Molecular Layer Of Cerebellum Research Articles

PTU-induced hypothyroidism modulates antioxidant defence status in the developing cerebellum

The objective of the present study was to evaluate the effect of 6-n-propylthiouracil (PTU)-induced hypothyroidism on oxidative stress parameters, expression of antioxidant defence enzymes, cell proliferation and apoptosis in the developing cerebellum. PTU challenged neonates showed significant decrease in serum T3 and T4 levels and marked increase in TSH levels. Significantly elevated levels of cerebellar H2O2 and lipid peroxidation were observed in 7 days old hypothyroid rats, along with increased activities of superoxide dismutase and glutathione peroxidase and decline in catalase activity. In 30 days old hypothyroid rats, a significant decline in cerebellar lipid peroxidation, superoxide dismutase and glutathione peroxidase activity and expression was observed along with an up-regulation in catalase activity and expression. Expression of antioxidant enzymes was studied by Western blot and semi-quantitative rt-PCR. A distinct increase in cell proliferation as indicated by proliferating cell nuclear antigen (PCNA) immunoreactivity was observed in the internal granular layer of cerebellum of 7 days old hypothyroid rats and significant drop in PCNA positive cells in the cerebellar molecular layer and internal granular layer of 30 days old PTU treated rats as compared to controls. In situ end labeling by TUNEL assay showed increased apoptosis in cerebellum of hypothyroid rats in comparison to controls. These results suggest that the antioxidant defence system of the developing cerebellum is sensitive to thyroid hormone deficiency and consequent alterations in oxidative stress status may play a role in regulation of cell proliferation of the cerebellum during neonatal brain development.

In vivoimaging of climbing fibers after laser nanosurgery

Event Abstract Back to Event In vivo imaging of climbing fibers after laser nanosurgery Anna Letizia Allegra Mascaro1, Paolo Cesare2, Leonardo Sacconi1, Giorgio Grasselli2, Piergiorgio Strata3 and Francesco S. Pavone1* 1 University of Florence, European Lab. For Non-Linear Spectroscopy (LENS), Italy 2 Santa Lucia Fndn. (IRCCS), EBRI, Italy 3 Univ. of Turin, Dept. of Neurosci. and Natl. Inst. of Neurosci, Italy Cerebellar climbing fibers (CFs) are able to undergo structural remodeling during adulthood (Rossi et al., 2001), but the real dynamics of these plastic rearrangements is not characterized at all. We set up a model of singularly axotomized CF within an intact network to investigate the reparative properties of these axons in the adult CNS in vivo. Time lapse two photon imaging has been combined to laser nanosurgery to define a temporal pattern of the degenerative event and to follow the structural rearrangement after injury. GFP expression was induced in the CF by lentiviral vectors injected into the inferior olive (IO). The open skull technique (Holtmaat et al., 2009) has been exploited to obtain optical access to the sparsely GFP-labeled CFs in the cerebellar molecular layer of the treated mice; the neuritis were visualized with daily frequency through the optical window. Laser axotomy was performed on the distal portion of the CF. Time lapse imaging of the wounded region shows that there may be a structural rearrangement of the dissected neuron itself or of the surrounding fibers. Moreover we followed the dynamics of subcellular structures whose motility may be altered by the injury. The structural rearrangements of CFs varicosities were analyzed to characterize synaptic plasticity after damage. Furthermore, as their structural meaning is still not defined, we compared the dynamics of the highly motile transverse branches in physiological and pathological conditions. The remodeling potential of mature CFs is due to their basal expression of several growth-associated proteins, like GAP-43 (Kruger et al., 1993). This model of in vivo labeling allows, in addition, to investigate the role of proteins associated with the remodeling processes after damage through manipulation of the viral vector. The plasticity of CFs, both in terms of remodeling of the main arbor and of altered dynamics of subcellular components, was characterized in a GAP-43 silenced model. Time lapse two photon imaging coupled to viral manipulation and laser induced nanosurgery represent a powerful combination to investigate the mechanisms of degeneration and regeneration in the days that follows the injury events in the CNS of adult mice. Keywords: Cerebellum Conference: The Cerebellum: from neurons to higher control and cognition, Pavia, Italy, 8 Jul - 9 Jul, 2010. Presentation Type: Short Communication Topic: The Cerebellum: from neurons to higher control and cognition Citation: Allegra Mascaro A, Cesare P, Sacconi L, Grasselli G, Strata P and Pavone FS (2010). In vivo imaging of climbing fibers after laser nanosurgery. Front. Neurosci. Conference Abstract: The Cerebellum: from neurons to higher control and cognition. doi: 10.3389/conf.fnins.2010.83.00012 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Oct 2010; Published Online: 13 Oct 2010. * Correspondence: Dr. Francesco S Pavone, University of Florence, European Lab. For Non-Linear Spectroscopy (LENS), Sesto Fiorentino, Italy, pavone@lens.unifi.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anna Letizia Allegra Mascaro Paolo Cesare Leonardo Sacconi Giorgio Grasselli Piergiorgio Strata Francesco S Pavone Google Anna Letizia Allegra Mascaro Paolo Cesare Leonardo Sacconi Giorgio Grasselli Piergiorgio Strata Francesco S Pavone Google Scholar Anna Letizia Allegra Mascaro Paolo Cesare Leonardo Sacconi Giorgio Grasselli Piergiorgio Strata Francesco S Pavone PubMed Anna Letizia Allegra Mascaro Paolo Cesare Leonardo Sacconi Giorgio Grasselli Piergiorgio Strata Francesco S Pavone Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A new Purkinje cell antibody (anti-Ca) associated with subacute cerebellar ataxia: immunological characterization

