Hipnic modulation of cerebellar information processing: implications for the cerebro-cerebellar dialogue

Recent evidence indicates that during the sleep-waking cycle the forebrain and the cerebellum show parallel changes of their operating capabilities and suggest that cooperation between these two structures plays a different role in the different behavioral states. In particular, a high degree of cerebro-cerebellar cooperation is expected in waking and in paradoxical sleep when enhanced information processing within the cerebellum and the cortex is associated with effective reciprocal cerebro-cerebellar signal transmission. We first speculate that during waking, a state in which a wide range of behaviors is produced by the interaction with the external world, the cerebellum might assist the cortex to develop the neural dynamic patterns which underlie behaviors and that this could be accomplished via cerebellar modulation of both short- and long-range cortical synchronization. In particular, we propose that the cerebellum might favour the automatic triggering of the patterns already acquired, when requested by the context, as well as the acquisition of novel patterns, when found to be of adaptive value, and might even modulate the access to consciousness of brain operations, if producing unpredicted results, by regulating pattern complexity. This proposal is based on the experimental evidence that oscillatory activity may flow within the cerebro-cerebellar loops and that stimulation or lesion of the cerebellar structures affects cortical synchronization. Then we report evidence indicating that during paradoxical sleep, when brain activation occurs in the absence of sensory inflow and motor output, cerebro-cerebellar cooperation mainly favours consolidation of newly acquired waking patterns and/or savings of old patterns from disruption possibly through a non-utilitarian replay process. Finally, we propose that cerebro-cerebellar cooperation weakens during slow wave sleep, given that in this sleep state neuronal activity and excitability decrease both in the cerebellum and in the forebrain and cerebello-cortical signal transmission is at least partially gated at the thalamic level.     

Aneuploid mosaicism in the developing and adult cerebellar cortex

Neuroprogenitor cells (NPCs) in several telencephalic proliferative regions of the mammalian brain, including the embryonic cerebral cortex and postnatal subventricular zone (SVZ), display cell division "defects" in normal cells that result in aneuploid adult progeny. Here, we identify the developing cerebellum as a major, nontelencephalic proliferative region of the vertebrate central nervous system (CNS) that also produces aneuploid NPCs and nonmitotic cells. Mitotic NPCs assessed by metaphase chromosome analyses revealed that 15.3% and 20.8% of cerebellar NPCs are aneuploid at P0 and P7, respectively. By using immunofluorescent analysis of cerebellar NPCs, we show that chromosome segregation defects contribute to the generation of cells with an aneuploid genomic complement. Nonmitotic cells were assessed by fluorescence-activated cell sorting (FACS) coupled with fluorescence in situ hybridization (FISH), which revealed neuronal and nonneuronal aneuploid populations in both the adult mouse and human cerebellum. Taken together, these results demonstrate that the prevalence of neural aneuploidy includes nontelencephalic portions of the neuraxis and suggest that the generation and maintenance of aneuploid cells is a widespread, if not universal, property of central nervous system development and organization.     

Synaptophysin expression during synaptogenesis in the rat cerebellar cortex

In order to study the mechanisms of synaptogenesis in the rat cerebellar cortex, a library of monoclonal antibodies has been generated against proteins of the isolated synapse. One recognizes a glycosylated 38 kDa protein that is concentrated in the synaptic vesicle fraction and resembles synaptophysin biochemically in its molecular weight, charge, and pattern of glycosylation. In the adult cerebellar cortex, the antisynaptophysin(mabQ155) immunoreactivity is codistributed with synapses. Immunoreactivity is strongest in the molecular layer where punctate deposits of reaction product outline the Purkinje cell dendrites. Discrete small profiles, consistent with the distribution of basket cell axon terminals, surround the Purkinje cells, and in the granular layer the synaptic glomeruli are intensely stained. There is no immunoreactivity in the white matter axon tracts. Electron microscope immunocytochemistry confirms the synaptic location of the antigen and suggests that the reaction product is associated with synaptic vesicles. Both round and flat vesicle populations are immunoreactive. Antisynaptophysin(mabQ155) has been used to follow synaptogenesis in the developing rat cerebellum. In the newborn rat (P0), despite the paucity of synapses, there is some specific immunoreactivity, especially in the subcortical white matter. Electron microscopy shows that the antigenicity is associated with vesicles within growth cones, filopodia, and immature axon profiles. During development, antisynaptophysin immunoreactivity increases progressively, along with the maturing cell populations, for both the granule cell-Purkinje cell and the mossy fiber-granule cell synapses. Quantitative biochemical analysis confirms the cytochemical results. These data suggest that neuronal growth cones express a synapse-specific antigen before complete morphological synapses are present.     

Thiamine-like molecules in the cerebellar cortex of the rat: light and electron microscopic immunocytochemical investigation

Immunocytochemical methods were used for studying the location of thiamine-like molecules in the cerebellar cortex of the rat. A positive reaction was observed in several synapses and in several dendrites. This reaction was associated with tubular formations and synaptic vesicles. A positive reaction was also found in several mitochondria and in glial cells. The possible role of thiamine-like molecules in cerebellar cortex is discussed.     

Activity of muscle spindles, motor cortex and cerebellar nuclei during action tremor

Repeated electrode penetration of the dentate and interpositus nuclei in a rhesus monkey transformed an 11-13 Hz physiologic tremor into a much larger action tremor at 5-7 Hz. This tremor was associated with muscle spindle spike train modulation and reflexly evoked tremor modulation of interpositus and motor cortex neurons as well as electromyogram (EMG). No tremor modulation was observed in spike trains recorded from dentate. The timing relationships of the spindle, EMG, and interpositus tremor discharges suggest that the interpositus plays a direct role in tremor suppression. Dentate, by contrast, may function indirectly by setting optimal transcortical long loop reflex dynamics concerned with intended voluntary movement.     

Stumbler, a new mutant mouse with cerebellar disease

A new mutant mouse named Stumbler (stu) displays clinical features suggesting a cerebellar lesion. The main light microscopic findings, based on a Golgi technique and on sections of plastic embedded material, are that Purkinje cells in the mutant cerebellum have small dendritic arborizations and exhibit immature spines on their somata. Purkinje cells also contain an increased number of mitochondrial profiles both in cell bodies and in swellings on dendrites.     

Antigenic compartmentation in the mouse cerebellar cortex: Zebrin and HNK-1 reveal a complex,overlapping molecular topography

Two monoclonal antibodies-anti-zebrin I and anti-HNK-1-have been used to study the compartmentation of the mouse cerebellar cortex. As in other species, the pattern of localization of the Purkinje cell specific antigen zebrin I is confined to a subset of Purkinje cells that are organized into parasagittal bands. The basic pattern consists of two abutting paramedian bands (P1+) and up to three additional vermal bands on either side (P2⁺? P4⁺) This patern is altered in the vermal regions of lobules X and VI-VII where all Purkinje cells are immunoreactive. In the hemisphere there are three additional bands present (P5⁺? P7⁺) plus two shorter bands in the paravermal area (P4b⁺ and P5a⁺) that extend from the paramedian lobule through the lobulus simplex. This pattern is very similar, but perhaps not identical, to that previously described for the rat. These results suggest a common mammalian plan for the expression and localization of zebrin I. By using a monoclonal antibody to an epitope associated with HNK-1, we have now identified a novel pattern of compartmentation in mouse cerebellum. The HNK-1 epitope is expressed most notably on Purkinje cells and Golgi cells. The molecular layer immunoreactivity associated with the Purkinje cell dendrites varies in intensity in a systematic and reproducible fashion. This reveals a novel cerebellar compartmentation that is sometimes complementary, sometimes overlapping, to that revealed by anti-zebrin. As a result, it is now possible to subdivide the cerebellar cortex into a still finer mosaic of antigenic patches and bands than was possible by using zebrins alone.© 1993 Wiley-Liss, Inc.     

Reduced cerebellar neurodegeneration after combined therapy with cyclodextrin/allopregnanolone and miglustat in NPC1: A mouse model of Niemann‐Pick type C1 disease

Niemann‐Pick type C1 (NPC1) disease is a lysosomal storage disease characterized by a deficiency of NPC1 gene function. The malfunction of protein results in a progressive accumulation of lipids in many organs. A combined approach with substrate‐reduction therapy (SRT) and byproduct therapy (BPT) has been shown to ameliorate the disease course in a mutant mouse model (NPC1^–/–). The present study examines the morphological parameters underlying these changes. For the combined SRT/BPT treatment, NPC1^–/– mutant mice (NPC1^{–/–SRT/BPT}) were injected with allopregnanolone/cyclodextrin weekly, starting at postnatal day (P) 7. Starting at P10, a miglustat injection was administered daily until P23. Thereafter, miglustat was added to the powdered chow. For the sham treatment, both mutant NPC1^–/– (NPC1^–/–sham) and wild‐type (NPC1^+/+sham) mice received an NaCl injection and were fed powdered chow without miglustat. Analysis was performed on cerebellar slices by histology and immunohistochemistry. The volumes and cell counts of cerebellar structures were quantified. Additionally, ultrastructural analysis was performed with transmission electron microscopy. In agreement with previous studies, the current study demonstrates Purkinje cell degeneration in the mutant mice, which was partially abrogated by SRT/BPT. The volumes of cerebellar white matter and molecular layer were reduced as well. Also, the number of neurons was reduced in granular and molecular layers. However, only the molecular layer benefited from the therapy, as shown by an increase in the volume and the amount of neurons. The volume and number of neurons of the deep cerebellar nuclei were significantly decreased in mutant mice; an appreciable therapeutic benefit could be demonstrated for the nucleus interpositus. © 2014 Wiley Periodicals, Inc.     

Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex

We have examined the effects of a genetically programmed target cell death on the noradrenergic afferent projection to mouse cerebellar cortex. We have observed that the noradrenergic axon terminals originating in in locus coeruleus are maintained in the cerebellar cortex of the Purkinje cell degeneration (pcd) mutant mouse in spite of the absence of Purkinje cells, the targets for the noradrenergic projection. The number of noradrenergic terminals in the atrophic mutant cerebellar cortex is approximately normal as assessed by counts of fibers exhibiting catecholamine fluorescence and by measurement of high affinity uptake of tritium-labeled norepinephrine (NE) by synaptosomes prepared from cerebellar cortex. An increased density of NE fibers is observed which appears to be a consequence of reduced cerebellar mass in the mutant. Although the number of noradrenergic terminals is unaffected, morphological and biochemical alterations are observed in this system. The fibers are more brightly fluorescent in mutant than in normal mice and their pattern is less orderly. The content of the endogenous transmitter, NE, is increased from 150 to 170% whereas the activity of the rate-limiting enzyme tyrosine hydroxylase (TH) is reduced to about 60% of normal values. These changes appear to be permanent as they are still present in 6 month-old mutant animals, the oldest studied. No alterations in either NE content or TH activity are found in pcd/pcd hippocampus, another target for the locus coeruleus axons.     

Monoclonal antibodies reveal sagittal banding in the rodent cerebellar cortex

We have produced two monoclonal antibodies against polypeptide-associated antigens of developing rat cerebellum. One antibody recognizes an antigen associated with synaptic vesicles and another binds to a polypeptide which is restricted to the cytoplasm of a subset of cerebellar Purkinje cells. Both antibodies reveal the biochemical differentiation of the rodent cerebellar cortex into antigenically distinct sagittal zones.     

Anisotropic representation of forelimb position in the cerebellar cortex and nucleus interpositus of the rat

The relationship between the spatial location of limb and the activity of cerebellar neurons has received little attention and its nature still remains ambiguous. To address this question we studied the activity of Purkinje and nucleus interpositus cells in relation to the spatial location of rat forelimb. A computer-controlled robot arm displaced the limb passively across 15 positions distributed on a parasagittal plane. The limb was upheld for 8 s in each position, which was identified by the Cartesian coordinates of the forepaw. We selected the neurons whose activities were significantly modulated by forepaw position and found that the majority represented preferentially one spatial dimension of the Cartesian plane both in the cerebellar cortex and nucleus interpositus. In particular, the antero-posterior axis was best represented in cerebellar neuronal discharges. This result suggests that the intermediate part of the cerebellum might encode limb position by way of an anisotropic representation of the spatial coordinates of the limb end-point.     

Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Structure-function relations of two somatotopically corresponding regions of the rat cerebellar cortex: olivo-cortico-nuclear connections

The somatotopical organization of the climbing fiber input to the paravermal region of lobulus simplex (LS, lobule Vla) was charted in the cerebellar cortex of anaesthetized rats. From medial to lateral in LS, zones a2, c1, c2 and c3 were identified. Forelimb responses were found in both LS and the paramedian lobule (PML) and simultaneous recordings from the c1 zone in both lobules showed that trial-by-trial fluctuations in climbing fiber field size evoked by ipsilateral forelimb stimulation did not occur in synchrony, suggesting that the two parts of the same zone are not closely linked by their climbing fiber input. Electrophysiological mapping in combination with a double fluorescent axonal tracing strategy (mix of Fluoro-Emerald and green beads, and mix of Fluoro-Ruby and red beads) revealed that the two parts of the c1 zone receive climbing fiber input from similar territories in the medial and dorsal accessory olives, but that only 4% of the total population of labelled cells have axons that branch to supply climbing fiber afferents to both regions of cortex. The corticonuclear output of the two parts of the zone was found in mainly overlapping regions of the transitional region between the anterior and posterior divisions of nucleus interpositus. Overall, the results suggest that the olivocerebellar and corticonuclear projections of cerebellar zones are similarly organized in rat and cat, implying that the function of individual zones is conserved between species.     

Neuronal maturation in the normal and hypothyroid rat cerebellar cortex studied with monoclonal antibody MIT-23

By immunocytochemistry we have studied the expression of the mitochondria-associated polypeptide MIT-23 during the postnatal development of the normal and hypothyroid rat cerebellar cortex, in afferent fibers, and also in neurons of the cerebellar nuclei. The glial cells are never immunoreactive. In all neurons of the cerebellar cortex, MIT-23 expression always occurs after the final mitosis and migration are complete, and persists throughout adult life. Almost all MIT-23 expression begins postnatally. A few Purkinje cells are already immunoreactive at birth and the rest begin expression during the following two days. Immunoreactive Golgi and granule cells are found from postnatal day 4 (P4), basket cells from P10, and stellate cells from P16. Purkinje cells from different anteroposterior regions of the vermis express different levels of MIT-23 with higher staining intensities in lobules I to IV. These differences appear early in development and are retained in the adult. MIT-23 expression in the hypothyroid cerebellar cortex differs from that in control animals only in minor ways. However, sections immunoperoxidase-stained with anti-MIT-23 antibody reveal that, in addition to previously reported alterations in cerebellar development due to a shortage of thyroid hormones, Purkinje cell axonal development is slowed down in the hypothyroid condition, and occasional Purkinje cells in normal and especially in hypothyroid animals have their somata and or dendrites in ectopic locations. Analysis of these cells reveals a preferential direction of dendritic trunk growth in the direction of the molecular layer. Furthermore, secondary branching of ectopic dendrites is confined exclusively to the developing molecular layer, as in normal Purkinje cells, thus suggesting that neither the mature nor immature granule cell environment is sufficient to sustain normal dendritic development.     

Development of glutamic acid decarboxylase-immunoreactive elements in the cerebellar cortex of normal and lurcher mutant mice.

The development of glutamic acid decarboxylase-immunoreactivity (GAD-IR) in cells, fibers, and varicosities of the cerebellar cortex has been examined by light microscopy in normal and lurcher mutant mice between postnatal day 3 and 30 (P3-P30). Purkinje cell morphology was demonstrated in adjacent sections by using an antiserum to the 28Kd vitamin D-dependent calcium binding protein (CaBP). In early postnatal lurcher mice, but not in normal littermates, GAD-IR fibers, presumably Purkinje cell pseudopodia, invade the external granular layer. The plexus of CaBP-IR axons in the internal granular layer is much less complex in lurcher mice than in normal littermates, even before the onset of lurcher Purkinje cell degeneration at P8. In normal mice, GAD-IR fibers encapsulate Purkinje cell somata by P15. Lurcher Purkinje cells, in contrast, receive scattered contacts by GAD-IR puncta and possess a "cap" of such elements surrounding the primary dendrite and apical soma. Pinceau formations, visible as a knot of GAD-IR puncta hanging from the base of Purkinje cells in normal P15 mice, are not present in lurcher littermates. "Empty baskets" or collapsed pinceau formations in regions devoid of Purkinje cells are not revealed by anti-GAD immunohistochemistry in the P17-P30 lurcher cerebellar cortex.     

Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction

It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.     

Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm²) and less rapidly between PN days 30 to 48 (up to 9,300 μm²), followed by a moderate decrease at PN day 90 (about 6,600 μm²). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm³ at PN day 6,93,000 μm³ at PN day 25, and 100,000 μm³ at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 10⁴ spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 10⁵ spines/Purkinje cell), which then declined to 6.3 × 10⁴ spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley-Liss, Inc.     

Synaptogenesis in the cerebellar cortex: differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.     

Activation of the cerebellar cortex and the dentate nucleus in a prism adaptation fMRI study

During prism adaptation two types of learning processes can be distinguished. First, fast strategic motor control responses are predominant in the early course of prism adaptation to achieve rapid error correction within few trials. Second, slower spatial realignment occurs among the misaligned visual and proprioceptive sensorimotor coordinate system. The aim of the present ultra‐highfield (7T) functional magnetic resonance imaging (fMRI) study was to explore cerebellar cortical and dentate nucleus activation during the course of prism adaptation in relation to a similar visuomotor task without prism exposure. Nineteen young healthy participants were included into the study. Recently developed normalization procedures were applied for the cerebellar cortex and the dentate nucleus. By means of subtraction analysis (early prism adaptation > visuomotor, early prism adaptation > late prism adaptation) we identified ipsilateral activation associated with strategic motor control responses within the posterior cerebellar cortex (lobules VIII and IX) and the ventro‐caudal dentate nucleus. During the late phase of adaptation we observed pronounced activation of posterior parts of lobule VI, although subtraction analyses (late prism adaptation > visuomotor) remained negative. These results are in good accordance with the concept of a representation of non‐motor functions, here strategic control, within the ventro‐caudal dentate nucleus. Hum Brain Mapp 35:1574–1586, 2014. © 2013 Wiley‐Liss, Inc.