Rat Cerebellar Cortex Research Articles

Projection patterns of single mossy fiber axons originating from the dorsal column nuclei mapped on the aldolase C compartments in the rat cerebellar cortex

Although cerebellar mossy fibers are the most abundant cerebellar afferents and are deeply involved in cerebellar function, the organization of their projection has remained obscure, particularly in relation to cerebellar compartmentalization. The dorsal column nuclei (DCN) are a major source of cerebellar mossy fibers and possess distinct somatotopic representations of specific somatosensory submodalities. We reconstructed individual dextran-labeled DCN axons completely from serial sections and mapped their terminals on the longitudinal cerebellar compartments that were visualized by aldolase C immunostaining to clarify their projection pattern. Individual axons branched and formed about 100 rosette terminals in the cerebellar cortex, but infrequently projected to the cerebellar nuclei (1 out of 15 axons). Cortical terminals were clustered in multiple areas in the vermis and pars intermedia mostly, but not exclusively, ipsilateral to the origin of the axon. The gracile, cuneate, and external cuneate nuclei (ECuN) mainly projected to the copula pyramidis and lobule V, paramedian and simple lobules, and lobules I-V and VIII-IX, respectively, although there was some overlap. The majority of terminals were located within aldolase C negative or lightly positive compartments. However, terminals of a single axon can be located on aldolase C-negative as well as on aldolase C-positive compartments. In particular, the rostral ECuN, which is responsive to shoulder movements, projected consistently to lobule IX, which were mostly aldolase C-positive. In sum, DCN-cerebellar axons project to multiple compartments with terminals clustered mainly in the conventional spinocerebellar region with a coarse topography, which shows some relationship to the cortical compartments defined by aldolase C.

Immediate early genes regulation in rat cerebellar cortex during long-term synaptic plasticity induction

The cerebellum is one of the brain areas involved in learning and memory formation. Long-term synaptic plasticity is thought to play a pivotal role in supporting these functions. Moreover Immediate Early Genes (IEGs) expression and de novo protein synthesis and/or modification have been strictly associated with maintenance of Long-Term Potentiation (LTP) as well as memory consolidation and storage. Two highly conserved signalling cascades, PKA and MAPK, seem to be involved in early- to late-LTP conversion; both pathway can activate CREB transcription factor through phosphorylation and P-CREB has been suggested to initiate the protein synthesis leading to late-LTP induction. The transcription factor c-fos is known to be rapidly and transiently induced in the Nervous System by a variety of stimuli and is thought to be directly involved in processes of neuronal plasticity including LTP. We used rat parasagittal cerebellar slices as a model system in which specific patterns of stimulation delivered to the mossy fibers can induce both Long-Term Potentiation and Long-Term Depression (LTD), depending on local inhibition and other regulating factors. Using Voltage Sensitive Dye (VSD) imaging we obtained high-resolution maps of the spatial distribution of LTP/LTD induced from a Teta Burst Stimulus (TBS) application. Control and stimulated slices were fixed at different times from the TBS application and processed for in situ hybridization or immunohystochemistry in order to detect IEGs mRNA expression patterns and protein expression/modifications. The expression pattern of c-fos and CREB mRNAs and their protein distribution and/or phosphorylation were then correlated with LTP/LTD maps generated by VSD imaging. Preliminary data indicate a significant increase of P-CREB in the granular layer suggesting that CREB phosphorylation is induced as early as 15 minutes post TBS application. In situ hybridization experiments indicate a good correlation between c-fos and CREB mRNAs up-regulation and LTP distribution at 120 minutes post TBS. At the protein level, the comparison of immunofluorescence signals and VSD immaging data indicate a clear correlation between c-Fos and P-CREB distribution and synaptic plasticity patterns. We are planning further experiments to confirm these data and to test our experimental system in the presence of drugs that could interfere with the transcription, translation or post-translational protein regulation.

A JIP3-regulated GSK3β/DCX signaling pathway restricts axon branching.