We report on a newly discovered serum and cerebrospinal fluid (CSF) reactivity to Purkinje cells (PCs) associated with subacute inflammatory cerebellar ataxia. The patient, a previously healthy 33-year-old lady, presented with severe limb and gait ataxia, dysarthria, and diplopia two weeks after she had recovered from a common cold. Immunohistochemical studies on mouse, rat, and monkey brain sections revealed binding of a high-titer (up to 1:10,000) IgG antibody to the cerebellar molecular layer, Purkinje cell (PC) layer, and white matter. The antibody is highly specific for PCs and binds to the cytoplasm as well as to the inner side of the membrane of PC somata, dendrites and axons. It is produced by B cell clones within the CNS, belongs to the IgG1 subclass, and activates complement in vitro. Western blotting of primate cerebellum extract revealed binding of CSF and serum IgG to an 80-97 kDa protein. Extensive control studies were performed to rule out a broad panel of previously described paraneoplastic and non-paraneoplastic antibodies known to be associated with cerebellar ataxia. Screening of >9000 human full length proteins by means of a protein array and additional confirmatory experiments revealed Rho GTPase activating protein 26 (ARHGAP26, GRAF, oligophrenin-1-like protein) as the target antigen. Preadsorption of the patient's serum with human ARHGAP26 but not preadsorption with other proteins resulted in complete loss of PC staining. Our findings suggest a role of autoimmunity against ARHGAP26 in the pathogenesis of subacute inflammatory cerebellar ataxia, and extend the panel of diagnostic markers for this devastating disease.

Electrophysiological evidence of cerebellar fiber system involvement in the Miller Fisher syndrome

Background In the Miller Fisher syndrome (MFS), ataxia may be due involvement of Ia afferents and the cerebellum. Transcranial magnetic stimulation (TMS) over the cerebellum is known to interfere transiently with normal function. Methods In this study, we utilized a previously described TMS protocol over the cerebellum in combination with ballistic movements to investigate cerebellar dysfunction in MFS patients. Results The agonist (biceps) reaction time in MFS patients during a motor cancellation task was not significantly reduced during the initial TMS study. However, during the repeat TMS study, significant reduction was seen for all patients, in tandem with clinical recovery. There was significant correlation between anti-GQ1b IgG titers and change in agonist reaction time between the initial and repeat TMS studies. Conclusions TMS likely affected horizontally orientated parallel fibers in the cerebellar molecular layer. During disease onset, antibody binding may have interfered with facilitation of reaction time during motor cancellation tasks seen in normal subjects. Normalization of reaction time facilitation corresponded to resolution of antibody-mediated interference in the molecular layer. Our study has provided evidence suggesting parallel fiber involvement in MFS, and suggested a role of anti-GQ1b IgG antibody in these changes.

Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain

The neurosteroid allopregnanolone (AP) is a GABAergic agonist that suppresses central nervous system (CNS) activity in the adult brain, and by reducing excitotoxicity is considered to be neuroprotective. A role for neurosteroids in the developing brain, particularly in late gestation, is still debated. The aim of this study was to investigate effects on proliferation and cell death in the brain of late gestation fetal sheep after inhibition of AP synthesis using finasteride, a 5α-reductase type 2 (5α-R2) inhibitor. Catheters were implanted in fetal sheep at ∼125 days of gestation. At 3–4 days postsurgery, fetuses received infusions of either finasteride (20 mg/kg/h; n=5), the AP analogue alfaxalone (5 mg/kg/h; n=5), or finasteride and alfaxalone together ( n=5). Brains were obtained at 24 h after infusion to determine cell death (apoptotic or necrotic) and cell proliferation in the hippocampus and cerebellum, areas known to be susceptible to excitotoxic damage. Finasteride treatment significantly increased apoptosis (activated caspase-3 expression) in hippocampal CA3 and CA1, and cerebellar molecular and granular layers, an effect abolished by co-infusion of alfaxalone and finasteride. Double-label immunohistochemistry showed that both neurons and astrocytes were caspase-3 positive. Finasteride treatment also increased the number of dead (pyknotic) cells in the hippocampus and cerebellum (Purkinje cells), but not when finasteride+alfaxalone was infused. Cell proliferation (Ki-67-immunoreactivity) increased after finasteride treatment; double-labeling showed the majority of Ki-67-positive cells were astrocytes. Thus, steroids such as AP appear to influence the constitutive rate of apoptosis and proliferation in the hippocampus and cerebellum of the fetal brain, and suggest an important role for neurosteroids in the development of the brain.

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 +/- 4% of the GABAergic synapses and 25 +/- 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.

Protective role of dark soy sauce against acrylamide-induced neurotoxicity in rats by antioxidative activity

Dark soy sauce (DSS) exerts antioxidant activity in vitro or in vivo. To test the possibility of chemoprevention by such dietary supplements on acrylamide (ACR)-induced subacute toxicity, Sprague-Dawley male rats were administered DSS dissolved in water (0.5 ml/kgbw•d) for 2 weeks after, before or during ACR exposure (0.2 mg/L in distillate water). DSS significantly improved the body weight gain, relative brain weights, and the gait abnormalities of ACR-treated rats when DSS treated at the same time as ACR-exposure (p < 0.05). DSS significantly improved the axonal degeneration, the ratio of myelinated nerves < 3 μm in diameter, degree of central chromatolysis of the ganglion neurons in peripheral nerves, and numbers of SYP (+) aberrant dots per mm cortex in the cerebellar molecular layer of ACR-treated rats no matter before, after, or during ACR-exposure (p < 0.05). DSS significantly decreased the malondialdehyde level and increased the superoxide dismutase activity in brain of ACR-treated rats when DSS treated during ACR-exposure (p < 0.05). These findings suggest that DSS plays a protective role against ACR-developed neurotoxicity and, partly at least, through an anti-oxidative mechanism. And it is worthy to note that DSS treatment at the same time as ACR exposure plays a more effective protective role than before or after ACR exposure.

Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ

Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABAA receptors in neuronal compartments, the activation kinetics and pharmacology of GABAA receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABAA receptors with much lower affinity, reducing peak GABA-evoked responses with an IC50 of approximately 0.5mM. Most importantly, the kinetics of receptor activation, determined as 10–90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABAA receptors. With a laser spot of 1μm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2μm laterally and 7.5μm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABAA receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

P09: Susceptibilities to acrylamide-induced neurotoxicity and testicular toxicity between young and adult rats

Exposure to acrylamide (ACR) in foodstuffs has become a worldwide concern, and mean intake of ACR in infants and children is estimated to be 2- to 3-fold higher than that in adults when expressed on a body weight basis. To compare the susceptibilities to ACR-induced neurotoxicity and testicular toxicity between young and adult rats, 3- or 7-week-old male SD rats were given ACR at 0, 50, 100 or 200 ppm in the drinking water for 4 weeks, and histopathological examinations of the nervous and male reproductive systems were performed. In addition to scoring of gait abnormalities, neurotoxicity was quantitatively assessed with reference to nerve fiber density, percentages of degenerated axons in the sciatic nerves, and numbers of aberrant dot-like structures immunoreactive to synaptophysin in the cerebellar molecular layer. When compared to adult animals, mean daily intake of ACR per Kg body weight was higher in young animals at each dose. In both young and adult animals, gait abnormalities and histopathological changes suggestive of neurotoxicity including central chromatolysis of ganglion cells in the trigeminal nerves and an increase in degenerated axons in the sciatic nerves were evident from 100 ppm. Although signs of neurotoxicity were generally higher in young animals, the higher severity might reflect the larger amount of ACR intake in young animals. In the testis, marked degeneration and exfoliation mainly of spermatids were observed from 100 ppm onwards in young animals, whereas small numbers of exfoliated germ cells were found in adults cases at 200 ppm. These results suggest that the susceptibilities to ACR-induced neurotoxicity in young and adult rats are similar and that the neurotoxicity is correlated to intake of ACR per body weight. Regarding testicular toxicity, young animals are more susceptible than adults. It is evident that total ROS production increased with enhanced inflammatory status, suggesting a plausible mechanistic pathway between radiation-induced persistent inflammation and cancer.

Density of cerebellar basket and stellate cells in autism: evidence for a late developmental loss of Purkinje cells.

Alterations in the cerebellum have been described as a neuropathological feature of autism. Although numerous studies have focused on the Purkinje cell (PC), the projection neuron of the cerebellar cortex, PC function is critically dependent on their innervation by the GABAergic basket cells (BCs) and stellate cells (SCs) in the cerebellar molecular layer. The present study was designed to determine whether there are differences in the packing density of these inhibitory interneurons or whether the ratio of these interneurons to PCs differs in autistic and age-matched control brains. The GABAergic interneurons were identified by using immunohistochemistry for parvalbumin (PV) in serial sections from the posterior cerebellar lobe of six autistic and four control brains and counted using stereological principles. Prior PC counts in the same area on adjacent sections (Whitney et al., 2008) were available and were used to calculate the number of BCs and SCs per PC. In this sample of brains, no statistically significant difference was detected between the autistic and the control groups in the density of BCs or SCs (P = 0.44 and P = 0.84, respectively) or in the number of BCs or SCs per PC (P = 0.47 and P = 0.44, respectively). The preservation of BCs and SCs, in the presence of the reduced PC numbers as found in at least two, and possibly three, of these six autistic cases (Whitney et al., 2008) suggests that PCs were generated, migrated to their proper location in the PC layer, and subsequently died in the autistic cases that showed a reduction in PCs.

DPNI-caged Gaba As A Tool For Investigating The Kinetic Properties And Distribution Of Gaba Receptors And For Silencing Neurons In Situ

The nitroindoline caged amino acids are hydrolytically stable and have photochemical and pharmacological properties suitable for characterising neurotransmitter receptors. Although the NI and MNI - caged glutamates show no interference in the activation of glutamate receptors at 10 mM concentration range, the corresponding GABA and glycine NI derivatives showed evidence of binding and interference in the amplitude and kinetics of activation at their respective receptors at 0.1 mM concentrations. Similarly interference with GABAergic transmission by 50 μM CNB-caged GABA has also been reported. We describe here a nitroindoline caged GABA modified by addition of a biphosphate on the caging group with the idea that the high negative charge will impede binding of the cage to the GABA receptor. Experiments to test the inhibition of miniature GABA mediated synaptic events in cerebellar molecular layer interneurons showed much reduced inhibition, approximately 66% inhibition of amplitude at 1 mM DPNI-GABA. However, with laser pulses of 0.1 ms at 1 mM DPNI-GABA the 10-90% risetimes were comparable with synaptically evoked currents, indicating that equilibration of the cage with the receptor is fast and interferes little with kinetic measurements. The decline of laser evoked currents was prolonged compared with spontaneous events suggesting that the effective volume of GABA release in the laser spot is larger than synaptic release. The precision of locating GABA ‘hot spots’ in the focal plane was estimated to decline 50% in 1.75 microns, permitting detailed mapping of the distribution of receptors over neuronal compartments. Furthermore, at 100 μM DPNI-GABA, where there is no interference with receptor activation, photolysis localised to the soma of Purkinje neurons or interneurones suppressed spiking with millisecond precision, providing a useful tool for network analysis.

