Developmental expression of polypeptide PEP-19 in cerebellar cell suspensions transplanted into the cerebellum of pcd mutant mice

Summary Cerebellar cell suspensions were prepared from normal mouse embryos and implanted into the cerebellum of Purkinje cell degeneration (pcd) mutant mice, which are characterized by a virtually complete degeneration of Purkinje cells between postnatal day (P) 17 and P45. The expression of immunoreactivity for PEP-19, a developmentally-regulated brain-specific polypeptide, was analyzed in normal mouse cerebellum, as well as in pcd mutants with or without grafts. In the normal cerebellum, PEP-19 immunoreactivity was present in Purkinje cells. In unoperated mutants, 45 days of age or older, Purkinje cells were absent. In grafted pcd mice, numerous PEP-19 immunoreactive, neuroblast-like cells were seen in the graft at 5 days after transplantation. By 9 days, large PEP-19 immunoreactive neurons were found in the host molecular layer; by 17 days after transplantation, such neurons displayed an extensive dendritic tree and resembled differentiated Purkinje cells. The vast majority of PEP-19 immunoreactive cells was located in the molecular layer of the host at 9 days after transplantation and beyond; nonetheless, the same cells extended axonal processes toward the graft, indicating an affinity for co-grafted (possibly deep nuclei) neurons. These results point to the ability of donor Purkinje cells for survival, migration into the host brain and morphological and chemical differentiation following transplantation to the degenerated cerebellar cortex of the recipient mutants.On sabbatical leave from the Institute of Neurosciences, National Yang-Ming Medical College, Taipei, Taiwan 112, ROC     

Electrophysiological demonstration of a synaptic integration of transplanted Purkinje cells into the cerebellum of the adult Purkinje cell degeneration mutant mouse

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from "Purkinje cell degeneration" mutants 1-2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted "Purkinje cell degeneration" mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of "Purkinje cell degeneration" mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.     

Reinstatement of synaptic connectivity in the striatum of weaver mutant mice following transplantation of ventral mesencephalic anlagen

Summary Ventral mesencephalic anlagen survive following grafting to the striatum of weaver mutant mice and reinnervate the dopamine-depleted basal ganglia of the recipients. The aim of the present study was to examine the pattern of connectivity established by graft-deriving dopamine afferents in the host striatum. Grafts were obtained from normal embryos at a gestational age of 14–15 days and implanted into a surgical cavity overlying the dorsal striatum of adult weaver recipients. Tissue was processed for electron microscopic immunocytochemistry using a primary antiserum against tyrosine hydroxylase. At the time of examination, recipient weaver mutants were 8.5 months old and the grafts had survived for 4.5 months. Grafts were found to contain an estimated 100–1000 tyrosine hydroxylase immunoreactive neurons. Tyrosine hydroxylase immunoreactive fibres, displaying characteristic varicosities, innervated the dorsal striatum to a depth of 1000 µm. In the non-grafted striatum, 8% of the contacts of tyrosine hydroxylase immunoreactive nerve terminals were junctional. That proportion contrasted with the corresponding value of normal animals, which is 27%. In the grafted striatum, 29% of the contacts were junctional. That percentage approximated the value found in normal animals. By applying a stereological correction, it can be estimated from those numbers that thetrue proportion of junctional contacts in the non-grafted striatum of 8.5-month-old mutants may be 26%, whereas that in the grafted side may be 91%, which is close to the normal situation. The majority of contacts in the reinnervated striatum (84%) were made with dendrites and spines. However, the proportion of total axosomatic contacts in the reinnervated striatum was twice as high as that found in the striatum of normal animals, and the proportion of junctional synapses was three times higher than that found normally.We conclude that: (1) in spite of a genetically determined degenerative process, the dorsal neostriatum of weaver mutant mice is receptive to synaptic investment by dopamine afferents originating in normal donor tissue. (2) In repopulating the denervated weaver striatum, graft-deriving dopamine afferents display a connectional selectivity, i.e. they establish synaptic relations preferentially with those cellular domains that are normally innervated by dopamine nerve terminals. In this context, it is possible that dopamine fibres originating in the grafts invest postsynaptic sites that had either been vacated from the intrinsic dopamine input or had never received such an input. (3) The striatal connectivity following transplantation may retain features of immaturity as suggested by the increased incidence of axosomatic contacts, a feature of the developing nigrostriatal projection.     

