Remodeling of monoplanar Purkinje cell dendrites during cerebellar circuit formation

Dendrite arborization patterns are critical determinants of neuronal connectivity and integration. Planar and highly branched dendrites of the cerebellar Purkinje cell receive specific topographical projections from two major afferent pathways; a single climbing fiber axon from the inferior olive that extend along Purkinje dendrites, and parallel fiber axons of granule cells that contact vertically to the plane of dendrites. It has been believed that murine Purkinje cell dendrites extend in a single parasagittal plane in the molecular layer after the cell polarity is determined during the early postnatal development. By three-dimensional confocal analysis of growing Purkinje cells, we observed that mouse Purkinje cells underwent dynamic dendritic remodeling during circuit maturation in the third postnatal week. After dendrites were polarized and flattened in the early second postnatal week, dendritic arbors gradually expanded in multiple sagittal planes in the molecular layer by intensive growth and branching by the third postnatal week. Dendrites then became confined to a single plane in the fourth postnatal week. Multiplanar Purkinje cells in the third week were often associated by ectopic climbing fibers innervating nearby Purkinje cells in distinct sagittal planes. The mature monoplanar arborization was disrupted in mutant mice with abnormal Purkinje cell connectivity and motor discoordination. The dendrite remodeling was also impaired by pharmacological disruption of normal afferent activity during the second or third postnatal week. Our results suggest that the monoplanar arborization of Purkinje cells is coupled with functional development of the cerebellar circuitry.     

Principles governing the operation of synaptic inhibition in dendrites

Synaptic inhibition plays a key role in shaping the dynamics of neuronal networks and selecting cell assemblies. Typically, an inhibitory axon contacts a particular dendritic subdomain of its target neuron, where it often makes 10-20 synapses, sometimes on very distal branches. The functional implications of such a connectivity pattern are not well understood. Our experimentally based theoretical study highlights several new and counterintuitive principles for dendritic inhibition. We show that distal "off-path" rather than proximal "on-path" inhibition effectively dampens proximal excitable dendritic "hotspots," thus powerfully controlling the neuron's output. Additionally, with multiple synaptic contacts, inhibition operates globally, spreading centripetally hundreds of micrometers from the inhibitory synapses. Consequently, inhibition in regions lacking inhibitory synapses may exceed that at the synaptic sites themselves. These results offer new insights into the synergetic effect of dendritic inhibition in controlling dendritic excitability and plasticity and in dynamically molding functional dendritic subdomains and their output.     

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     

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.     

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.     

Postnatal development of the shape of cerebellar Purkinje cells 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.     

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.     

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.     

Effect of harmaline on the complex spike shape and depression time in cerebellar Purkinje cell discharge in rat postnatal ontogenesis

Functional relationship between wave form of complex spike (CS) and depression time of simple spike (SS) in discharge of cerebellar Purkinje cells was studied after their activation with afferent climbing fiber at different terms of postnatal ontogenesis in norm and after treatment with harmaline. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), the adult (4–6 months), and the old animals (22–26 months). It was established that the SS duration in norm was approximately equal in rat pups, adult, and old animals, whereas it markedly decreased form the young to the old animals during the SS depression in the Purkinje cell discharge. Frequency of small action potential (lAP) and their number in the Purkinje cell discharge were approximately equal in young rat pups and adult animals, while in old animals these parameters were higher, on average, by 30%. After administration of harmaline, all CS parameters in rat pups and old animals increased in parallel with the depression time elongation. In adult rats, harmaline did not produce statistically significant changes of the mean values of CS parameters, but an increase of the simple spike depression time was observed. The obtained results allow concluding that the SS wave form and the simple spike depression time in norm are functionally coupled and change with age. The effect of harmaline on the CS wave forms as well as on interrelation of the CS duration and the CS depression time in the Purkinje cell discharge was more pronounced at the early and the late stages of Wistar rat postnatal ontogenesis.     

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.     

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.     

Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss

Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.