Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.     

Bistability in cerebellar Purkinje cell dendrites modelled with high-threshold calcium and delayed-rectifier potassium channels

 Phase-plane analysis of the ionic currents underlying dendritic plateau potentials was carried out to study the nonlinear dynamics and steady-state transfer properties of the dendritic tree in cerebellar Purkinje cells. The results of an analysis of the P-type calcium and delayed rectifier potassium channel system are presented in this study. These channels constitute a simple system that can support bistability and plateau potentials. By requiring both the steady-state current-voltage curve and nullclines to mimic basic plateau potential properties, we obtained well-defined ranges of specific conductance that can support bistability. Hysteresis was found to be surprisingly prevalent in this simple ion-channel system. Using the steady-state current voltage relationship, we derive concise, algebraic expressions for the voltage and current thresholds of state transitions as functions of specific conductance. The significance of bistability in this ion-channel system is discussed with respect to the generation of plateau potentials in Purkinje cells dendrites and the role of the cerebellum in motor control. Received: 13 October 1993/Accepted in revised form: 21 March 1995     

Anomalous diffusion in Purkinje cell dendrites caused by spines

We combined local photolysis of caged compounds with fluorescence imaging to visualize molecular diffusion within dendrites of cerebellar Purkinje cells. Diffusion of a volume marker, fluorescein dextran, within spiny dendrites was remarkably slow in comparison to its diffusion in smooth dendrites. Computer simulations indicate that this retardation is due to a transient trapping of molecules within dendritic spines, yielding anomalous diffusion. We considered the influence of spine trapping on the diffusion of calcium ions (Ca(2+)) and inositol-1,4,5-triphospate (IP(3)), two synaptic second messengers. Diffusion of IP(3) was strongly influenced by the presence of dendritic spines, while Ca(2+) was removed so rapidly that it could not diffuse far enough to be trapped. We conclude that an important function of dendritic spines may be to trap chemical signals and thereby create slowed anomalous diffusion within dendrites.     

P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells.

The pharmacological and single-channel properties of Ca2+ channels were studied in the somata and dendrites of adult cerebellar Purkinje cells. The Ca2+ channels were exclusively of the high threshold type: low threshold Ca2+ channels were not found. These high threshold channels were not blocked by omega-conotoxin GVIA and were inhibited rather than activated by BAY K 8644. They were therefore pharmacologically distinct from high threshold N- and L-type channels. Funnel web spider toxin was an effective blocker. The channels opened to conductance levels of 9, 14, and 19 pS (in 110 mM Ba2+). These slope conductances were in the range of those reported for N- and L-type channels. Our results are in agreement with previous reports suggesting that Ca2+ channels in Purkinje cells can be classified as P-type channels according to their pharmacology. The results also suggest that distinctions among Ca2+ channel types based on the single-channel conductance are not definitive.     

Dying-back of Purkinje cell dendrites with synapse loss in aging rats

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.     

Optogenetic manipulation of cerebellar Purkinje cell activity in vivo

Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum.     

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.     

Calcium-dependent chloride current in rat cerebellar Purkinje cell membranes

The presence of calcium-dependent potential-activated chloride currents in the membranes of freshly isolated rat cerebellar Purkinje cells (12–15 days) was shown by the whole-cell patch clamp technique. Chloride currents appeared in a sodium-free external solution and reversibly disappeared in the absence of external chloride and calcium ions.     

Transient biochemical compartmentalization of Purkinje cells during early cerebellar development

It has recently been observed that during early cerebellar development--from embryonic Day 17 to postnatal Day 3 in the rat--only certain discrete clusters of Purkinje cells (PCs) are immunoreactive to cyclic GMP-dependent protein kinase (cGK). In contrast, at later stages and in the adult, all the PCs are immunoreactive. These results obtained with cGK suggest a transitory intrinsic heterogeneity in the immature cerebellar cortex. It seemed therefore interesting to investigate the distribution of other PC markers during early development in the rat and in other species. The results presented here were obtained with two other antibodies--against vitamin D-dependent calcium binding protein and against Purkinje cell specific glycoprotein--which, like cGK, label all adult PCs. Each antibody gave a different and reproducible mosaic of positive and negative clusters of PCs in the perinatal cerebellum, thus indicating a transient biochemical compartmentalization resulting from the differential expression of parts of the same genotype by clusters of PCs. This compartmentalization in concomitant with the ingrowing of the cerebellar afferents. Once synaptogenesis starts, the biochemical heterogeneity of PCs disappears.     

