Monoaminergic nerve terminals in the cerebellar cortex of Purkinje cell degeneration mutant mice: fine structural integrity and modification of cellular environs following loss of Purkinje and granule cells

The cerebellar cortex of normal and Purkinje cell degeneration mutant mice was examined by electron microscopy after fixation with potassium permanganate for the demonstration of small granular vesicles in monoaminergic nerve terminals. In control mice, monoaminergic terminals were found mainly in apposition to Purkinje cell dendrites. After the degeneration of Purkinje cells, which constitute the major target for monoaminergic fibres in the cerebellum, monoaminergic terminals persisted in the cerebellar cortex of Purkinje cell degeneration mutant mice. They were ensheathed by astroglial processes in most of the instances. They were also apposed to boutons that contained agranular vesicles, and to stellate cells in the molecular layer. Clear synaptic specializations in the form of thickening of the synaptic membranes were not observed in either control or mutant mice. It is hypothesized that the survival of monoaminergic axons following loss of their target cells may be attributed to the lack of intimate adhesion to their target elements, to a possible functional interaction with the glia, or to the integrity of the extracerebellar terminal fields of the monoamine axon collaterals.     

Noradrenergic innervation of the cerebellar cortex in normal and in Purkinje cell degeneration mutant mice: evidence for long term survival following loss of the two major cerebellar cortical neuronal populations

Purkinje cell degeneration mutant mice were examined during the course of Purkinje cell death (26 and 35 days old) and at 3, 5, 9 and 12 months of age. Glyoxylic acid fluorescence histochemistry for catecholamines was used to investigate possible alterations or reorganization of the noradrenergic fibers from the coeruleo-cerebellar system in response to the degeneration of two major cell types in the cerebellar cortex, of which one, the Purkinje cell, is reported to be the major target neuron. In control mice, noradrenergic fibers traveled in linear and tortuous profiles through the granule cell layer, formed pericellular arrays alongside Purkinje cell somata, and branched profusely into both radially oriented and longitudinally oriented chains. The density of noradrenergic varicosities diminished in the molecular layer, there was with age. In the mutants, concomitant with the progressive shrinkage of the molecular layer, there was a progressive increase in the density of noradrenergic varicosities. This was most conspicuous at 9 and 12 months of age, at which time the molecular layer has been depleted not only of Purkinje cell dendrites, but also of parallel fibers. Noradrenergic fibers in these zones formed dense parallel bundles of varicose profiles whose density reached 621.3 +/- 122.8% (mean +/- SD, n = 4) at 9-12 months of age, compared with age-matched controls. Neurochemical measurement of norepinephrine content in whole cerebellum of the Purkinje cell degeneration mutants revealed no change compared with age-matched controls. We conclude that noradrenergic innervation persists in the cerebellar cortex despite the death of Purkinje cells and most of the granule cells. Although we found an increased density of varicosities in the molecular layer of mutant mice, progressing with age, we believe that this can be explained on the basis of the resultant geometry of the altered cerebellar cortex. It appears that the health of the environment surrounding the noradrenergic fibers in cerebellar cortex has little influence on their anatomical integrity.     

Reconstruction of the defective cerebellar circuitry in adult Purkinje cell degeneration mutant mice by Purkinje cell replacement through transplantation of solid embryonic implants

Solid pieces of cerebellar primordia taken from 12-day-old C57BL embryos were implanted into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice and analysed 2-3 months later. Purkinje cell replacement was followed by means of immunocytochemistry with antisera against either cyclic guanosine monophosphate-dependent protein kinase or vitamin D-dependent calcium-binding protein, which allows the complete staining of these neurons. Although all solid graft implants survived, their fate within the mutant cerebellum varied in three ways: Often, a more or less large fragment of the solid graft remained in the white matter, close to the cortex or even partially replacing it. These remnants contained a few distorted Purkinje cells and a region corresponding to the transplanted deep nuclei, composed of numerous immunostained axons and axon terminals surrounding immunonegative neurons. Less frequently remnants of the graft were extruded to an extracerebellar location, between two adjacent folia. They contained a few Purkinje cells intermixed with granule cells and other neurons. In a few cases corresponding to superficial deposition, the implants developed lobulated and trilaminated minicerebella which were located outside the mutant cerebellum but integrated into it. In all three situations, a large number of grafted Purkinje cells succeeded in moving out of the implants and in invading the host molecular layer. These Purkinje cells develop flattened dendritic trees perpendicular to host bundles of parallel fibres. Ultrastructural examination of the synaptic investment of Purkinje cells which have reached the host molecular layer revealed that they acquire normal synaptic inputs although complex pericellular baskets and pinceau formation do not develop. Axons from molecular layer interneurons synapse on perikaryal and smooth dendritic membranes, climbing fibres synapse on stubby spines emerging from thick dendritic branches, and parallel fibres contact almost exclusively the long-necked spines of the distal spiny branchlets. Finally, Purkinje cells which succeed in migrating to molecular layer regions no further than 0.6 mm from the host deep nuclei are able to grow axons which reach appropriate target areas and establish synaptic connections on nuclear neurons. The results obtained from this series of long-term survival cerebellar transplantations point to the possibility of fulfilling most of the conditions necessary for functional restoration of neural grafts in systems in which neurons are connected in a point-to-point manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Intraparenchymal grafting of cerebellar cell suspensions to the deep cerebellar nuclei of pcd mutant mice, with particular emphasis on re-establishment of a Purkinje cell cortico-nuclear projection.