Axonal competition in the synaptic wiring of the cerebellar cortex during development and in the mature cerebellum

Purkinje cell (PC) dendrites are made by a proximal dendritic domain, which is provided with scattered clusters of spines innervated by a single climbing fiber (CF) and by a distal domain with a high density of spines innervated by parallel fibers (PFs). Following block of electrical activity a spine increase occurs in the proximal domain and the new spines are innervated by the PFs while the number of synaptic contacts formed by the CF is reduced. Also the GABAergic input expands its territory of innervation on the proximal domain, which undergoes a profound restructuring of the glutamate and GABA receptors. Excitatory-like postsynaptic assemblies appear not only on the new spines, but also on the smooth region of the dendrite and both of them may be innervated by GABAergic terminals. In this case GABA receptors coexist with the glutamate receptors leading to the formation of hybrid synapses. In contrast, PF synapses contain solely glutamate receptors. Thus, the expression of glutamate receptors appears to be an intrinsic property of the PC, while the expression of the GABA receptors is induced by the presence of GABAergic terminals. The data highlight an important feature of the CF input; its electrical activity, in addition to inducing a powerful phasic excitation and a tonic inhibition, controls the finer architecture of the cerebellar cortex.     

First month of development of fetal neurons transplanted as a cell suspension into the adult CNS

It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents. In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e. during the first month after transplantation. Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations. Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting. The final size of the transplant thus depended upon the size of the lesion. At seven days post-grafting, the neurons were small in size and the cellular density was high. At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces. At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased. Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed. All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics. Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous. At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized. Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones. At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal. It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.     

Regional differences in cytoarchitecture of the weaver cerebellum suggest a new model for weaver gene action

This paper examines the structure and cytoarchitecture of the cerebellum of the weaver mutant mouse with particular emphasis on regional differences along the mediolateral and anterior-posterior axes. We have uncovered several, previously undescribed features of the weaver cerebellar phenotype. Perhaps the most dramatic example of our findings is the severe disruption of the folial structure of the hemispheres of the weaver cerebellum. A dorsal overgrowth of tissue occurs in the hemispheres that forms a finger-like projection superficial to an atrophic but structurally more normal cerebellar mass underneath. While this folial abnormality is most evident in the homozygote (wv/wv) the antecedents of its appearance are already apparent in the heterozygote (+/wv). At the level of the cytoarchitectonics of the mutant brain, we find substantial variation in the positioning, numbers and density of both Purkinje and granule cells. As a whole, Purkinje plus Golgi II cell numbers are down by over 40%, but this reduction occurs almost exclusively in the medial half of the cerebellum. The hemispheric region contains a nearly normal number of cells per sagittal section (although their positions are predominantly incorrect). The granule cells also show numerical variation; they are nearly absent at the midline, but a substantial number of them survive in the lateral cerebellar cortex. In the paraflocculus, for example, granule cells can be observed in a modest internal granule cell layer as late as 38 postnatal days. These results are discussed in terms of a model of wv gene action in which we propose that the effect of the mutation is a general disruption of cellular distribution in the cerebellar cortex, affecting both Purkinje and granule cells and beginning prenatally.     

Effect of ether-soluble serum componentsin vitro on the behavior of immature cerebellar cells in weaver mutant mice

The reassociation and migration behaviors of dissociated cerebellar cells from weaver mutant mice were investigated with a new microwell tissue culture system. Single cell suspensions from 6- and 7-day-old homozygous weaver (wv/wv) and normal littermates (+/+) reassembled at comparable rates into reaggregates of similar size. Whereas normal cerebellar cells subsequently extended cables consisting of fiber bundles between the cellular reaggregates, few such cables were formed by reaggregated weaver cells. In addition, granule cell degeneration and a relative absence of migrating granule cells along flat cells attached to the culture substratum were characteristic of weaver cerebellar cell microwell cultures. These altered cell behaviors of weaver cerebellar cells were corrected in a medium supplemented with ethanol-ether extracted horse serum.     