Axon branching plays a critical role in establishing the accurate patterning of neuronal circuits in the brain. However, the mechanisms that control axon branching remain poorly understood. Here we report that knockdown of the brain-enriched signaling protein JNK-interacting protein 3 (JIP3) triggers exuberant axon branching and self-contact in primary granule neurons of the rat cerebellar cortex. JIP3 knockdown in cerebellar slices and in postnatal rat pups in vivo leads to the formation of ectopic branches in granule neuron parallel fiber axons in the cerebellar cortex. We also find that JIP3 restriction of axon branching is mediated by the protein kinase glycogen synthase kinase 3β (GSK3β). JIP3 knockdown induces the downregulation of GSK3β in neurons, and GSK3β knockdown phenocopies the effect of JIP3 knockdown on axon branching and self-contact. Finally, we establish doublecortin (DCX) as a novel substrate of GSK3β in the control of axon branching and self-contact. GSK3β phosphorylates DCX at the distinct site of Ser327 and thereby contributes to DCX function in the restriction of axon branching. Together, our data define a JIP3-regulated GSK3β/DCX signaling pathway that restricts axon branching in the mammalian brain. These findings may have important implications for our understanding of neuronal circuitry during development, as well as the pathogenesis of neurodevelopmental disorders of cognition.

Glutamate transporter EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter mediating inhibition of GABA release from interneurons

Neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to exert concerted control of the synaptic strength within neural pathways that converge on single target neurons. The excitatory transmitter released from climbing fibers (CFs), presumably glutamate, is shown to inhibit γ-aminobutyric acid (GABA) release at basket cell (BC)-Purkinje cell (PC) synapses in the rat cerebellar cortex through its extrasynaptic diffusion and activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on BC axon terminals. This study aimed at examining how the CF transmitter-diffusion-mediated presynaptic inhibition is controlled by glutamate transporters. Pharmacological blockade of the PC-selective neuronal transporter EAAT4 markedly enhanced CF-induced inhibition of GABAergic transmission. Tetanic CF-stimulation elicited long-term potentiation of glutamate transporters in PCs, and thereby attenuated the CF-induced inhibition. Combined use of electrophysiology and immunohistochemistry revealed a significant inverse relationship between the level of EAAT4 expression and the inhibitory action of CF-stimulation on the GABA release at different cerebellar lobules - the CF-induced inhibition was profound in lobule III, where the EAAT4 expression level was low, whereas it was minimal in lobule X, where EAAT4 was abundant. The findings clearly demonstrate that the neuronal glutamate transporter EAAT4 in PCs plays a critical role in the extrasynaptic diffusion of CF transmitter - it appears not only to retrogradely determine the degree of CF-mediated inhibition of GABAergic inputs to the PC by controlling the glutamate concentration for intersynaptic diffusion, but also regulate synaptic information processing in the cerebellar cortex depending on its differential regional distribution as well as use-dependent plasticity of uptake efficacy.

Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a pre-embedding immunogold method

A proper motor activity relies on a correct cerebellar function. The Kv3.1 and Kv3.3 voltage-gated potassium channels are key proteins involved in cerebellar function and dysfunction, as the lack of these causes severe motor deficits. Both channel subunits are coexpressed in granule cells and are rapidly activated at relatively positive potentials to support the generation of fast action potentials. However, the contribution of each subunit to the molecular architecture of the parallel fibers, the granule cell axons, is so far unknown. The goal of this study was to elucidate the relative distribution of Kv3.1b and Kv3.3 in specific compartments of the rat parallel fibers by using a pre-embedding immunocytochemical method for electron microscopy. Numerous Kv3.1b and Kv3.3 silver-intensified gold particles were associated with membranes of parallel fiber synaptic terminals and their intervaricose segments. Kv3.1b was found in about 85% of parallel fiber synaptic terminals and in about 47% of their intervaricose portions. However, only 28% of intervaricosities and 23% of parallel fiber presynaptic boutons were Kv3.3 immunopositive. The analysis also revealed that 54% of Purkinje cell dendritic spines localized Kv3.3. Although both potassium channel subunits share localization in the same presynaptic parallel fiber compartments, the present results with the method used indicate that there are a higher percentage of parallel fibers labeled for Kv3.1b than for Kv3.3, and that the labeling intensity for each subunit is higher in specific subcompartments analyzed than in others.

A FOXO–Pak1 transcriptional pathway controls neuronal polarity

Neuronal polarity is essential for normal brain development and function. However, cell-intrinsic mechanisms that govern the establishment of neuronal polarity remain to be identified. Here, we report that knockdown of endogenous FOXO proteins in hippocampal and cerebellar granule neurons, including in the rat cerebellar cortex in vivo, reveals a requirement for the FOXO transcription factors in the establishment of neuronal polarity. The FOXO transcription factors, including the brain-enriched protein FOXO6, play a critical role in axo-dendritic polarization of undifferentiated neurites, and hence in a switch from unpolarized to polarized neuronal morphology. We also identify the gene encoding the protein kinase Pak1, which acts locally in neuronal processes to induce polarity, as a critical direct target gene of the FOXO transcription factors. Knockdown of endogenous Pak1 phenocopies the effect of FOXO knockdown on neuronal polarity. Importantly, exogenous expression of Pak1 in the background of FOXO knockdown in both primary neurons and postnatal rat pups in vivo restores the polarized morphology of neurons. These findings define the FOXO proteins and Pak1 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal polarity, thus identifying a novel function for the FOXO transcription factors in a unique aspect of neural development.

T-SNARE and v-SNARE in the GABAergic and glutamatergic synapses of the rat cerebellar cortex

Using light microscope immunohistochemistry, the distributions of the t-SNARE, SNAP-25 and syntaxin-1a, and v-SNARE, VAMP-2 (or synaptobrevin-2), have been analyzed in the rat cerebellar cortex and compared with those of GAD-65/67, marker of GABAergic neurons, and vGLUT-1 and vGLUT-2, markers of glutamatergic neurons. SNAP-25 immunoreactivity was detected throughout the cortex, highly diffused in the neuropil. In the molecular and Purkinje layers, the immunoreactivity surrounded dendrite trunks of the Purkinje neurons, and bodies of stellate, basket and Purkinje neurons, which appeared all immunonegative. In the granular layer, the immunoreactivity was less diffuse and formed small grains localized within the protoplasmic islands of Held (synaptic glomeruli). Immunoreactivity was also observed in the white matter. The distribution of syntaxin-1a immunoreactivity appeared superimposable to that of SNAP-25. VAMP-2 immunoreactivity was observed in small dots finely dispersed in the neuropil of the molecular/Purkinje layer; a moderate labelling of the Purkinje neuron bodies was also observed. In the granular layer, VAMP-2 immunoreactivity formed larger deposits, presumably corresponding to mossy fibre terminals. Experiments of double labelling immunohistochemistry revealed no co-localization between t-SNARE (SNAP-25) or v-SNARE (VAMP-2) and GAD-65/67. On the contrary, co-localization between SNAP-25 and VAMP-2, on one side, and v-GLUT-1 or vGLUT-2, on the other side, was frequently revealed. Briefly, in the molecular layer, v-GLUT-1 and vGLUT-2-positive terminals only partly displayed also SNAP-25 immunoreactivity; whereas in the granular layer, all v-GLUT-1 and vGLUT-2-positive terminals displayed this co-localization. The results of this study indicate that t-SNARE and v-SNARE largely characterize the glutamatergic synapses in the rat cerebellar cortex. The same proteins are lacking in the GABAergic synapses.

Distribution of the SNAP25 and SNAP23 synaptosomal-associated protein isoforms in rat cerebellar cortex

Synaptosome-associated protein of 25 kDa (SNAP25) is a component of the fusion complex that mediates synaptic vesicle exocytosis, regulates calcium dynamics and neuronal plasticity. Despite its crucial role in vesicle release, SNAP25 is not distributed homogenously within the brain. It seems to be virtually absent in mature inhibitory terminals and is observed in a subtype of excitatory neurons defined by the expression of vesicular glutamate transporter 1 (VGluT1). Since a complementary distribution of VGluT1 and VGluT2 in excitatory synapses is correlated with different probabilities of release (Pr), we evaluated whether SNAP25 localization is associated with specific synaptic properties. In the cerebellum, climbing fiber (CF) and parallel fiber (PF) inputs, which impinge onto the same Purkinje cell (PC), have very different functional properties. In the cerebellum of adult rats, using confocal and electron microscopy, we observed that VGluT2-positive CFs, characterized by a high Pr, only weakly express SNAP25, while VGluT1-positive PFs that show a low Pr abundantly express SNAP25. Moreover, SNAP25 was less profuse in the VGluT2-positive rosettes of mossy fibers (MFs) and was almost absent in inhibitory terminals. We extended our analysis to the SNAP23 homolog; this is expressed at different levels in both γ-aminobutyric acid-containing terminals (GABAergic) and glutamatergic terminals of the cerebellar cortex. In conclusion, the preferential localization of SNAP25 in specific synaptic boutons suggests a correlation between SNAP25 and the Pr. This evidence supports the hypothesis that SNAP25 has a modulatory role in shaping synaptic responses.