Summary In transplanting embryonic cerebellar grafts to the cerebellar cortex of Purkinje cell degeneration (pcd) mutant mice to replace missing Purkinje cells (PC), donor PC leave the graft and migrate to the molecular layer of the host. However, PC axons do not always reach the deep cerebellar nuclei of the host, which would be a key element in restoring much of the necessary inhibitory cortico-nuclear projection associated with normal cerebellar function. Rather, grafted PC axons often innervate a region containing deep cerebellar nuclei neurons inside the transplant, while the perikaryon migrates to the host molecular layer. In the present study, aimed at re-establishing a PC innervation of the deep nuclei, we implanted E12 cerebellar cell suspensions intraparenchymally to the deep cerebellar mass of the hosts. The development of grafted PC was monitored with 28-kDa calcium-binding protein (CaBP) immunocytochemistry at various times after transplantation. At short survival times (5 days after grafting), grafts were confined to the site of the original injection. At longer survival times (7–32 days after grafting), grafted PC formed a migratory stream that reached the cerebellar cortex of the host. The most robust graft development was seen 1 month after grafting, the longest survival time allowed in this series of experiments. At that time, clusters of donor PC were found both in the deep nuclei parenchyma and aligned along cortical folia. The orientation of the dendritic trees of PC that had migrated to the cortex was toward the pia. A CaBP-immunoreactive fibre plexus innervated the host deep cerebellar nuclei. The stream of grafted PC extended from the deep cerebellar nuclei to the cerebellar cortex of the host, indicating that donor PC could establish their axonal contacts in the deep nuclei and then move to their final cortical locality, thus recapitulating a migratory path normally taken during cerebellar ontogeny. It appears therefore that both from the pathophysiological and ontogenetic standpoints, the deep cerebellar nuclei represent the appropriate site for PC implantation in cerebellocortical atrophy.     

Prolonged responses in rat cerebellar Purkinje cells following activation of the granule cell layer: an intracellular in vitro and in vivo investigation

We obtained intracellular recordings of 84 Purkinje cells in vitro from guinea pig slices and of 35 cells in vivo from ketamine-anesthetized rats in order to assess detailed properties of synaptic responses in Purkinje cells following granule cell activation. In vitro, electrical stimulation of the granule cell layer underlying recorded Purkinje cells was used in sagittal slices to predominantly activate synapses on ascending granule cell axons. In vivo, stimulation of the upper lip was used to activate Purkinje cells overlying the upper lip patch in the granule cell layer of crus IIa. In the presence of a GABA_A antagonist, Purkinje cells at resting membrane potential responded to both electrical stimulation in vitro and peripheral stimulation in vivo, with a depolarization of 1–10 mV amplitude that lasted for 100–300 ms in the absence of climbing fiber input. Similar prolonged depolarizations could also be induced by brief depolarizing current pulses delivered through the recording electrode, demonstrating that either synaptic or direct depolarization may activate inward currents leading to a sustained response. In support of this hypothesis we found that prolonged depolarizations were shortened significantly when stimulation in the granule cell layer or intracellular current pulses were delivered during hyperpolarizing current steps. Stimulation in the granule cell layer or intracellular current pulses delivered during periods of spontaneous somatic spiking resulted in prolonged depolarizations in dendritic recordings, which were accompanied by an increase in somatic spiking frequency. Following upper lip stimulation in vivo, this increase in somatic spiking was interrupted by an inhibition of 10–50 ms duration. In a majority of recordings, this inhibition did not completely abolish prolonged depolarizations, however, and a delayed increase in somatic spike frequency was still observed. These results suggest that prolonged increases in Purkinje cell spike frequency following peripheral stimulation are due to an underlying prolonged dendritic depolarization induced by granule cell input. Further, a single, short burst of input via ascending granule cell axons appears to be sufficient to induce these responses.     

