Age-related quantitative changes in the organelles of rat neocerebellar Purkinje cells.

A quantitative study regarding the age-related changes occurring in the somatic organelles of the neocerebellar Purkinje cell is carried out, using female rats aged 2 to 24 months. Standard manual morphometric techniques are used to calculate the following parameters: somatic volume, volumetric fractions and absolute volumes of the intracellular structures as well as the membrane profile concentration, the membrane surface concentration and the mean surface of the rough endoplasmic reticulum cisternae per cell (RER-S). From a statistical point of view, all the cell components significantly modify their volumetric fractions (except the multivesicular bodies and nucleolus; the latter in relation to the nucleus) and their absolute volumes (except the mitochondria and the multivesicular bodies); the parameters regarding the reticulum are also modified during ageing. There is a linear trend between the age and either the somatic volume of the RER-S or the absolute volumes of the following structures: mitochondria, dense bodies, ground substance and total cytoplasm. A linear correlation is also observed between the cell volume and either the RER-S or the absolute volume of intracellular structures (the Golgi apparatus, the multivesicular bodies and nucleolus being excluded). Anatomophysiological considerations about the findings are discussed. The role of the ground substance as the major modulator of the volumetric plasticity of the Purkinje cell during ageing, is emphasized as a conclusion.     

Cytophotometric Re-investigation of DNA content in Purkinje cells of the rat cerebellum

Summary Stage scanning cytophotometry of Feulgen-stained cell nuclei of the cerebellum of young adult rats revealed that the absorbance values of the majority of the Purkinje cells show a Gaussian distribution with a low coefficient of variance. The peak absorbance of this population is the same as that of the granule cells. About 1% of the Purkinje cells measured, were found to have a stain content which indicates a 4C amount of DNA. For both the granular and the Purkinje cell population, a very small number of nuclei possesses absorbance values intermediate between 2C and 4C. The present data suggest prevalent diploidy of the Purkinje cells, and are at variance with those postulating a tetraploid and/or hyperdiploid status of this population.     

Tetraethylammonium and 4-aminopyridine block calcium-dependent chloride current in rat cerebellum Purkinje cells

Using patch-clamp method (whole cell configuration), it was shown that tetraethylammonium (TEA) and 4-aminopyridine (4-AP) block calcium-dependent chloride currents in the membrane of freshly isolated cerebellar Purkinje cells of rats (12–15 days). In the concentration range studied (50 μM–10 mM TEA and 100 μM–1 mM 4-AP), both compounds blocked the chloride current at IC₅₀ 130 μM for TEA and 110 μM for 4-AP. TEA blockade was reversible after washing. The effect of 4-AP at concentrations greater than 100 μM was irreversible: both outward and inward chloride currents were blocked even after the removal of 4-AP from the incubation medium.     

DNA content in the Purkinje cells of the rat cerebellum spontaneously infected with Kilham virus]

The DNA content in the cerebellum Purkinje cells of rats naturally contaminated with the Kilham virus and in the animals, in which no infection was observed, was determined cytospectrophotometrically on the Feulgen-stained preparations. The incidence of the cells with hyperdiploid nuclei was shown to be equal in the groups compared. It was concluded that the nonmultiple increase of the DNA content in the Purkinje cells of rat cerebellum was apparently unrelated to the virus infection.     

A new neurological mutant rat with symmetrical calcification of Purkinje cells in cerebellum.

A new neurological mutant has been found in the inbred F344 strain of rats. The mutation is inherited as an autosomal recessive trait and is manifest clinically by a hesitant and wobbling gait with asynergic limbs and slight tremor. These symptoms begin at 16-18 days of age and remain essentially constant thereafter. Histologic examination revealed severe degeneration of the Purkinje cells and symmetrical calcification in these and in their dendritic branches in the cerebellar cortex. Such calcified Purkinje cells were intensely stained with the periodic acid-Schiff (PAS) method. PAS-positive substances in the Purkinje cells and extending diffusely over the lesioned sites in the molecular layer were also evident before calcification took place. We have named this neurological mutant the Cerebellar Calcification (CC) rat with the gene symbol cc. This offers a new animal model for the study of the Purkinje cell degeneration and intracranial calcification.     

Purkinje cells of rat and chicken cerebellum contain calreticulin (CaBP3).

In this report data are presented which firmly establish that by treating isolated F0 with the thiol reagent diamide, two 25 kDa F0 subunits react to form a dimer of 45 kDa apparent molecular mass. This dimerising effect is correlated to the impairment of the binding of F1 to F0, both at microM and mM diamide concentrations. Under the latter condition, modification of other F0 subunits also occurs. Passive proton conductance through F0, as well as its sensitivity to N,N'-dicyclohexylcarbodiimide, are affected at low diamide concentration. Thus perturbation of the cysteine residue of the 25 kDa F0 subunit is sufficient for altering the ATP synthase proton channel.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.     

Network analysis of dendritic fields of pyramidal cells in neocortex and Purkinje cells in the cerebellum of the rat.

The connectivity within the dendritic array of Purkinje cells in the cerebellum and pyramidal cells of the neocortex of the rat, stained by the Golgi-Cox method, has been quantified by the method of network analysis. Connectivity was characterized either by applying the system of Strahler ordering, which assigns a relative order of magnitude to each branch of the arborescence or by the identification of unique topological branching patterns within the tree. The former method has been used to define the entire dendritic array of the Purkinje cell and the apical system of neocortical pyramids. It has been shown that the relation between the numbers of branches of successive Strahler order in Purkinje cells form an inverse geometric series in which the highest order is unity and the ratio between successive orders approximates to 3. On the other hand, the apical dendrites of neocortical pyramids exhibit two bifurcation ratios, i.e. a ratio of 3 between low orders and a ratio of 4 between higher orders. A computer simulation technique was used to generate networks of a size comparable with the Purkinje cell networks and grown according to two hypotheses namely, a 'terminal growth model' in which additional segments were added randomly to the terminal branches only and a 'segmental growth model' in which additional segments were added randomly to any branch within the array including terminal branches. Subsequent ordering of the simulated trees revealed that the relation between the numbers of successive orders for networks generated according to the 'segmental model' tended towards an inverse geometric series with a ratio of 4 and that generated according to the 'terminal model' tended towards a ratio of 3. This result showed that the dendritic tree of Purkinje cells grow in a manner indistinguishable from a system adding branches to random terminal segments and that neocortical apical dendrites add their collateral branches to random segments of the apical shaft but that the collateral branches themselves grow by random terminal branching. The possibility that such conclusions may be influenced by loss of branches incurred by either a failure of impregnation, by sectioning, or by environmental influences was investigated by means of a computer technique...     

Subsurface cisterns in the Purkinje cells of cerebellum of Syrian hamster

Summary Three types of subsurface cisterns were observed in Purkinje cells of the cerebellum of the Syrian hamster. The type-1 cisterns are subsynaptic, related to axosomatic synapses, and are separated from the postsynaptic cell membranes with distances of 400–800 Å. These are probably modified rough surfaced endoplasmic reticulum. The type-2 cisterns are closely apposed to the surface membranes of Purkinje cells, and have very little intracisternal space except at the dilated lateral edges. The type-3 cisterns are similar in structure to the type-2 cisterns but in addition are closely associated with mitochondria. The type-2 and type-3 cisterns appear between one and two weeks after birth and are still present in adults, having almost the same frequency of occurrence. Thin cell processes opposite the type-2 and type-3 cisterns are considered to be glial cell processes. The morphological details of these types of subsurface cisterns are described here, and their possible functional significance is briefly discussed.This work was carried out at the Department of Anatomy, University of Minnesota, Minneapolis, USA, and was supported by grants from the China Medical Board of New York and Anatomical Training Grant GM114 from the USPHS.Dr. Takahashi wishes to express his sincere thanks to Dr. A. Lazarow and Dr. R. L. Wood of the Department of Anatomy, University of Minnesota, who enabled him to use facilities for electron microscopy.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.     

The electro-dynamics of the dendritic space in Purkinje cells of the cerebellum

The functional geometry of the reconstructed dendritic arborization of Purkinje neurons is the object of this work. The combined effects of the local geometry of the dendritic branches and of the membrane mechanisms are computed in passive configuration to obtain the electrotonic structure of the arborization. Steady-currents applied to the soma and expressed as a function of the path distance from the soma form different clusters of profiles in which dendritic branches are similar in voltages and current transfer effectiveness. The locations of the different clusters are mapped on the dendrograms and 3D representations of the arborization. It reveals the presence of different spatial dendritic sectors clearly separated in 3D space that shape the arborization in ordered electrical domains, each with similar passive charge transfer efficiencies. Further simulations are performed in active configuration with a realistic cocktail of conductances to find out whether similar spatial domains found in the passive model also characterize the active dendritic arborization. During tonic activation of excitatory synaptic inputs homogeneously distributed over the whole arborization, the Purkinje cell generates regular oscillatory potentials. The temporal patterns of the electrical oscillations induce similar spatial sectors in the arborization as those observed in the passive electrotonic structure. By taking a video of the dendritic maps of the membrane potentials during a single oscillation, we demonstrate that the functional dendritic field of a Purkinje neuron displays dynamic changes which occur in the spatial distribution of membrane potentials in the course of the oscillation. We conclude that the branching pattern of the arborization explains such continuous reconfiguration and discuss its functional implications.     

Vertex analysis of Purkinje cell dendritic trees in the cerebellum of the rat

All networks are made up of vertices (points interconnected by segments), which include terminals interconnected by terminal segments, nodes interconnected by link segments and the root point connected to the tree by the root segment. All nodes may be classified into unique types according to the number of terminal and link segments they drain. For example, there are three distinct dichotomous nodes, a 'primary' node draining two terminal segments, a 'secondary' node draining one terminal segment and a link segment, and a 'tertiary' node draining two link segments. The numbers of primary and tertiary nodes approximate to equality in large networks and thus the ratio of primary to secondary nodes defines topology. All higher order nodes ( trichotomous and beyond) may be resolved into dichotomous forms and incorporated into the analysis. Different forms of growth may thus be analysed by comparing the frequency distributions of nodes with those generated by computer simulated growth models. Moreover, all vertices can be ordered so that metrical parameters are easily incorporated and the hierarchical arrangements of vertices of different order discerned. The dendritic trees of 48 Purkinje cells, taken from folia along the primary fissure, were analysed using vertex analysis. The mean number of segments in Purkinje cell trees was 881 +/- 23 (s.e.) and mean total dendritic length 7959 +/- 233 (s.e.) micrometers. Segment lengths were longest over proximal segments but over most of the tree segment lengths were constant at 10 +/- 0.2 (s.e.) micrometers. Vertex, segment and terminal frequency distributions of equivalent orders were all normal with a slight positive skew. Peak frequencies were recorded at the 12th equivalent order. The mean primary/secondary nodal vertex ratio was 0.93 and the proportion of trichotomous branch points in the tree was 5%. Comparison of the frequency distribution of all vertices with computer generated models showed that growth of the Purkinje cell was most closely simulated by a random terminal growth model, incorporating 5% trichotomy , in which the branching of high order terminals was more likely than low order terminals. It was concluded that growth of the Purkinje cell tree could proceed by random terminal branching with growth occurring preferentially over a front composed of terminals that are ascending through a corridor in the molecular layer whose margins are defined by neighbouring trees.     

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: an immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.