Direct immunohistochemical detection of binding sites for beta-blocker within rat cerebellum

The direct immunohistochemical detection of the potent beta-blocker (?)-alprenolol was accomplished in rat cerebellum. (?)-Alprenolol binding sites were found in Purkinje cells and in portions of the molecular and granular layers lying close to the Purkinje cells.     

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a “time-sequential effect” of weaver mutation.     

Purkinje cell compartmentalization in the cerebellum of the spontaneous mutant mouse dreher

The cerebellar morphological phenotype of the spontaneous neurological mutant mouse dreher (Lmx1a ^dr-J ) results from cell fate changes in dorsal midline patterning involving the roof plate and rhombic lip. Positional cloning revealed that the gene Lmx1a, which encodes a LIM homeodomain protein, is mutated in dreher, and is expressed in the developing roof plate and rhombic lip. Loss of Lmx1a causes reduction of the roof plate, an important embryonic signaling center, and abnormal cell fate specification within the embryonic cerebellar rhombic lip. In adult animals, these defects result in variable, medial fusion of the cerebellar vermis and posterior cerebellar vermis hypoplasia. It is unknown whether deleting Lmx1a results in displacement or loss of specific lobules in the vermis. To distinguish between an ectopic and absent vermis, the expression patterns of two Purkinje cell-specific compartmentation antigens, zebrin II/aldolase C and the small heat shock protein HSP25 were analyzed in dreher cerebella. The data reveal that despite the reduction in volume and abnormal foliation of the cerebellum, the transverse zones and parasagittal stripe arrays characteristic of the normal vermis are present in dreher, but may be highly distorted. In dreher mutants with a severe phenotype, zebrin II stripes are fragmented and distributed non-symmetrically about the cerebellar midline. We conclude that although Purkinje cell agenesis or selective Purkinje cell death may contribute to the dreher phenotype, our data suggest that aberrant anlage patterning and granule cell development lead to Purkinje cell ectopia, which ultimately causes abnormal cerebellar architecture in dreher.     

Bone formation and resorption around developing teeth transplanted into the femur

Seven of 24 newborn hamsters developing maxillary molars which were transplanted to the femur for 28 days showed growth and development of crowns and roots. Enamel, dentin, pulp, cementum, periodontal ligament and alveolar bone proper developed in their normal locations just as they had done previously in molars transplanted into subcutaneous connective tissues. Several relationships were observed between alveolar bone proper, developed in the foreign body environment and under the inductive influence of the tooth root, and femoral bone. Femoral bone was continuous with alvolar bone and supported the tooth socket. In some areas near transplanted molar roots, femoral bone was built out to join and support alveolar bone. In other areas, femoral bone was resorbed by the development of a molar root or the molar root was diverted from its normal direction of development. Despite the effort to orient transplanted molars for eruption, the orientation was altered and no evidence for tooth eruption was observed.     

Vermectomy enhances parvalbumin expression and improves motor performance in weaver mutant mice: an animal model for cerebellar ataxia.

In the Weaver mutant mouse (wv/wv), an animal model for hereditary cerebellar ataxia, electrophysiological experiments have revealed a disorganized output of cerebellar Purkinje cells (the latter using GABA as an inhibitory transmitter) which, by a cascade of mechanisms, was thought to be the cause of the poor motor abilities. In Purkinje cell degeneration mice (pcd/pcd) lacking nearly all Purkinje cells and displaying milder motor deficiencies than wv, in comparison to wild-type mice, a strong increase in parvalbumin- and (co-localized with parvalbumin) glycine-immunopositive somata in the deep cerebellar and vestibular nuclei has recently been found. It was therefore intriguing to investigate whether motor performance in weaver mutants could be ameliorated by applying cerebellar lesions to eliminate the faulty output and to look for a change in transmitter weighting, indicated by a strong increase in parvalbumin-positive somata in areas (the respective target areas) which were formerly devoid of it. Ten Weaver mutants were subjected to cerebellar lesions. After removal of the vermis a total abolition of tremor, a definite improvement in the balance of affected body parts, an increase in locomotor activity when tested in an open-field matrix, and a strong increase in parvalbumin expression in Weaver mutant deep cerebellar and vestibular nuclei in comparison to wild-types have indeed been found. Increase in motor activity (or explorative behaviour) has been placed in relation to learning mechanisms. The increase in parvalbumin expression and the observed improvement in motor abilities and mechanisms probably related to learning underline the hypothesis that any change in the physiological equilibrium of the brain function by removal of input or output related to an assembly of nerve cells leads to a cascade of changes at the transmitter and neuronal level in near or distant connected brain structures.     

