the firing pattern (simple spikes) of Purkinje cells become more subtained and is formed by a more or less regular succession of spikes. Experimental conditions significantly change for all ages studied this firing pattern, and also change the time course of the evolution of all the considered parameters excepted for the modal interspike interval. 3) As a whole, Purkinje cells look immature until 8 days after birth. They rapidly mature between days 10 and 20. After the end of the 3rd postnatal week they show no significant differences when compared to the spontaneous firing of adult Purkinje cells, at least for the unanaesthetized preparations.At all ages a good correlation exist between Purkinje cells spontaneous firing (simple spikes) and data from the litterature concerning synaptogenesis in the cerebellum (parallel fibers Purkinje cells synapses).     

Histamine excites rat cerebellar Purkinje cells via H2 receptors in vitro

Recent neuroanatomical studies have revealed a direct hypothalamocerebellar histaminergic pathway. However, the functional significance of the histaminergic fibers in the cerebellum is not yet clear. In this study, the effects of histamine on the firing of cerebellar Purkinje cells (PCs) were investigated in vitro. Histamine predominantly produced excitatory (106/111, 95.5%) and in a few cases inhibitory (5/111, 4.5%) responses in PCs. The histamine-induced excitation was not blocked by perfusing the slice with low Ca2+ high/Mg2+ medium (n = 8), supporting a direct postsynaptic action of histamine. The histamine H2 receptor antagonist ranitidine effectively blocked the excitatory response of PCs to histamine (n = 20), but triprolidine, an H1 receptor antagonist, could not significantly block the histamine-induced excitation, or only very slightly decreased the excitatory effect of histamine on the cells (n = 13). On the other hand, the highly selective H2 receptor agonist dimaprit mimicked the excitatory effect of histamine on PCs and this dimaprit-induced excitation was also blocked by ranitidine (n = 20), but not triprolidine (n = 8). However, the H1 receptor agonists betahistine and 2-thiazolylethylamine did not show any effect on the PCs (n = 9 and 14). These results reveal that histamine excites cerebellar PCs via H2 receptors and suggest that the hypothalamocerebellar histaminergic fibers may play an important role in functional activities of the cerebellum.     

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

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

Correlations among climbing fiber responses of nearby cerebellar Purkinje cells in the immature rat

Summary Correlations between pairs of spontaneous climbing fiber responses (CFRs) recorded from couples of nearby Purkinje cells (PCs) were studied in immature rats by using cross-correlograms between CFR pairs, and compared to those in adult animals. Correlations were found as early as day 3. Some days later, on PN days 7–9, these correlations were higher than in the adult. In most cases, this was apparently not due to the multiple innervation of PC by climbing fibers (CFs) which normally occurs during this immature stage since: 1) temporal relationships between the paired CFRs varied by more than 30 ms and 2) thresholds for pairs of graded CFRs and additional components of the responses evoked in the 2 PCs by juxtafastigial or olivary stimulation were different. Synchronizing mechanisms were therefore likely to be already located at the olivary level. However, in 3 couples of multiply innervated PCs whose spontaneous CF activities were highly correlated, stimulation experiments revealed a common innervation of the 2 cells by branches of the same CF. In multiply innervated cells, spontaneous responses mediated through distinct CFs were also synchronized, suggesting that these fibers originate from neighboring neurons of the inferior olive. Finally, in 7 to 9-day-old rats, correlations among CFR pairs were much more restricted in the longitudinal axis of the folium than in the transverse one. On the whole, the present study indicates that correlations among CFRs of nearby PCs exist as soon as CF-PC synapses are established and the latter are already organized in sagittal strips at early stages of development.This work was supported by INSERM (C.R.L. Nr. 78.1.003.6)     

5-Lipoxygenase in mouse cerebellar Purkinje cells

It has been suggested that the enzymatic pathway of 5-lipoxygenase (5-LOX) influences brain functioning and pathobiology. The mRNAs for both the enzyme 5-LOX and its activating protein FLAP have been found in the cerebellum. In this work, we investigated the cellular expression of 5-LOX in the adult mouse cerebellar cortex. We used the in situ mRNA hybridization assay, immunocytochemistry, laser capture microdissection, and our previously developed method for assaying the DNA methylation status of a putative mouse 5-LOX promoter. Since both 5-LOX mRNA in situ hybridization signal and FLAP immunoreactivity co-localize with calbindin 28 kD immunoreactivity (a Purkinje cell marker) but not with S-100β immunoreactivity (a Bergmann glia marker), the suggestion is that the 5-LOX pathway is expressed in cerebellar Purkinje cells. We found that methylation in the sites targeted by methylation-sensitive restriction endonucleases AciI and HinP1I but not BstUI and HpaII was greater in DNA samples obtained from a high-5-LOX-expressing cerebellar region (Purkinje cells) versus a low-5-LOX-expressing region (the molecular cell layer), suggesting a possible epigenetic contribution to the cell-specific 5-LOX expression in the cerebellum. We propose that Purkinje cell-localized 5-LOX and FLAP expression may be involved in the cerebellar synthesis of leukotrienes and/or could influence the Dicer-mediated microRNA formation and processes of neuroplasticity.     