Principal Cell Spiking, Postsynaptic Excitation, and Oxygen Consumption in the Rat Cerebellar Cortex

One contention within the field of neuroimaging concerns the character of the depicted activity: Does it represent neuronal action potential generation (i.e., spiking) or postsynaptic excitation? This question is related to the metabolic costs of different aspects of neurosignaling. The cerebellar cortex is well suited for addressing this problem because synaptic input to and spiking of the principal cell, the Purkinje cell (PC), are spatially segregated. Also, PCs are pacemakers, able to generate spikes endogenously. We examined the contributions to cerebellar cortical oxygen consumption (CMRO2) of postsynaptic excitation and PC spiking during evoked and ongoing neuronal activity in the rat. By inhibiting excitatory synaptic input using ionotropic glutamate receptor blockers, we found that the increase in CMRO2 evoked by parallel fiber (PF) stimulation depended entirely on postsynaptic excitation. In contrast, PC spiking was largely responsible for the increase in CMRO2 when ongoing neuronal activity was increased by gamma-aminobutyric acid type A receptor blockade. In this case, CMRO2 increased equally during PC spiking with excitatory synaptic activity as during PC pacemaker spiking without excitatory synaptic input. Subsequent inhibition of action potential propagation and neurotransmission by blocking voltage-gated Na+-channels eliminated the increases in CMRO2 due to PF stimulation and increased PC spiking, but left a large fraction of CMRO2, i.e., basal CMRO2, intact. In conclusion, whereas basal CMRO2 in anesthetized animals did not seem to be related to neurosignaling, increases in CMRO2 could be induced by all aspects of neurosignaling. Our findings imply that CMRO2 responses cannot a priori be assigned to specific neuronal activities.

Age-related changes of monoaminooxidases in rat cerebellar cortex.

Age-related changes of the monoaminoxidases, evaluated by enzymatic staining, quantitative analysis of images, biochemical assay and statistical analysis of data were studied in cerebellar cortex of young (3-month-old) and aged (26-month-old) male Sprague-Dawley rats. The enzymatic staining shows the presence of monoamino-oxidases within the molecular and granular layers as well as within the Purkinje neurons of the cerebellum of young and aged animals. In molecular layer, and in Purkinje neurons the levels of monoaminooxidases were strongly increased in old rats. The granular layer showed, on the contrary, an age-dependent loss of enzymatic staining. These morphological findings were confirmed by biochemical results. The possibility that age-related changes in monoaminooxidase levels may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs is discussed.

Evidence for oxidative stress in the developing cerebellum of the rat after chronic mild carbon monoxide exposure (0.0025% in air).

BackgroundThe present study was designed to test the hypothesis that chronic very mild prenatal carbon monoxide (CO) exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Studies at this chronic low CO exposure over the earliest periods of mammalian development have not been performed to date. Pregnant rats were exposed chronically to CO from gestational day E5 to E20. In the postnatal period, rat pups were grouped as follows: Group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and group D, controls (air without CO). At P20, immunocytochemical analyses of oxidative stress markers, and structural and functional proteins were assessed in the cerebellar cortex of the four groups. Quantitative real time PCR assays were performed for inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) nitric oxide synthases.ResultsSuperoxide dismutase-1 (SOD1), SOD2, and hemeoxygenase-1 (HO-1) immunoreactivity increased in cells of the cerebellar cortex of CO-exposed pups. INOS and nitrotyrosine immunoreactivity also increased in blood vessels and Purkinje cells (PCs) of pups from group-A, B and C. By contrast, nNOS immunoreactivity decreased in PCs from group-B. Endothelial NOS immunoreactivity showed no changes in any CO-exposed group. The mRNA levels for iNOS were significantly up-regulated in the cerebellum of rats from group B; however, mRNA levels for nNOS and eNOS remained relatively unchanged in groups A, B and C. Ferritin-H immunoreactivity increased in group-B. Immunocytochemistry for neurofilaments (structural protein), synapsin-1 (functional protein), and glutamic acid decarboxylase (the enzyme responsible for the synthesis of the inhibitory neurotransmitter GABA), were decreased in groups A and B. Immunoreactivity for two calcium binding proteins, parvalbumin and calbindin, remained unchanged. The immunoreactivity of the astrocytic marker GFAP increased after prenatal exposure.ConclusionWe conclude that exogenously supplied CO during the prenatal period promotes oxidative stress as indicated by the up-regulation of SOD-1, SOD-2, HO-1, Ferritin-H, and iNOS with increased nitrotyrosine in the rat cerebella suggesting that deleterious and protective mechanisms were activated. These changes correlate with reductions of proteins important to cerebellar function: pre-synaptic terminals proteins (synapsin-1), proteins for the maintenance of neuronal size, shape and axonal quality (neurofilaments) and protein involved in GABAergic neurotransmission (GAD). Increased GFAP immunoreactivity after prenatal CO-exposure suggests a glial mediated response to the constant presence of CO. There were differential responses to prenatal vs. postnatal CO exposure: Prenatal exposure seems to be more damaging; a feature exemplified by the persistence of markers indicating oxidative stress in pups at P20, following prenatal only CO-exposure. The continuation of this cellular environment up to day 20 after CO exposure suggests the condition is chronic. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.

γ-Aminobutyric Acid Modulates Local Brain Oxygen Consumption and Blood Flow in Rat Cerebellar Cortex

γ-Aminobutyric Acid Modulates Local Brain Oxygen Consumption and Blood Flow in Rat Cerebellar Cortex

Diversity in the G i/o-coupled receptor-mediated presynaptic inhibition at granule cell-interneuron synapses in rat cerebellar cortex

Diversity in the G i/o-coupled receptor-mediated presynaptic inhibition at granule cell-interneuron synapses in rat cerebellar cortex

Climbing fiber coupling between adjacent Purkinje cell dendrites in vivo

Climbing fiber discharges within the rat cerebellar cortex have been shown to display synchrony, especially for climbing fibers terminating in the same parasagittal bands. In addition, Purkinje cells which have the smallest rostrocaudal separation also seem to have the highest degree of synchrony. But this has so far only been investigated for distances down to 250 μm. In the present study, we wanted to investigate whether Purkinje cells that are located immediately next to each other display a particularly pronounced synchrony in their climbing fiber discharges. To this end, we used a previously undescribed type of electrophysiological recording, a single electrode, loose patch, dual dendritic recording, from pairs of adjacent Purkinje cells in the decerebrated, non-anesthetized cat. From each recorded dendrite, this technique provided well isolated, unitary calcium spikes, which we found to have a spontaneous activity that was essentially identical with the pattern of spontaneous climbing fiber discharges. By calculating the coupling in firing between the adjacent dendrites, we found that most climbing fiber responses occurred independently of each other and that the probability of coupled discharges was less than 8%. These values are comparable to those obtained in previous studies for Purkinje cells located within the same parasagittal band and show that climbing fiber coupling within a microzone exists also in non-rodent mammalian species. However, since the degree of synchrony of climbing fiber discharge was not particularly pronounced in adjacent Purkinje cells, it seems unlikely that climbing fiber synchrony has pronounced systematic regional variations within the same microzone.

Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development

Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development

A Centrosomal Cdc20-APC Pathway Controls Dendrite Morphogenesis in Postmitotic Neurons

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. We report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. We also find that the centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity.

Immunohistochemistry of GluR1 subunits of AMPA receptors of rat cerebellar nerve cells

The localization of GluR1 subunits of ionotropic glutamate receptors in the glial cells and inhibitory neurons of cerebellar cortex and their association with the climbing and parallel fibers, and basket cell axons were studied. Samples of P14 and P21 rat cerebellar cortex were exposed to a specific antibody against GluR1 subunit(s) ofAMPA receptors and were examined with confocal laser scanning microscopy. GluR1 strong immunoreactivity was confined to Purkinje cell and the molecular layer. Weak GluR1 immunoreactivity was observed surrounding some Golgi cells in the granule cell layer. Intense GluR1 immunoreactivity was localized around Purkinje, basket, and stellate cells. Purkinje cells expressed strong GluR1 immunoreactivity surrounding the cell body, primary dendritic trunk and secondary and tertiary spiny dendritic branches. Marked immunofluorescent staining was also detected in the Bergmann glial fibers at the level of middle and outer third molecular layer. Positive immunofluorescence staining was also observed surrounding basket and stellate cells, and in the capillary wall. These findings suggest the specific localization of GluR1 subunits ofAMPA receptors in Bergmann glial cells, inhibitory cerebellar neurons, and the associated excitatory glutamatergic circuits formed by climbing and parallel fibers, and by the inhibitory basket cell axons.

Loss of connexin36 in rat hippocampus and cerebellar cortex in persistent Borna disease virus infection

Neonatal Borna disease virus (BDV) infection of the Lewis rat leads to progressive degeneration of dentate gyrus granule cells, and cerebellar Purkinje neurons. Our aim here was to clarify whether BDV interfered with the formation of electrical synapses, and we, therefore, analysed expression of the neuronal gap junction protein connexin36 (Cx36) in the Lewis rat hippocampal formation, and cerebellar cortex, 4 and 8 weeks after neonatal infection. Semiquantitative RT-PCR, revealed a BDV-dependent decrease in Cx36 mRNA in the hippocampal formation 4 and 8 weeks post-infection (p.i.), and in the cerebellar cortex 8 weeks p.i. Correspondingly, immunofluorescent staining revealed reduced Cx36 immunoreactivity in both dentate gyrus, and ammons horn CA3 region, 4 and 8 weeks post-infection. In the cerebellar cortex, Cx36 immunoreactivity was detected only 8 weeks post-infection in the molecular layer, where it was down regulated by BDV. Our findings demonstrate, for the first time, distinct BDV-dependent reductions in Cx36 mRNA and protein in the rat hippocampal formation and cerebellar cortex, suggesting altered neuronal network properties to be an important feature of persistent viral brain infections.

Modeling neuro-vascular coupling in rat cerebellum: Characterization of deviations from linearity

We investigated the quantitative relation between neuronal activity and blood flow by means of a general parametric mathematical model which described the neuro-vascular system as being dynamic, linear, time-invariant, and subjected to additive noise. The model was constructed from measurements by means of system identification methods and validated across experiments. We sought to cover the system response to multiple stimulation frequencies and durations by a single model. We used the model to investigate the transport delay, the linear order, the deviations from linearity, and conditions for linearizability. We exercised the model on data from rat cerebellar cortex. In anesthetized rats, stimulation of the inferior olive caused climbing fiber activity and blood flow changes. Field potential amplitudes were used as an indicator of neuronal activity and blood flow was measured by laser-Doppler flowmetry. In one set of experiments, stimulation frequencies were in the range 2-20 Hz and the stimulation durations were 60 s and 600 s. The transport delay was estimated to be nearly zero, the linear order to be two. The deviations from linearity were consistently characterized as frequency saturation and dips in blood flow responses to stimulation for 60 s, and overgrowth of blood flow responses to stimulation for 600 s. In another set of experiments, stimulation frequencies were in the range 0.5-10 Hz and the stimulation duration was 15 s. The neuro-vascular system could be approximated by the linear model when the stimulation frequencies were restricted to the range 0.5-7 Hz. In conclusion, our model could predict blood flow responses to stimuli of low frequency and short duration.

Layer specific changes of astroglial gap junctions in the rat cerebellar cortex by persistent Borna Disease Virus infection

Neonatal Borna Disease Virus (BDV) infection of the Lewis rat brain, leads to Purkinje cell degeneration, in association with astroglial activation. Since astroglial gap junctions (GJ) are known to influence neuronal degeneration, we investigated BDV dependent changes in astroglial GJ connexins (Cx) Cx43, and Cx30 in the Lewis rat cerebellum, 4, and 8 weeks after neonatal infection. On the mRNA level, RT-PCR demonstrated a BDV dependent increase in cerebellar Cx43, and a decrease in Cx30, 8, but not 4 weeks p.i. On the protein level, Western blot analysis revealed no overall upregulation of Cx43, but an increase of its phosphorylated forms, 8 weeks p.i. Cx30 protein was downregulated. Immunohistochemistry revealed a BDV dependent reduction of Cx43 in the granular layer (GL), 4 weeks p.i. 8 weeks p.i., Cx43 immunoreactivity recovered in the GL, and was induced in the molecular layer (ML). Cx30 revealed a BDV dependent decrease in the GL, both 4, and 8 weeks p.i. Changes in astroglial Cxs correlated not with expression of the astrogliotic marker GFAP, which was upregulated in radial glia. With regard to functional coupling, primary cerebellar astroglial cultures, revealed a BDV dependent increase of Cx43, and Cx30 immunoreactivity and in spreading of the GJ permeant dye Lucifer Yellow. These results demonstrate a massive, BDV dependent reorganization of astroglial Cx expression, and of functional GJ coupling in the cerebellar cortex, which might be of importance for the BDV dependent neurodegeneration in this brain region.