Serotoninergic fibres form dense synaptic contacts with Purkinje cells in the mouse cerebellar cortex--an immunohistochemical study.

The distribution of serotonin immunoreactivity in the mouse cerebellar cortex was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger (1979) on epoxy embedded semithin sections. The great majority of serotonin-positive afferents distribute throughout the Purkinje cell layer and form dense synaptic contacts with the somata of the Purkinje neurons. Only a sparse immunostaining of serotoninergic fibres could be detected at the granular cell and molecular layers. The microanatomical organization of the serotoninergic projections to the mouse cerebellar cortex is quite different from that observed in other animal species. These findings suggest that in the mouse cerebellar cortex, the Purkinje cell population represents the main target for serotoninergic afferents. Our histochemical data provide morphological support for a series of electrophysiological observations which indicate serotonin as a potential modulatory neurotransmitter for Purkinje cell firing activity.     

Alterations in Clarke's column secondary to granule cell degeneration in the neurological mutant mice,weaver and staggerer

In the neurological mutants weaver and staggerer, the granule cells either do not successfully migrate into the granular (weaver) or, having migrated, die during the second postnatal week (staggerer). We wished to determine if the resulting agranular cortex might produce retrograde transneuronal changes in the spinal cord similar to the changes observed after neonatal hemicerebellectomy. Golgi analysis of Clarke's column indicates that neurons in weaver are affected; neurons in staggerer are not. These observations support the view that neuronal maturation requires the presence of a target nucleus during ‘critical’ postnatal growth periods.     

The development of swimming behavior in the neurological mutant weaver mouse

Weaver (wv/wv) mice have well-specified ontogenetic defects in both the cerebellum and striatum, but have not previously been evaluated systematically for patterns of motor development. In this study, the effects of the weaver mutation were evaluated through an examination of swimming behavior over the first 3 postnatal weeks. Detailed movement analyses of individual limb movements as well as interlimb coordination were used to evaluate the effects of the weaver mutation. Weaver mutant mice displayed a developmental lag in terms of swimming style relative to controls. They also displayed a generalized slowness in limb movements during the swim, which correlated with the developmental onset of use of a particular limb during the swim. However, basic motor patterns in weaver swimming continue to exhibit good overall coordination through the 3rd postnatal week, even though locomotor ataxia has become pronounced by this time. Our results indicate that specific and limited alterations in movement can be traced to very early in development (postnatal Day 3) in weaver mutant mice, a time at which the earliest biochemical and neuroanatomical deficits in these animals have been established. Our results also emphasize the need for systematic contextual analyses of movement to understand interlocking processes both in movement ontogeny and its disorders. © 1996 John Wiley & Sons, Inc.     