Aging of cerebellar Purkinje cells

Cerebellar Purkinje cells (PCs), the sole output neurons in the cerebellar cortex, play an important role in the cerebellar circuit. PCs appear to be rather sensitive to aging, exhibiting significant changes in both morphology and function during senescence. This article reviews such changes during the normal aging process, including a decrease in the quantity of cells, atrophy in the soma, retraction in the dendritic arborizations, degeneration in the subcellular organelles, a decline in synapse density, disorder in the neurotransmitter system, and alterations in electrophysiological properties. Although these deteriorative changes occur during aging, compensatory mechanisms exist to counteract the impairments in the aging PCs. The possible neural mechanisms underlying these changes and potential preventive treatments are discussed.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Abnormal Purkinje cell dendrites in Lurcher chimeric mice result from a deafferentation-induced atrophy

Previous studies of Purkinje cell dendrites in lurcher ↔ wild-type mouse chimeras (lurcher chimeras) have documented the surprising occurrence of unusual atrophic dendritic morphologies among the wild-type cells of the mosaic cerebella. We have hypothesized that these aberrant morphologies arise from a process of developmental deafferentation that is due to the unique loss of mutant Purkinje cells in these chimeras. These earlier studies left unanswered the question of whether the abnormal dendrites were the result of a blocked developmental process (agenesis) or regressive events that deform a previously well-developed dendritic arbor (atrophy). Using a set of simple morphometric measures, we now examine wild-type Purkinje cells in young lurcher chimeras. At postnatal day 20, normal Purkinje cell development is nearly but not fully complete. In lurcher chimeras, the morphologies of the wild-type Purkinje cell dendrites are similar to those in wild-type controls of the same age. This means that they are larger in height, width, and cross-section than their counterparts in adult lurcher chimeras. The younger cells exhibit almost none of the atrophic morphologies described in mature animals. We conclude that the aberrant morphologies found in adult lurcher chimeras arise from atrophy rather than through a failure in development. Furthermore, consideration of the details of the wild-type dendrites in the lurcher chimeras leads to the proposal that the height and width of the Purkinje cell dendritic tree are controlled by two independent mechanisms. © 1996 John Wiley & Sons, Inc.     

Spatial and temporal analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites

We have used the calcium indicator dye arsenazo III, together with a photodiode array, to record intracellular calcium changes simultaneously from all regions of individual guinea pig cerebellar Purkinje cells in slices. The optical signals, recorded with millisecond time resolution, are good indicators of calcium-dependent electrical events. For many cells the sensitivity of the recordings was high enough to detect signals from each array element without averaging. Consequently, it was possible to use these signals to follow the complex spatial and temporal patterns of plateau and spike potentials. Calcium entry corresponding to action potentials was detected from all parts of the dendritic field including the fine spiny branchlets, demonstrating that calcium action potentials spread over the entire arbor. Usually, the entire dendritic tree fired at once. But sometimes only restricted areas had signals at any one moment with transients detected in different regions at other times. In one cell, six separate zones were distinguished. These results show that calcium action potentials could be regenerative in some dendrites and could fail to propagate into others. Signals from plateau potentials were also detected from extensive areas in the dendritic field but were always smaller than those caused by a burst of action potentials.     

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.     

Cooperative forms of granule cell and Purkinje cell activity in the frog cerebellar cortex

Spatial and temporal characteristics of functional relations between granule cells and Purkinje cells were studied by multimicroelectrode recording of activity of two or more neurons and statistical analysis of the results. On the arrival of mono- and polymodal afferent volleys, excited granule cells and Purkinje cells were shown to organize themselves into cooperative groups (elementary ensembles) of neurons measuring 200–300 and 300–400 µ, respectively. Elementary ensembles of these neurons are regarded as components of functional units which process information in the frog cerebellar cortex. Some of the special features connected with the cooperative principle of their organization and activity were investigated.Rostov State University. Institute of Cybernetics, Hanoi. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 171–176, March–April, 1977.     

Postnatal development of the cerebellar Purkinje cell shape in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals - guinea pigs - was performed using Nissl's procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.