Effect of excitatory amino acids on Purkinje cell dendrites in cerebellar slices from normal and staggerer mice

The sensitivity of Purkinje cells to short pulse applications of L-aspartate, L-glutamate and related derivatives in their dendritic fields was tested in normal and staggerer mutant mice using cerebellar slices maintained in vitro. In normal mice, the response of Purkinje cells to L-aspartate and L-glutamate consisted of a transient and dose-dependent increase of their firing of simple spikes. The potency of L-aspartate in exciting Purkinje cells was lower than that of L-glutamate when the two drugs were released from adjacent barrels of the same iontophoretic electrode. Quisqualate was an even more potent excitant of these cells than L-aspartate and L-glutamate, whereas N-methyl-DL-aspartate had little or no effect. In staggerer mutant mice, the sensitivity of Purkinje cells to L-aspartate, L-glutamate and quisqualate was not significantly altered. On the contrary, N-methyl-DL-aspartate had a much stronger potency than normal in exciting Purkinje cells although this was still smaller than that of the other agonists tested. These results suggest that the sensitivity of Purkinje cells to L-aspartate and L-glutamate, i.e. the putative neurotransmitters of the climbing and parallel fibers respectively, remains largely normal in staggerer mice. In contrast, in the mutant, N-methyl-D-aspartate receptors are likely to be much more developed than normal.     

Fluoro-jade identification of cerebellar granule cell and purkinje cell death in the alpha1A calcium ion channel mutant mouse,leaner

Cell death is a critical component of normal nervous system development; too little or too much results in abnormal development and function of the nervous system. The leaner mouse exhibits excessive, abnormal cerebellar granule cell and Purkinje cell death during postnatal development, which is a consequence of a mutated calcium ion channel subunit, alpha(1A). Previous studies have shown that leaner cerebellar Purkinje cells die in a specific pattern that appears to be influenced by functional and anatomical boundaries of the cerebellum. However, the mechanism of Purkinje cell death and the specific timing of the spatial pattern of cell death remain unclear. By double labeling both leaner and wild-type cerebella with Fluoro-Jade and terminal deoxynucleotide transferase-mediated, deoxyuridine triphosphate nick-end labeling or Fluoro-Jade and tyrosine hydroxylase immunohistochemistry we demonstrated that the relatively new stain, Fluoro-Jade, will label neurons that are dying secondary to a genetic mutation. Then, by staining leaner and wild-type cerebella between postnatal days 20 and 80 with Fluoro-Jade, we were able to show that Purkinje cell death begins at approximately postnatal day 25, peaks in the vermis about postnatal day 40 and in the hemispheres at postnatal day 50 and persists at a low level at postnatal day 80. In addition, we showed that there is a significant difference in the amount of cerebellar Purkinje cell death between rostral and caudal divisions of the leaner cerebellum, and that there is little to no Purkinje cell death in the wild type cerebellum at the ages we examined.This is the first report of the use of Fluoro-Jade to identify dying neurons in a genetic model for neuronal cell death. By using Fluoro-Jade, we have specifically defined the temporospatial pattern of postnatal Purkinje cell death in the leaner mouse. This information can be used to gain insight into the dynamic mechanisms controlling Purkinje cell death in the leaner cerebellum.     

Horizontal vestibuloocular reflex (VOR) head velocity estimation in Purkinje cell degeneration (pcd/pcd) mutant mice

The horizontal vestibuloocular reflex (VOR) of Purkinje cell degeneration (pcd/pcd) mutant mice, which lack a functional cerebellar cortex, was compared in darkness to that of wild-type animals during constant velocity yaw rotations about an earth-horizontal axis and during sinusoidal yaw rotations about an earth-vertical axis. Both wild-type and pcd/pcd mice showed a compensatory average VOR eye velocity, or bias, during constant velocity horizontal axis rotations, evidence of central neural processing of otolith afferent signals to create a signal proportional to head angular velocity. Eye velocity bias was greater in pcd/pcd mice than in wild-type mice at a low rotational velocity (32 degrees/s), but less at higher velocities (128 and 200 degrees/s). Lesion of the medial nodulus severely attenuated eye velocity bias in two wild-type mice, without attenuating VOR during sinusoidal vertical axis yaw rotations at 0.2 Hz. These results show that while head velocity estimation in mice, as in primates, depends on the cerebellum, pcd/pcd mutant mice develop velocity estimation without a functional cerebellar cortex. We conclude that neural circuits that exclude cerebellar cortex are capable of the signal processing necessary for head angular velocity estimation, but that these circuits are insufficient for normal estimation at high velocities.     

Morphological evidence for a monosynaptic connection between cerebellar Purkinje cells and vestibulospinal tract neurons in the larval clawed toad, Xenopus laevis.