Application of a Translational Profiling Approach for the Comparative Analysis of CNS Cell Types

Comparative analysis can provide important insights into complex biological systems. As demonstrated in the accompanying paper, translating ribosome affinity purification (TRAP) permits comprehensive studies of translated mRNAs in genetically defined cell populations after physiological perturbations. To establish the generality of this approach, we present translational profiles for 24 CNS cell populations and identify known cell-specific and enriched transcripts for each population. We report thousands of cell-specific mRNAs that were not detected in whole-tissue microarray studies and provide examples that demonstrate the benefits deriving from comparative analysis. To provide a foundation for further biological and in silico studies, we provide a resource of 16 transgenic mouse lines, their corresponding anatomic characterization, and translational profiles for cell types from a variety of central nervous system structures. This resource will enable a wide spectrum of molecular and mechanistic studies of both well-known and previously uncharacterized neural cell populations.

Calcium‐permeable presynaptic AMPA receptors in cerebellar molecular layer interneurones

Axons of cerebellar molecular layer interneurones (MLIs) bear ionotropic glutamate receptors. Here, we show that these receptors elicit cytosolic [Ca2+] transients in axonal varicosities following glutamate spillover induced by stimulation of parallel fibres (PFs). A spatial profile analysis indicates that these transients occur at the same locations when induced by PF stimulation or trains of action potentials. They are not affected by the NMDAR antagonist AP-V, but are abolished by the AMPAR inhibitor GYKI-53655. Mimicking glutamate spillover by a puff of AMPA triggers axonal [Ca2+]i transients even in the presence of TTX. Addition of specific voltage-dependent Ca2+ channel (VDCC) blockers such as omega-AGAIVA and omega-conotoxin GVIA or broad range inhibitors such as Cd2+ did not significantly inhibit the signal indicating the involvement of Ca2+-permeable AMPARs. This hypothesis is further supported by the finding that the subunit specific AMPAR antagonist IEM-1460 blocks 75% of the signal. Bath application of AMPA increases the frequency and mean peak amplitude of GABAergic mIPSCs, an effect that is blocked by philanthotoxin-433 (PhTx) and reinforced by facilitating concentrations of ryanodine. By contrast, a high concentration of ryanodine or dantrolene reduced the effects of AMPA on mIPSCs. Single-cell RT-PCR experiments show that all GluR1-4 subunits are potentially expressed in MLI. Taken together, the results suggest that Ca2+-permeable AMPARs are colocalized with VDCCs in axonal varicosities and can be activated by glutamate spillover through PF stimulation. The AMPAR-mediated Ca2+ signal is amplified by Ca2+-induced Ca2+ release from intracellular stores, leading to GABA release by MLIs.

Calretinin-immunoreactive systems in the cerebellum and cerebellum-related lateral-line medullary nuclei of an elasmobranch, Scyliorhinus canicula

Calretinin immunohistochemistry was used to study the organization of some cerebellar structures and lateral line medullary nuclei of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula. In the cerebellar molecular layer, stellate cells are strongly calretinin-immunoreactive (CR-ir). Perikarya and dendrites of Purkinje cells are contacted by numerous stellate cell small CR-ir boutons. Some Purkinje cell perikarya are contacted by CR-ir climbing fibers forming complex axo-somatic contacts. In the granular layer, numerous CR-ir mossy fibers exhibited large swellings. Notable differences in density and diameter of mossy fibers are observed between the auricles and cerebellar body. Thin beaded CR-ir fibers are also present in the granular layer of the body. The lateral line nuclei of the octavolateralis region are comprised of a molecular-like cerebellar crest that covers the dorsal (electroreceptive) and the medial octavolateralis nuclei (mechanoreceptive). The cerebellar crest exhibited numerous CR-ir stellate cells. In the dorsal octavolateralis nucleus, the presence of conspicuous CR-ir cells and neuropil closely associated to the region of primary fiber terminals distinguishes it clearly from the medial nucleus, revealing major differences between the electroreceptive and mechanoreceptive primary nuclei of elasmobranchs. Moreover, CR distribution in the dogfish cerebellum showed interesting differences with those reported in cerebella of other vertebrates, indicating a high variability of cerebellar CR expression in phylogeny.