Experimental and immunohistochemical studies on the cerebellar substance P of the rat: localization, postnatal ontogeny and ways of entry to the cerebellum

With the indirect immunofluorescence technique, the localization (including the postnatal ontogeny) of substance P in the cerebellum, and the ways of entry of substance P-containing fibers into the cerebellum were explored. In the newborn rat cerebellum, dense fiber bands of axons with substance P-like immunoreactivity which can be traced to the lower brain stem are found. These fibers are also traceable to the developing granular cell layer. Two weeks after birth, however, substance P-containing structures seen in the cerebellum begin to decrease progressively and in the cerebellum of the adult rats, only a small amount of substance P-containing structures is observed. The present study established that substance P-containing fibers are mostly derived from extracerebellar substance P-containing cells and demonstrated the presence of three sites of entry of these substance P-containing fibers to the cerebellum, via (1) the inferior cerebellar peduncle, (2) the fasciculus uncinatus and (3) the middle cerebellar peduncle, respectively. Following deafferentation of the cerebellum, substance P-accumulating fibers are observed only ventral to the lesion (i.e. on the brain stem side), while in the cerebellum a remarkable decrease of substance P-containing fibers is seen and no substance P-accumulating fibers are found dorsal to the lesion (cerebellar side).     

Depression and potentiation of the synaptic transmission between a granule cell and a Purkinje cell in rat cerebellar culture

Both the depression and the potentiation of synaptic transmission between a cerebellar granule cell and a Purkinje cell, which are considered the cellular basis of motor learning, were established in a simple culture preparation. The repetitive stimulation of both a granule cell and an inferior-olivary neuron depressed the synaptic transmission, and the repetitive stimulation of only a granule cell potentiated the transmission. Thus, a simple model system, where detailed analysis of molecular and cellular mechanisms of synaptic plasticities can be performed, has been established.     

Neurological dysfunction expressed in the grooming behavior of developing weaver mutant mice

The present study provides the first quantitative developmental analysis of movement in the weaver (wv/wv) mutant mouse. This autosomal recessive mutation affects both striatal and cerebellar circuitries that are related to motor performance. We report data on postswim grooming behavior in 14 mutant and 14 control animals on alternate postnatal Days 13–20. Mutant animals showed a greater number, but shorter duration, of grooming bouts across this developmental period. The mutant animals also used external support during grooming, expressed various forms of ataxia, performed a higher proportion of smaller forelimb strokes than did the control animals, and failed to complete as many full stereotypic grooming sequences. These differences between mutant and control animals followed distinctive developmental courses. Our data demonstrate that previous anatomical and physiological characterizations of the weaver mutation have overt motor correlates.     

Dopamine D1 binding sites in the striatum of the mutant mouse weaver

In the weaver mouse there is a major abnormality in the dopamine-containing innervation of the striatum. Dopamine islands from during development, along with some innervation of the non-islandic matrix; but during the first postnatal month much of the islandic innervation degenerates and there is a failure of the normal postnatal development of the diffuse nigrostriatal innervation. In the experiments reported here we analysed the distribution of D1 dopamine receptor-related binding sites in the weaver striatum in an effort to test the relationship between the dopamine-containing innervation of the striatum and the synthesis and distribution of dopamine receptors there. Dopamine D1 receptor binding sites labeled by the D1 specific antagonist [3H]SCH 23390 were studied in the striatum of 7-day and adult homozygous weaver (wv/wv) and homozygous control (+/+) mice. Saturation analysis of [3H]SCH 23390 binding in adult animals suggested that the dissociation constants of the binding sites are similar in mutants and controls. The Bmax values in the striatum of weavers were 16% higher than in the controls when the data were expressed as fmoles/mg protein. The protein content of the adult weaver's striatum was decreased by 15 to 30%, however, so that when values were expressed as fmoles/section, no significant difference between values in weavers and homozygous controls were found. Quantitative autoradiography supported the results of saturation analysis. We conclude that the apparent increase of [3H]SCH23390 binding sites in the mutants occurred as the result of shrinkage of the weaver's caudoputamen and that dopamine D1 receptor binding sites in the caudoputamen, as assessed with [3H]SCH 23390, are normal. The studies of regional distribution of [3H]SCH 23390 binding sites in 7-day and adult mice indicated that the characteristic postnatal transition of the [3H]SCH 23390 binding pattern from islandic to a diffuse distribution occurred normally in the weaver's caudoputamen. Thus, in spite of the degeneration and failure of development of the nigrostriatal innervation in weaver mice, D1 binding in the weaver's striatum undergoes the elaborate change in distribution of these sites that is a hallmark of normal striatal development.