Microtubule bundles in fish cerebellar Purkinje cells

Summary The initial axon segments and the cell bodies of Purkinje cells were examined in electron microscopic serial sections and toluidine blue semithin sections of goldfish cerebellum. We observed two characteristic cytoplasmic features different from those of other vertebrate neurons. 1. The areas of Nissl substance and Golgi apparatus are sharply divided in the periphery and center of the cytoplasm. 2. Microtubules fasciculated by cross-bridges in the axon hillock and initial axon segment remain bundled in the perikaryon, pass near the eccentric nucleus, and enter into the Golgi area of the central cytoplasm, where they are surrounded by mitochondria. We suggest that the intracellular fasciculated microtubules may establish a prepared pathway for fast anterograde and retrograde transport to and from the Golgi area of the cell body.     

Axonal abnormalities in cerebellar Purkinje cells of the ''hyperspiny Purkinje cell'' mutant mouse

Summary The hyperspiny Purkinje cell (hpc) is a murine, autosomal recessive mutation affecting cerebellar Purkinje cells. Axonal abnormalities in these neurons have been revealed by selective silver impregnation, specific immunohistochemical staining and electron microscopy. The main pathological feature consists of a massive axonal degeneration in the terminal domains of the Purkinje cell projection. This process starts approximately ten days postnatally, simultaneously with the onset of cerebellar symptoms, and evolves very rapidly. By 21 days, the vast majority of the terminal arbors have degenerated, resulting in an almost complete disruption of the corticonuclear projection. Axonal degeneration, although proceeding in a dying-back fashion, only provokes retrograde death in a small percentage of Purkinje cells (less than 15%).Purkinje cells exhibit other signs of axonal damage and axonal reaction: (a) Almost all of them bear gigantic varicosities (spheroids or torpedoes) along their transit through the granular layer. (b) In a small percentage of cases, a dendritic segment is inserted between the axon hillock and the initial segment (meganeurite). These ectopic dendrites receive a normal contingent of synaptic inputs, and are transient structures observed in four- to six-week-old mice, (c) The infra- and supraganglionic plexuses, formed by recurrent collaterals of Purkinje cell axons, have increased density and terminal domains, (d) In mice aged over 50 days, many Purkinje cells have developed arciform axons, which is evidence of a compensatory reaction.The definite axonal pathology ofhpc Purkinje cells confers to this mutation its own specificity, which differs from all other known mutations primarily affecting this neuronal population. Therefore, thehpc mutation offers a valuable tool to analyse some of the genetic factors involved in the differentiation and maintenance of cerebellar Purkinje cells.     

The action of antidromic impulses on the cerebellar Purkinje cells

1. Antidromic impulses have been set up in the axons of Purkinje cells of the cerebellar vermis by stimulation in the juxta-fastigial (J.F.) region. Most experiments were performed on the normal cat cerebellum, but in nine the cerebellum was chronically deafferented by bilateral pedunculotomy 9-23 days previously.2. Intra- and extracellular recording from Purkinje cells both showed a characteristic inflexion on the rising phase of the spike potential (the characteristic IS-SD inflexion) that presumably signals a delay in invasion between the axon and the large soma-dendritic expansion.3. Laminar field analysis of the antidromic spike potentials showed that the antidromic impulses invaded at least 200 mu of the main dendrites as well as the soma, there being then a steep decrement to the surface. At superficial levels there was even an inverse antidromic spike potential. There appeared to be a synchronous invasion of the soma-dendritic complex, perhaps due to trigger zones of low threshold on the dendrites.4. Antidromic soma-dendritic invasion was modified in the expected manner by a volley in the parallel fibres; there was inhibition of transmission into the soma and up the main dendrites (maximum effect at 200-300 mu depth) due to the inhibitory action of the basket and superficial stellate cells that are excited by the parallel fibres; there was facilitation of transmission in the dendrites at levels superficial to 200 mu due to the direct excitatory action of parallel fibres. Both the inhibitory and excitatory actions had a duration in excess of 100 msec.5. In the chronically deafferented cerebellum a second J.F. stimulation evoked a full size antidromic spike potential at an interval of 3 msec. There was a gradual decline in size down to intervals of about 2 msec, and at briefer intervals, to 1 msec, there was a small residual spike potential that possibly is due to transmission into the Purkinje cell axon collaterals at intervals too brief for soma-dendritic invasion. With repetitive stimulation there was a well maintained soma-dendritic invasion at a frequency as high as 300/sec.6. In the chronically deafferented cerebellum an antidromic volley in the Purkinje cell axons caused a brief inhibitory silence of rhythmically discharging Purkinje cells. It is suggested that this is a direct inhibitory action of the Purkinje axon collaterals, that parallels their direct inhibitory action that has been demonstrated by Ito and collaborators (1964) on the intracerebellar nuclei and Deiters nucleus.7. In the chronically deafferented cerebellum an antidromic volley in the Purkinje axons produced not only the large negative spike potential indicative of antidromic soma-dendritic invasion, but also a later small and slow positive wave that appeared to be closely linked with the negative spike. It is shown how this would arise by current flow into the dendrites that had been depolarized but not excited by the initial antidromic invasion.