Productive Simian Virus 40 Infection of Neurons in Immunosuppressed Rhesus Monkeys

There currently is no animal model of JC virus-associated progressive multifocal leukoencephalopathy (PML). Reactivation of simian virus 40 (SV40) in immunosuppressed rhesus monkeys, however, rarely causes a PML-like illness. We sought to isolate a neurotropic clone of SV40 and determine its pathogenic potential in monkeys. The clone SV40CNS1 was amplified by polymerase chain reaction from the brain DNA of a simian/human immunodeficiency virus-infected monkey that had developed PML and meningoencephalitis. Compared with the SV40 prototype 776, SV40CNS1 had a small number of single-amino-acid mutations and caused a productive infection in monkey fibroblasts. It was inoculated into 2 SV40-negative, simian/human immunodeficiency virus-immunosuppressed monkeys. Both animals developed meningoencephalitis with productive SV40 infection of cerebral cortical neurons and glia in the superficial layers of the cortex and at the gray-white junction. Focal SV40-infected cells were also found in the cerebellar molecular and granule cell layers and white matter. Both animals also developed disseminated SV40 infection with nephritis and pneumonitis. Thus, SV40CNS1 is infectious and pathogenic in immunosuppressed monkeys, but it induces encephalitis with fulminant productive infection in cortical neurons and systemic disease, rather than PML. These findings shed new light on SV40 neurotropism and expand the host cell range of this virus.

Distribution of ADP-ribosylation factor-related protein 1 in mouse brain.

ADP-ribosylation factor (Arf)-related protein 1 (ARFRP1) is a membrane-associated GTPase, which inhibits the Arf/Sec7-dependent activation of phospholipase D and belongs to the Arf-like (Arl) GTPases. Although ARFRP1 is involved in post-Golgi membrane trafficking and its lack leads to embryonic lethality, little is known about its possible function in the central nervous system. To obtain more knowledge about ARFRP1, we have characterized its mRNA distribution in adult mouse brain by in situ hybridization and real-time PCR. We observed a widespread distribution of ARFRP1-mRNA, with the highest levels in cerebral cortex, thalamic nuclei, colliculus, substantia nigra and granule cell layer of cerebellum. Moderate levels were observed in some amygdaloid nuclei, CA2 area and dentate gyrus of hippocampus, endopiriform nuclei, globus pallidus, striatum, molecular layer of cerebellum, and locus coeruleus, whereas no expression was detected in hypothalamic nuclei, CA1 and CA3 areas of hippocampus, zona incerta. A significant decrease of ARFRP1-mRNA was observed in cerebral cortex following sleep deprivation, whereas no change was observed in cerebellar cortex, locus courelus, brainstem, hippocampus and pontine nuclei. This study provides the first detailed analysis of the regional distribution of ARFRP1 in the mouse brain and a quantitative view of its changes following sleep deprivation.

A new pathway for lactate production in the CNS

A new pathway for lactate production in the CNS

Sustained granule cell activity disinhibits juvenile mouse cerebellar stellate cells through presynaptic mechanisms

