Changes in the content of glutamate and GABA in the cerebellar vermis and hemispheres of the Purkinje cell degeneration (pcd) mutant

The contents of glutamate and GABA, as well as aspartate, glycine, and alanine, were examined in the cerebellar vermis and hemispheres of normal and Purkinje cell degeneration (pcd) mutant mice at 6, 9, and 12 months of age. Relative to normal values, the content of glutamate was approximately 50% lower in the vermis for the 3 age groups. In the hemispheres, the content of glutamate was also lower than control values and showed a progressive loss from 30 to 47% with age. On the other hand, in the case of GABA in the vermis, the level was 39% lower in the pcd mutant at 6 months of age but no different from control values at 12 months. However, relative to data for normal mice, the content of GABA in the hemispheres was consistently lower (20%) for all age groups. The level of aspartate was approximately 60% lower in the cerebellar vermis and 45 to 55% lower in the hemispheres of the mutant with respect to control data for all three age groups. Likewise, alanine showed a reduced content in the hemispheres (36–46%) and vermis (24%) in the mutant relative to normal values at 6, 9, and 12 months of age. On the other hand, the level of glycine was 43–64% higher in the vermis and 77–100% greater in the hemispheres of the mutant than in the control group. The higher values for glycine were observed at the two oldest ages. In conclusions, the data are consistent with the idea that glutamate and GABA are present in high concentrations in granule and Purkinje cells, respectively, and provide additional support for a transmitter function for both amino acids in the cerebellum.     

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.     

Genetic ablation of PLA2G6 in mice leads to cerebellar atrophy characterized by Purkinje cell loss and glial cell activation

Infantile neuroaxonal dystrophy (INAD) is a progressive, autosomal recessive neurodegenerative disease characterized by axonal dystrophy, abnormal iron deposition and cerebellar atrophy. This disease was recently mapped to PLA2G6, which encodes group VI Ca(2+)-independent phospholipase A(2) (iPLA(2) or iPLA(2)β). Here we show that genetic ablation of PLA2G6 in mice (iPLA(2)β(-/-)) leads to the development of cerebellar atrophy by the age of 13 months. Atrophied cerebella exhibited significant loss of Purkinje cells, as well as reactive astrogliosis, the activation of microglial cells, and the pronounced up-regulation of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Moreover, glial cell activation and the elevation in TNF-α and IL-1β expression occurred before apparent cerebellar atrophy. Our findings indicate that the absence of PLA2G6 causes neuroinflammation and Purkinje cell loss and ultimately leads to cerebellar atrophy. Our study suggests that iPLA(2)β(-/-) mice are a valuable model for cerebellar atrophy in INAD and that early anti-inflammatory therapy may help slow the progression of cerebellar atrophy in this deadly neurodegenerative disease.     

Rb and p107 are required for normal cerebellar development and granule cell survival but not for Purkinje cell persistence

The involvement of the retinoblastoma gene product (Rb) and its family members (p107 and p130) in cell cycle exit and terminal differentiation of neural precursor cells has been demonstrated in vitro. To investigate the roles of Rb and p107 in growth, differentiation and apoptosis in the developing and mature cerebellum, we selectively inactivated either Rb alone or in combination with p107 in cerebellar precursor cells or in Purkinje cells. In our mouse models, we show that (1) Rb is required for differentiation, cell cycle exit and survival of granule cell precursors; (2) p107 can not fully compensate for the loss of Rb function in granule cells; (3) Rb and p107 are not required for differentiation and survival of Purkinje cells during embryonic and early postnatal development; (4) Rb function in Purkinje cells is cell autonomous; and (5) loss of Rb deficient CNS precursor cells is mediated by p53-independent apoptosis.     

Besides Purkinje cells and granule neurons: an appraisal of the cell biology of the interneurons of the cerebellar cortex

Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the availability of distinct molecular genetic tools to identify and manipulate discrete cell types have suggested the cerebellar cortex as an excellent model to understand the formation and working of the central nervous system. However, the formulation of a unifying model of cerebellar function has so far proven to be a most cantankerous problem, not least because our understanding of the internal cerebellar cortical circuitry is clearly spotty. Recent research has highlighted the fact that cerebellar cortical interneurons are a quite more diverse and heterogeneous class of cells than generally appreciated, and have provided novel insights into the mechanisms that underpin the development and histogenetic integration of these cells. Here, we provide a short overview of cerebellar cortical interneuron diversity, and we summarize some recent results that are hoped to provide a primer on current understanding of cerebellar biology.     

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

The weaver granuloprival phenotype is due to intrinsic action of the mutant locus in granule cells: evidence from homozygous weaver chimeras

The weaver mutation (wv) causes a near total loss of midline granule cells in the mouse cerebellum. The cellular site of mutant locus action leading to the granuloprival phenotype was examined with experimental intraspecific and interspecific homozygous weaver chimeras. It was found that the granule cells which survived and successfully migrated to the internal granular layer of the chimeric cerebellum were all of the wild-type (non-wv) genotype. Using interspecies chimeras, it was determined that the genotype of Purkinje cells and Bergmann glia cells was apparently irrelevant to the survival of granule cells. It is concluded that granule cell death is most likely due to the wv locus acting intrinsically to the weaver granule cells, and not to another cellular site of gene action.     

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24 h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997     

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.     

Unequal patterns of development of succinate-dehydrogenase and acetylcholinesterase in Purkinje cell bodies and granule cells isolated in bulk from the cerebellar cortex of the immature rat

Abstract— –A preparative procedure for the isolation in bulk of two cellular populations of the cerebellar cortex of the immature rat, the granule cells and the Purkinje cell bodies, is described. The procedure is used to delineate the developmental pattern of succinate-INT-reduclase (EC 1.3.99.1) and acetylcholinesterase (EC 3.1.1.7) in the crucial period of cerebellar maturation, i.e. between 12 and 19 days postnatally. Although the overall yield of neuronal RNA diminished with age, the proportion of RNA in the Purkinje cell body fraction increased while that in the granule cells decreased and microscopic examination of the fractions confirmed this result. The yields of succinate-INT-reductase and of acetylcholinesterase in the fractions paralleled the yields of RNA. A significant finding was the trend toward diminishing specific activities (units/μg of RNA) with age of both enzymes in the Purkinje cell bodies as against the opposite, upward trend of their specific activities in the granule cells. An additional finding of interest was the different ratio of true acetylcholinesterase/total cholinesterase activity in the two cell types, with the granule cells consistently exhibiting higher true acetylcholinesterase values than the Purkinje cell bodies. The present report thus supplements the histoenzymological data on the developing rat cerebellum in that it reveals specific differences in the enzymatic development of two different cerebellar types, a finding which was greatly facilitated by the availability of the procedure for their bulk isolation.     

Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice

The Notch signaling pathway is important in regulating formation and anterior-posterior patterning of somites in vertebrate embryos. Here we show that distinct segmentation defects are displayed in embryos mutant for the Notch pathway genes Notch1, Lunatic fringe (Lfng), Delta-like 1 (Dll1), and Delta-like 3 (Dll3). Lfng-deficient mice and Dll3-deficient mice exhibit very similar defects, and marker analysis suggests that progression of the segmentation clock is disrupted in Dll3 mutants. We also show that Radical fringe (Rfng)-deficient mice exhibit no obvious phenotypic defects. To assess whether the absence of a phenotype in Rfng-deficient mice was the result of functional redundancy with the Lfng gene, we generated Lfng/Rfng double homozygous mutant mice. These mice exhibit the skeletal defects normally observed in Lfng-deficient mice, but we detected no obvious synergistic or additive effects in the double mutant animals.     

Antiapoptotic effects of roscovitine in cerebellar granule cells deprived of serum and potassium: a cell cycle-related mechanism

Neuronal apoptosis may be partly due to inappropriate control of the cell cycle. We used serum deprivation as stimulus and reduced potassium from 25 to 5mM (S/K deprivation), which induces apoptosis in cerebellar granule neurons (CGNs), to evaluate the direct correlation between re-entry in the cell cycle and apoptosis. Roscovitine (10 microM), an antitumoral drug that inhibits cyclin-dependent kinase 1 (cdk1), cdk2 and cdk5, showed a significant neuroprotective effect on CGNs deprived of S/K. S/K deprivation induced the expression of cell cycle proteins such as cyclin E, cyclin A, cdk2, cdk4 and E2F-1. It also caused CGNs to enter the S phase of the cell cycle, measured by a significant incorporation of BrdU (30% increase over control cells), which was reduced in the presence of roscovitine (10 microM). On the other hand, roscovitine modified the expression of cytochrome c (Cyt c), Bcl-2 and Bax, which are involved in the apoptotic intrinsic pathway induced by S/K deprivation. We suggest that the antiapoptotic effects of roscovitine on CGNs are due to its anti-proliferative efficacy and to an action on the mitochondrial apoptotic mechanism.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.