A double-labeling technique was used to trace synaptic connections between the efferent neurons of the cerebellum (Purkinje cells) and vestibulospinal tract neurons in larvae of the clawed toad, Xenopus laevis. The efferent cerebellar projection was labeled with horseradish peroxidase (HRP) while in the same preparations the brainstem neurons projecting to the spinal cord were labeled with cobaltous lysine. It was found that the distribution of the Purkinje cell terminal boutons overlaps significantly with the location of vestibulospinal neurons in the brainstem. Moreover, several close appositions were seen between Purkinje cell boutons and the dendrites and somata of these latter neurons. The close appositions seen in light microscopy were confirmed by subsequent electron microscopy. This study shows that early in development (at least well before metamorphosis) a cerebello-vestibulospinal connection exists in Xenopus laevis. This connection is likely to persist throughout metamorphosis to the adult state.     

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.     

Blockade of pilocarpine-induced cerebellar phosphoinositide hydrolysis with metabotropic glutamate antagonists: evidence for an indirect control of granule cell glutamate release by muscarinic agonists

The ability in vivo of the muscarinic agonist, pilocarpine, to increase phosphoinositol (PI) hydrolysis in lithium pretreated rats was investigated by measuring the accumulation of [(3)H]inositol phosphates (IP). As expected, 20 mg/kg s.c. pilocarpine, a muscarinic agonist, increased PI hydrolysis in the striatum, frontal cortex and hippocampus. Somewhat surprisingly, an increase in IP was also found in the cerebellar homogenates. In all four tissues the pilocarpine-induced effect could be completely inhibited by pretreatment with the muscarinic antagonist scopolamine (1.2 mg/kg i. p.). It was also found that the cerebellar but not the hippocampal pilocarpine-induced rise in PI hydrolysis could be blocked by the metabotropic glutamate (mGlu) receptor antagonist, LY341495 (100 nmol, i.c.v.). The same dose of LY341495 was found to also block both the cerebellar and hippocampal increase in IP formed by stimulation with the group I mGlu receptor agonist 3, 5-dihydroxyphenylglycine (1 micromol, i.c.v.). Given this data and the current information on the distribution of muscarinic and mGlu receptors in the cerebellum, it is suggested that these results may be a reflection of pilocarpine acting at M(2) receptors to indirectly increase glutamate release from parallel fibers by inhibition of gamma-aminobutyric acid-releasing Golgi cells.     

Exome sequencing reveals blended phenotype of double heterozygous FBN1 and FBN2 variants in a fetus

We report a 29 week fetus with arthrogryposis multiplex congenita, multiple joint dislocations, scoliosis and dysmorphism who was detected to be double heterozygote for putatively pathogenic FBN1 (NM_000138.4:c.6004C?>?T; p.Pro2002Ser) and FBN2 (NM_001999.3:c.2945G?>?T; p.Cys982Phe) variants on exome sequencing. The de-novo status of these variants is not confirmed as parental genotypes could not be ascertained. A comparison of the post-mortem findings of the fetus with reported phenotypes of Beals and Marfan syndromes indicated overlapping clinical features suggestive of a blended phenotype.     

An ultrastructural study of granule cell/Purkinje cell synapses in tottering (tg/tg), leaner (tg(la)/tg(la)) and compound heterozygous tottering/leaner (tg/tg(la)) mice

Homozygous tottering (tg/tg) and compound heterozygous tottering/leaner (tg/tg(la)) mutant mice exhibit juvenile onset of three abnormal neurological phenotypes: (i) petit mal-like epilepsy; (ii) ataxia; and (iii) an intermittent myoclonus-like movement disorder. Homozygous leaner mice (tg(la)/tg(la)) exhibit early onset of ataxia (postnatal days 10-12), and also exhibit the myoclonus-like movement disorder and evidence of absence seizure activity; the myoclonus-like disorder is most evident in the first month of life, then diminishes in severity and frequency. The ultrastructure of the cerebellar molecular layer was examined in adult (six to eight months) and juvenile (20-25 days) mice of all three mutant genotypes. In mice of all three genotypes and both ages, Purkinje cell dendritic spines were observed to make multiple contacts with individual parallel fiber varicosities in all sections analysed. These multiple synaptic units were observed in both anterior and posterior vermis and hemispheres of the cerebellum, and ranged from two to nine spines/parallel fiber varicosity. Occasionally, one of the postsynaptic spines belonged to an ectopic spine emerging from the proximal region of a Purkinje cell dendrite. This increase in the multiple synaptic index of some parallel fiber varicosities was observed in juvenile tottering mice before the onset of the symptoms of the neurological disorders. This is highly suggestive that the onset of the neurological phenotype is not a primary cause of multiple Purkinje cell dendritic spines synapsing with single parallel fiber varicosities in these mice, but on the contrary, that it could be the cause of the ataxic symptoms.