GABA release from cerebellar molecular layer interneurons can be modulated by presynaptic glutamate and/or GABA B receptors upon perfusing the respective agonists. However, it is unclear how release and potential spillover of endogenous transmitter lead to activation of presynaptic receptors. High frequency firing of granule cells, as observed in vivo upon sensory stimulation, could lead to glutamate and/or GABA spillover. Here, we established sustained glutamatergic activity in the granule cell layer of acute mouse cerebellar slices and performed 190 paired recordings from connected stellate cells. Train stimulation at 50 Hz reduced by about 30% the peak amplitude of IPSCs evoked by brief depolarization of the presynaptic cell in 2-week-old mice. A presynaptic mechanism was indicated by changes in failure rate, paired-pulse ratio and coefficient of variation of evoked IPSCs. Furthermore, two-photon Ca2+ imaging in identified Ca2+ hot spots of stellate cell axons confirmed reduced presynaptic Ca2+ influx after train stimulation within the granular layer. Pharmacological experiments indicated that glutamate released from parallel fibres activated AMPARs in stellate cells, evoking GABA release from surrounding cells. Consequential GABA spillover activated presynaptic GABA B Rs, which reduced the amplitude of eIPSCs. Two-thirds of the total disinhibitory effect were mediated by GABA B Rs, one-third being attributable to presynaptic AMPARs. This estimation was confirmed by the observation that bath applied baclofen induced a more pronounced reduction of evoked IPSCs than kainate. Granule cell-mediated disinhibition persisted at near-physiological temperature but was strongly diminished in 3-week-old mice. At this age, GABA release probability was not reduced and presynaptic GABA B Rs were still detectable, but GABA uptake appeared to be advanced, attenuating GABA spillover. Thus, sustained granule cell activity modulates stellate cell-to-stellate cell synapses, involving transmitter spillover during a developmentally restricted period.

Pathological assessment of the nervous and male reproductive systems of rat offspring exposed maternally to acrylamide during the gestation and lactation periods - a preliminary study

To evaluate the developmental effects of exposure to acrylamide (ACR) on the nervous and male reproductive systems, pregnant Sprague-Dawley rats were given ACR at 0, 50, 100 or 200 ppm in the drinking water from gestational day 10 to postnatal day 21 and histopathological assessment of offspring was performed at weaning and postnatal week 11. Neurotoxicity was quantitatively assessed with reference to nerve fiber density, percentages of degenerated and small caliber axons in the sciatic nerves, and numbers of aberrant dot-like structures immunoreactive for synaptophysin in the cerebellar molecular layer. Although maternal neurotoxicity was evident from 100 ppm, no changes suggestive of neurotoxicity or testicular toxicity were observed in offspring. However, lowering of body weights was dose-dependently observed from birth at the dose levels of > or =50 ppm in males and > or =100 ppm in females. Maternal malnutrition was apparent at >/=100 ppm during the lactation period. Therefore, poor lactational ACR-exposure due to maternal toxicity might account for the lack of ACR-induced offspring toxicity other than retarded body growth.

Lack of connexin43-mediated Bergmann glial gap junctional coupling does not affect cerebellar long-term depression, motor coordination, or eyeblink conditioning

Bergmann glial cells are specialized astrocytes in the cerebellum. In the mature cerebellar molecular layer, Bergmann glial processes are closely associated with Purkinje cells, enclosing Purkinje cell dendritic synapses with a glial sheath. There is intensive gap junctional coupling between Bergmann glial processes, but their significance in cerebellar functions is not known. Connexin43 (Cx43), a major component of astrocytic gap junction channels, is abundantly expressed in Bergmann glial cells. To examine the role of Cx43-mediated gap junctions between Bergmann glial cells in cerebellar functions, we generated Cx43 conditional knockout mice with the S100b-Cre transgenic line (Cx43fl/fl:S100b-Cre), which exhibited a significant loss of Cx43 in the Bergmann glial cells and astrocytes in the cerebellum with a postnatal onset. The Cx43fl/fl:S100b-Cre mice had normal cerebellar architecture. Although gap junctional coupling between the Bergmann glial cells measured by spreading of microinjected Lucifer yellow was virtually abolished in Cx43fl/fl:S100b-Cre mice, electrophysiologic analysis revealed that cerebellar long-term depression could be induced and maintained normally in their cerebellar slices. In addition, at the behavioral level, Cx43fl/fl:S100b-Cre mice had normal motor coordination in the rotarod task and normal conditioned eyelid response. Our findings suggest that Cx43-mediated gap junctional coupling between Bergmann glial cells is not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning.