Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm²) and less rapidly between PN days 30 to 48 (up to 9,300 μm²), followed by a moderate decrease at PN day 90 (about 6,600 μm²). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm³ at PN day 6,93,000 μm³ at PN day 25, and 100,000 μm³ at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 10⁴ spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 10⁵ spines/Purkinje cell), which then declined to 6.3 × 10⁴ spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley-Liss, Inc.     

The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep: A note on the ontogeny of the Purkinje cell

The development of the fetal sheep cerebellum at 80, 100, 120 and 140 days gestation (term = 146 days) and 3 months postnatally was studied using Nissl stained sections and rapid Golgi preparations. The most rapid expansion of the Purkinje cell dendritic tree occurred between 100 and 120 days of gestation (5-6 fold increase in area). By 140 days it had acquired its adult form after which time growth continued mainly in the vertical direction. The effects of intrauterine growth retardation on the growth of granule and Purkinje cell dendrites in the cerebellar cortex of fetal sheep (140 days) were investigated in Golgi preparations. Compared with control cerebella the length (but not the number) of granule cell dendrites was reduced by 14% (P less than 0.01); the area of the Purkinje cell dendritic field was reduced by 20% (P less than 0.01); the branching density was reduced by 8% (P less than 0.01); the total branch length was reduced by 27% (P less than 0.002); the density of dendritic spines per row was not affected. These factors resulted in a decrease of 26% (P less than 0.002) in the total number of dendritic spines per row per Purkinje cell. These findings show that the growth of granule cell dendrites and the Purkinje cell dendritic tree have been significantly affected by chronic intrauterine deprivation. Such structural abnormalities could affect the pattern of neuronal connectivity and could be associated with functional deficits.     

Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.     

Phocein: A potential actor in vesicular trafficking at Purkinje cell dendritic spines

Phocein is an intracellular protein highly expressed in neurons. It is the major partner of the striatin family members which are scaffolding proteins involved in signaling and trafficking. Due to its association with dynam in via direct interactions with nucleotide diphosphate kinase (NDPK) and EPS15, phocein has been implicated in vesicular trafficking, acting in particular in the endocytic process. This review focuses on immuno-cytochemical studies showing the strict localization of phocein in Purkinje cell dendritic spines involved in excitatory transmission in the cerebellum of postnatal and adult rodents. Immunogold labeling sometimes detects phocein in close vicinity with, endocytic-like membrane profiles suggesting that phocein plays a role in endocytosis. Furthermore, co-localization of phocein and SG2NA within spines suggests that their interactions have a functional significance in the molecular cascades that underly membrane trafficking in postsynaptic structures. As the striatin family members are highly concentrated in dendritic spines, their interactions with phocein might be involved in mediating synaptic plasticity through spine remodeling by endocytosis.     

Permanent alterations of the dendritic tree of cerebellar Purkinje neurons in the rat following postnatal exposure to cis-dichlorodiammineplatinum

Summary The aim of this study was to characterize further the effect of cis-dichlorodiammineplatinum (cisDDP) on cerebellar Purkinje neurons of the immature rat. Ten-day-old rats were treated with cisDDP subcutaneously and killed after 1, 7, 20 or 65 days. The cerebellar vermis was impregnated by the Golgi-Cox method to evaluate the extent of morphological maturation of the Purkinje cell dendritic tree. One day after treatment, the dendritic network of Purkinje cells of treated animals was poorly developed and the cell somata still showed numerous perisomatic processes. This indicates that cisDDP interferes with the organization of microtubules and microfilaments by the cell. Later, several abnormal shapes of the Purkinje cell dendritic tree were observed. These included: (1) elongated primary dendrites; (2) asymmetrical dendrites; (3) sprouting of secondary and spiny branches in two planes of the molecular layer; and (4) damming of spiny branchlets at the pial surface. Moreover, all the dendritic networks of Purkinje cells in treated animals were of a lower Strahler order than in controls. All these data suggest that the late anomalies of the dendritic trees are secondary to the general cisDDP-induced damage of the cerebellar cortex, rather than being a primary effect of the drug on the dendritic tree growth.Supported in part by C.N.R. and M.U.R.S.T. grants     

Development of Purkinje cell somatic spines in the weaver mouse

Summary The cerebellar cortices of weaver mice and their normal littermates ranging in age from six to thirteen days after birth were examined with the electron microscope. The initial development of spines, both somatic and dendritic, are the same in both the weaver and its normal littermates despite the extreme paucity of parallel fibers in the former.     

El aprendizaje motor induce cambios plásticos en las espinas dendríticas de las células de Purkinje del cerebelo de ratas

IntroductionThe paramedian lobule of the cerebellum is involved in learning to correctly perform motor skills through practice. Dendritic spines are dynamic structures that regulate excitatory synaptic stimulation. We studied plastic changes occurring in the dendritic spines of Purkinje cells from the paramedian lobule of rats during motor learning.MethodsAdult male rats were trained over a 6-day period using an acrobatic motor learning paradigm; the density and type of dendritic spines were determined every day during the study period using a modified version of the Golgi method.ResultsThe learning curve reflected a considerable decrease in the number of errors made by rats as the training period progressed. We observed more dendritic spines on days 2 and 6, particularly more thin spines on days 1, 3, and 6, fewer mushroom spines on day 3, fewer stubby spines on day 1, and more thick spines on days 4 and 6.ConclusionThe initial stage of motor learning may be associated with fast processing of the underlying synaptic information combined with an apparent “silencing” of memory consolidation processes, based on the regulation of the neuronal excitability.     

Dynamic imaging of cerebellar Purkinje cells reveals a population of filopodia which cross-link dendrites during early postnatal development

Two-photon microscopy was used to image dye-loaded filopodia of Purkinje cells in acute rat cerebellar slices. In the process of examining filopodia in Purkinje cells from a period of rapid dendritic growth (P10-21), we observed a small subset of filopodia which appeared to form connections between two dendrites of the same cell, usually between the tips of two adjacent dendrites or the tip of a dendrite and the shaft of another. There were fewer of these 'filopodial bridges' present at P18-21 than at an earlier stage in development (P10-12) and they were absent in mature Purkinje cells. Filopodial bridges do not appear to be an artifact of living brain slice preparation as they may also be seen by dye-loading Purkinje cells in slices prepared from perfusion-fixed brain. They have varied morphologies which are mostly similar to conventional, unattached filopodia. However, when measured over tens of minutes, filopodial bridges were observed to be less motile than conventional filopodia as indicated by a reduced index of expansion. While the functions of these novel structures are unknown it is attractive to speculate that they play an instructive role in Purkinje cell dendritic development.     

Focal axonal swellings in rat cerebellar Purkinje cells during normal development

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

Effects of chronic ethanol consumption beginning at adolescence: increased numbers of dendritic spines on cortical pyramidal cells in the adulthood

Summary To increase our understanding of the effects of chronic ethanol consumption beginning at adolescence, 25% ethanol in drinking water (v/v) was administered daily to young rats aged 45–50 days for 5 months. Increased numbers of dendritic spines on the apical dendrite of layer V pyramidal neurons of the somatosensory cortex (U-Mann-Whitney test,P<0.01–0.05) were found in almost every 50-m-long segment over a distance of 500 m from the cell body in ethanol-treated rats at the age of 195–200 days when compared with age-matched controls. Although the mechanisms leading to this unusual finding are not know, it is suggested that impairment of the naturally occurring elimination of redundant synapses can not be ruled out.     

Serotonergic interactions with rat cerebellar Purkinje cells

Recent immunocytochemical and histofluorescent studies have established the vastness of the serotonergic fibers arising from the raphe complex and projecting to the cerebellar cortex. Ultrastructurally, the indoleaminergic fibers are known to establish synaptic contacts as well as diffuse meandering nonsynaptic terminations near Purkinje cells. The objective of this study was to define the changes elicited by serotonin on spontaneous and chemically- and electrically-evoked excitatory synaptic activities on cerebellar Purkinje cells. Serotonin (10-50 nA) applied for 30-60 sec elicited predominantly a decrease in firing rate followed by rebound excitation (25 cells out of a total of 39 cells). Studies in which serotonin was continuously iontophoresed for prolonged periods, this amine produced an increase in the spontaneous firing rate in 55% of the Purkinje cells tested. A comparison of evoked excitation and spontaneous activity revealed that when serotonin was applied continuously there was a net decrease in evoked excitation to spontaneous activity ratio. Based on the evoked excitation studies, glutamate (5-30 nA) was applied for 30-40 sec in the presence of serotonin (10-30 nA). Seventy-seven percent of the cells displayed a decrease in responsiveness to glutamate while the remaining 23% were potentiated. More definitive explanation of these results are given in the body of the test.     

Axonal torpedoes in cerebellar Purkinje cells of two normal mouse strains during aging

The present study systematically investigated the proportional evolution of Purkinje cell (PC) axonal swellings, also termed torpedoes, during aging of the two unrelated mouse strains B6CBA and C57BL/6J. Torpedoes were identified using monoclonal antibodies against the calcium-binding protein calbindin D-28k in mice ranging in age from 8 days postnatally up to 32 months. The relative density of PCs bearing torpedoes in animals up to 6 months of age was less than 0.1%. The number increases between 6–8 months and rises further in older mice almost linearly up to 13.7% affected PCs in the oldest animal (32 months) studied. In contrast, PC loss, as indicated by parvalbumin-immunoreactive empty baskets, is only at a very moderate level (less than 0.5%) in these strains. While the proximal axonic segments often show two and occasionally up to five swellings and frequently appear to be hypertrophied as a whole, the dendritic trees and neuronal somata of the affected PCs exhibit normal morphology. On rare occasions adaptive reactions indicated by arciform axons and enlarged varicosities of recurrent collaterals were observed. The results demonstrate that in addition to age-related PC loss of whatever degree, axonal disturbances of PCs, indicated by torpedoes, are present, leading most probably to a graded loss of cerebellar cortico-fugal projections.     

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.     

Effects of chronic ibogaine treatment on cerebellar Purkinje cells in the rat

The present investigation assessed the chronic toxicity of ibogaine on cerebellar Purkinje cells in male Fischer 344 rats. A behaviorally active dose of ibogaine (10 mg/kg, i.p.) was administered to a group of six subjects every other day for 60 days while the control group received an equivalent volume of saline (1 ml/kg). Estimates of Purkinje cell number were determined using the optical dissector/fractionator technique. No significant differences in Purkinje cell number were observed between the ibogaine (243764[±32766]) and control groups (230813[±16670]).     

The puzzle of ploidy of Purkinje neurons

This paper places attention on the discrepancies existing in the literature on the ploidy of Purkinje neurons and focuses on the special case of partial replication of their genome and on the unequal frequency of polyploid Purkinje neurons in the cerebellar cortex of the lobes and the vermis. Owing to the compartmental structure of the cerebellum, this paper suggests the investigation with modern methods and techniques of other cerebellar regions such as the flocculus, with the aim of establishing whether increased ploidy correlates with cell hypertrophy and/or with stimulation of cerebellar functions.     

大鼠小脑薄肯野细胞内多巴胺B羟化酶和激活蛋白2a的共存

BACKGROUND： Tyrosine hydroxylase and phenylethanolamine-n-methyl transferase expression coexist in Purkinje cells of the rat cerebellum. Numerous reports have also been published addressing whether dopamine-beta-hydroxylase （DBH） expression exists in cerebellar Purkinje cells. OBJECTIVE： To investigate the coexistence of DBH and activator protein-2α expression in rat cerebellar Purkinje cells. DESIGN, TIME AND SETTING： A cell morphological study was performed at the Institute of Neuroscience, Chongqing Medical University, China in May 2007. MATERIALS： Ten healthy Wistar rats, of either gender, aged 14 weeks, served as experimental animals. Rabbit anti-mouse DBH, goat anti-mouse activator protein-2α and rabbit anti-mouse β-actin （Santa Cruz Biotechnology, Inc., USA）, horseradish peroxidase-labeled goat anti-rabbit IgG, FITC-labeled mouse anti-rabbit IgG, and Cy3-labeled mouse anti-goat IgG （Boster, Wuhan, China）, were used in this study. METHODS： Immunohistochemical staining was used to measure the expression of DBH or activator protein-2α, with double-label immunofluorescence being employed to determine coexpression of both, in the cerebellum of 5 randomly selected rats. Western blot assay was utilized to determine the expression of DBH and activator protein-2α in the cerebellum of the remaining 5 rats. MAIN OUTCOME MEASURES： Expression, localization and coexistence of DBH and activator protein-2α in the cerebellum were measured separately. RESULTS： Immunohistochemical staining demonstrated that cerebellar Purkinje cells stained positive for DBH and activator protein-2α. Western blot assay also demonstrated DBH and activator protein-2α expression in the cerebellum. Double-labeling immunofluorescence showed the coexistence of DBH and activator protein-2α in cerebellar Purkinje cells. CONCLUSION： Norepinephrine and activator protein-2α coexist in rat cerebellar Purkinje cells.     

Dynamic changes in CA1 dendritic spines associated with ischemic tolerance

Hippocampal CA1 neurons are particularly vulnerable to 5-10 min durations of global ischemia. These cells can develop tolerance to ischemia through prior exposure to brief episodes of ischemia (ischemic preconditioning, IP). Dendritic spines are implicated in various forms of neuroplasticity including memory and recovery of function. Here we characterized the changes in hippocampal CA1 dendritic spines during the development of ischemic tolerance and the subsequent postischemic recovery period. Gerbils received 5 min, bilateral carotid artery occlusions preceded by two 1.5 min occlusions each of which were 24 h apart (tolerance groups). Spine densities were calculated from CA1 apical and basilar dendrites in tolerant animals that survived 3 (IP3), 10 (IP10) or 30 (IP30) days as well as sham-operated animals and those that received only the two preconditioning episodes (PO). Habituation to a novel open-field was assessed 3, 7, 10 and 30 days after ischemia to gauge CA1 functional integrity. Dendritic spines were quantified from Golgi-Cox stained sections of the CA1 subfield. IP10, IP30 and PO animals had significantly higher CA1 basilar and apical spine densities than all other groups. Tolerant animals initially displayed open-field habituation impairments at a time when spine densities were reduced. Behavioral impairments gradually subsided over time in coincidence with an increase in CA1 spine densities. These findings suggest that dendritic spines may play a role in recovery of function associated with ischemic tolerance and stroke.