The organization of the corticonuclear and olivocerebellar climbing fiber projections to the rat cerebellar vermis: The congruence of projection zones and the zebrin pattern

The zonal organization of the corticonuclear and the olivocerebellar climbing fiber projections to the vermis of the cerebellum of the rat was compared to the pattern of zebrin-positive and zebrin-negative bands in material double-stained for zebrin II and for different anterograde tracers injected in subnuclei of the inferior olive, or retrograde tracers injected in the cerebellar and vestibular target nuclei of the Purkinje cells of the vermis. Projection zones A₁, A_X, X, B, C_X in the vermis and A₂ (accessory A zone) and C₂ in the hemisphere were defined by their efferent corticonuclear and their afferent climbing fiber connections, and were found to share the same topographical framework with the zebrin pattern.     

Hippocampal poly(ADP-Ribose) polymerase 1 and caspase 3 activation in neonatal bornavirus infection

Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.     

The Reelin receptors Apoer2 and Vldlr coordinate the patterning of Purkinje cell topography in the developing mouse cerebellum

The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI-VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (Apoer2(+/-): Vldlr(+/-)), but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum.     

Systematic Regional Variations in Purkinje Cell Spiking Patterns

In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions.     

Endoplasmic reticulum stress and neurodegeneration in rats neonatally infected with borna disease virus

Borna disease virus infection of neonatal rats results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus and cerebellum (neonatal Borna disease [NBD]). The cellular mechanisms leading to neurodevelopmental damage in NBD have not been fully elucidated. Insights into this model may have general implications for understanding the pathogenesis of virus-associated neurodevelopmental damage. Here we report the presence of endoplasmic reticulum (ER) stress markers and activation of the unfolded protein response in the NBD hippocampus and cerebellum. Specific findings included enhanced PERK-mediated phosphorylation of eif2alpha and concomitant regulation of ATF4 translation; IRE1-mediated splicing of XBP1 mRNA; and cleavage of the ATF6 protein in NBD rat brains. We found evidence for regional and cell type-specific divergence in the expression of ER stress-induced proapoptotic and quality control signals. Our results demonstrate that ER stress induction in death-susceptible Purkinje neurons in NBD is associated with the expression of the proapoptotic molecule CHOP in the absence of compensatory expression of the ER quality control molecules Bip and protein disulfide isomerase. In contrast, ER stress in death-resistant astrocytes is associated with complementary expression of CHOP and ER quality control signals. These results implicate an imbalance between ER stress-mediated apoptosis and survival signaling as a critical determinant of neural cell fate in NBD.     

Influence of experimentally induced agranularity on the synaptogenesis of serotonin nerve terminals in rat cerebellar cortex.

The serotonin (5-HT) innervation of the posterior vermis was studied by high resolution radioautography in both normal and X-ray-induced agranular rat cerebella, following 3 h topical superfusion with 10(-4) M 3H-5-HT. In the normal cerebellar cortex, 5-HT axonal varicosities are scarce and only rarely exhibit the membrane differentiations characterizing synaptic contacts. In the agranular cerebellum, 5-HT terminals appear to have a much higher density than in normal controls, although their absolute number may not be significantly different when the important reduction in volume of this experimental cerebellum is taken into account. These terminals frequently show typical synaptic contracts. Most of them are established on the branchlet spines of Purkinje cell dendrites, but some are also observed on the shafts of Golgi cell dendrites. The 5-HT innervation of the cerebellar cortex thus undergoes important changes in the absence of granule cells. It is suggested that these modifications may be part of the general reorganization process of the cerebellar circuitry consequent on the early destruction of the external granular layer. This new example of synaptic remodelling could imply that the formation of cerebellar connectivity is modulated, to a certain extent, by the local cellular environment.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in precocial- and altricial-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV-VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC represented by simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, from 1 to rarely 11 active PC are recorded, on average, in the single microelectrode track. At the one-week age, the highest number active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of the cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals exceeds again, although slightly of the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

Cerebellar cortical lamination and foliation require cyclin A2

The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.     

Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in mature- and immature-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV–VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC in the form of simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, on average, from 1 to rarely 11 active PC are recorded in the single microelectrode track. At the one-week age, the highest number of active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals it again exceeds, although slightly, the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

13-cis-retinoic acid causes patterning defects in the early embryonic rostral hindbrain and abnormal development of the cerebellum in the macaque

BACKGROUND: We have previously reported that exposure of embryos to 13-cis-retinoic acid (cRA) results in an abnormal phenotype of the fetal cerebellum. In this study, we analyzed early changes in the cerebellar anlagen (midbrain-hindbrain junction) as well as lesions of the fetal cerebellar vermis after a teratogenic dosing regimen of cRA in the macaque model. METHODS: We examined embryo coronal sections of the midbrain-hindbrain junction immunostained for Pax-2, Engrailed-2 (En-2) and Krox-20. To characterize the cerebellum foliation and fissure formation processes, we analyzed vermal cortical cell layer development and the number and depth of the major fissures on sagittal sections of fetal vermis. We also examined Purkinje cell development in vermal sections immunostained for CD3. RESULTS: Compared with controls, there was a consistent truncation of the midbrain-hindbrain region of early embryos exposed to cRA. The cRA-induced fetal vermis lesions included inhibition in its anteroposterior growth, altered folial patterning, a general loss of prominence of the fissures accompanied by a total loss of sublobular fissures, and changes in cortical cell layer development. CD3(+) Purkinje cells were abnormally dispersed deep into the molecular layer in the vermis. CONCLUSIONS: Our findings indicate that the effects of cRA on the developing cerebellum involve interference with the hierarchy of complex cellular and genetic interactions that lead to the growth and subdivision of the cerebellum into smaller units. The regional vermal defects may be related to early postnatal functional deficits.     

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.     

Overlapping functions of the cell adhesion molecules Nr-CAM and L1 in cerebellar granule cell development

The structurally related cell adhesion molecules L1 and Nr-CAM have overlapping expression patterns in cerebellar granule cells. Here we analyzed their involvement in granule cell development using mutant mice. Nr-CAM-deficient cerebellar granule cells failed to extend neurites in vitro on contactin, a known ligand for Nr-CAM expressed in the cerebellum, confirming that these mice are functionally null for Nr-CAM. In vivo, Nr-CAM-null cerebella did not exhibit obvious histological defects, although a mild size reduction of several lobes was observed, most notably lobes IV and V in the vermis. Mice deficient for both L1 and Nr-CAM exhibited severe cerebellar folial defects and a reduction in the thickness of the inner granule cell layer. Additionally, anti-L1 antibodies specifically disrupted survival and maintenance of Nr-CAM-deficient granule cells in cerebellar cultures treated with antibodies. The combined results indicate that Nr-CAM and L1 play a role in cerebellar granule cell development, and suggest that closely related molecules in the L1 family have overlapping functions.     

Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum.

Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.     

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.     

Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB

The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in brain tissue and upregulated by ischemia, neuronal excitation, and dehydration. The present study has been performed to elucidate the expression of SGK1 in cerebellar Purkinje cells and to explore whether it influences the colocalized glutamate transporter EAAT4. Intense SGK1 staining was observed in Purkinje cells following 48h of water deprivation. The kinase activates glutamate induced current (I(GLU)) in Xenopus oocytes heterologously expressing EAAT4, an effect mimicked by its isoforms SGK2, 3 and PKB. I(GLU) was decreased by the ubiquitin ligase Nedd4-2, an effect partially but not completely reversed by additional coexpression of the SGK kinase isoforms or PKB. According to immunohistochemistry EAAT4 protein abundance in the cell membrane was enhanced by SGK1 and decreased by Nedd4-2. In conclusion, SGK1 expression is upregulated by ischemia, excitation, and dehydration in cerebellar Purkinje cells. The upregulation of SGK1 may serve to stimulate EAAT4 and thus to reduce neuroexcitotoxicity.     

Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures

The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreased precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhanced the phenotype and accelerated its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 were not altered suggesting that specification and patterning of MHB tissue was not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium was shifted rostrally and medially demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice.