Agata Zappalà

Multiple Zonal Projections of the Basilar Pontine Nuclei to the Cerebellar Cortex of the Rat

This study revealed a sagittal zonal pattern of projections to the cerebellar cortex after hydraulic or iontophoretic injections of anterograde tracers (tritiated leucine, wheat germ agglutinin‐horseradish peroxidase, or biotinylated dextrane amine) in the basilar pontine nuclei of Wistar rats. The zonal pattern of projection was observed only after injections of small size, whereas large injections labeled diffusely wide areas of the cerebellar cortex, masking the zonal projection because the fusion of contiguous stripes. Diverging projections to discrete sets of sagittal stripes in the two sides of the cerebellar cortex arose from single injections. The stripes of fiber terminals were sharply delimited on both sides by areas, interstripes, either virtually void of labeling or with a much lower density of labeling. Thus, the areas of the cerebellar cortex were parceled in sets of sagittal compartments, stripes and interstripes, by the pontine projections. Up to five compartments (three stripes and two interstripes) were observed in the paraflocculus, in the copula pyramidis, and in vermal lobule IX. Up to nine compartments (five stripes and four interstripes) were found in the crus I, the lobulus simplex, the paramedian lobule, and vermal lobules VI–VIII. Up to seven compartments (four stripes and three interstripes) were found in the crus II. Single injections into the basilar pontine nuclei usually labeled symmetric areas of the cerebellar cortex, which, in some cases, showed similar number of stripes. When this was not the case, the stripes were usually more numerous in the contralateral than in the ipsilateral side. All areas of the cerebellar cortex were projected upon, with zonation patterns from different regions of the basilar pontine nuclei. The projections of the basilar pontine nuclei to the cerebellar cortex were arranged according to a fixed pattern specific for each cortical area, independently of the number of stripes labeled within. The mean width of the stripes visualized in the single cortical areas of different rats was similar, despite the different size of the injections. The length of the stripes ranged widely in the various areas of different rats. The data collected in this study are consistent with the idea that all the mossy afferents to the cerebellar cortex are arranged with a zonal pattern. J. Comp. Neurol. 430:471–484, 2001.

Expression of pannexin1 in the cns of adult mouse: Cellular localization and effect of 4-aminopyridine-induced seizures

The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.

Expression of pannexin2 protein in healthy and ischemized brain of adult rats

The expression pattern of the pannexin2 protein (Px2) in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat Px2 was generated in chicken and purified for affinity. This antibody was used to study by Western blot, Enzyme-Linked Immunosorbent Assay, and immunohistochemistry, the expression pattern of Px2 in healthy brain of adult rats and in the hippocampus of rats submitted to bilateral clamping of carotid arteries for 20 min, followed by different times of reperfusion (I/R) (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Px2 in the healthy brain. All Px2(+) positive cells were neurons which also showed no puncta on their cellular membranes. Both pyramidal cells and interneurons, the majority of which were positive to parvalbumin, were stained in healthy hippocampus. The number of Px2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R, with a negative peak of about -40% after 72 h from I/R. Interneurons which were positive for both Px2 and parvalbumin, represented about the 85% of all parvalbumin cells stained in the hippocampus. This percentage rested grossly unmodified at different time intervals after I/R in spite of the progressive neuronal depletion. Concomitantly, an intense astrogliosis occurred in the hippocampus. Most of the astroglial cells expressed de novo and for a transient time (from 24 h to 14 days from I/R), Px2. Primary co-cultures of hippocampal neurons and astrocytes were submitted to transient ischemia-like injury. This set of experiments further confirmed the in vivo results by showing that Px2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Px2 in the astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. In conclusion, our results showed the transient expression of Px2 in astrocytes of reactive gliosis occurring in the hippocampus following I/R injury. We hypothesize that Px2 expression in astrocytes following an ischemic insult is principally involved in the formation of hemichannels for the release of signaling molecules devoted to influence the cellular metabolism and the redox status of the surrounding environment.

Regulation of neuronal connexin-36 channels by pH

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pHi since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

Dynamic expression of Cx47 in mouse brain development and in the cuprizone model of myelin plasticity.

The study shows the dynamic expression of connexin47 (Cx47) in oligodendrocytes and myelin of mice, either in myelinogenesis occurring in early development or in an experimental model of new‐myelinogenesis of adult mice. Cx47 first appeared in the embryonic mouse brain at E10.5 successively the expression increased, principally in regions populated by developing oligodendrocytes. The expression declined postnatally toward adulthood and immunoreactivity was restricted to a few specific areas, such as the corpus callosum, the striatum, the cerebellum, and the spinal cord. Since the expression of Cx47 in developing oligodendrocytes preceded those of Cx32 and Cx29, a role of Cx47 in myelinogenesis was postulated. This hypothesis was tested in a model of re‐myelination, which principally involved the corpus callosum, occurring in adult mice by treatment with cuprizone. Cx47 was upregulated during demyelination and recovered during the remyelination phase. During demyelination, Cx47 was first over‐expressed in the corpus callosum and later, when the myelin virtually disappeared in the injured areas, Cx47 was expressed in astrocytes located inside and closely around the demyelinated areas. The remyelination of injured areas occurred after stopping the administration of cuprizone and continued to complete recovery. In this period the expression of Cx47 shifted from astrocytes to newly‐formed myelin. Thus, Cx47 exhibits in this model a transient and de novo expression in astrocytes with a topographic segregation in the injured areas, only when oligodendrocytes and the myelin were most severely affected. Taken as a whole the evidence suggests that Cx47 play a key role in myelination.

Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Projections of the basilar pontine nuclei and nucleus reticularis tegmenti pontis to the cerebellar nuclei of the rat

This study showed the precise projection pattern of the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) to the cerebellar nuclei (CN), as well as the different anatomic features of BPN and NRTP projections. The staining of BPN or NRTP with biotinylated dextran labeled projection fibers to complementary topographic areas in the CN. In fact, BPN principally project to a rostrocaudally oriented column of the nucleus lateralis (NL), which at the midcentral level shifts to the lateroventral part of the nucleus, as well as to the caudolateral part of the nucleus interpositus posterioris. The NRTP projects to a rostrocaudal column of the NL, which at the midcentral level shifts medially, as well as to the nucleus interpositalis and to the caudal part of the nucleus medialis. BPN axons in the CN usually branch into short collaterals of simple morphology that involve small terminal areas, whereas NRTP axons branch into longer collaterals of complex morphology involving terminal areas of different sizes. Each site of injection is at the origin of a set of terminal areas in the CN. The set of projections from different BPN or NRTP areas were partially, but never completely, overlapping. Thus, the set of terminal areas in the CN was specific for each area of both BPN and NRTP. Injection of tetramethyl‐rhodamine‐dextran‐amine into the CN stained cell bodies of BPN and NRTP with different repartition on the two sides. The study showed that CN are innervated by the contralateral BPN and not very much by the ipsilateral BPN, whereas they are innervated by NRTP bilaterally, even if with a contralateral prevalence. In conclusion, this study supports the hypothesis that both BPN and NRTP are concerned in the central program for skilled movements, even if they are probably involved in different functional roles. J. Comp. Neurol. 452:115–127, 2002.

Role of the Otx1 gene in cell differentiation of mammalian cortex.

This study analyses by immunohistochemical methods the effects of the deletion of the Otx1 gene on 12 areas of the cerebral cortex and on neurons expressing Ca‐binding proteins (CaBP), such as parvalbumin (Pv) and calbindin‐D28K (Cb). We found that the deletion of the Otx1 gene modified differently the various cortical areas. The decrease in cortical thickness ranged from 29.35 to 9.85% and the reduction in cellular population from 35.90 to 3.65% in the different cortical areas. The influence of the Otx1 gene concerns all cortical layers with variable effects on different cortical areas. The cellular population of cerebral cortex considered as a whole was reduced by 20.67%, Pv‐positive (Pv+) cells by 58.01% and Cb‐positive (Cb+) cells by 51.54%. The quantitative distribution of Pv+ and Cb+ cells varied independently in the different cortical areas. Topographic analysis of CaBP cells in Otx1‐null mice (Otx1−/−) showed that Pv+ cells were principally distributed in layers IV and V and Cb+ cells in layers V and VI. Given that in the development of wild‐type mice both cell types first appear in deep layers and later spread to superficial ones, the segregation of CaBP neurons in inner layers of Otx1−/− animals is an index of the immaturity of the cerebral cortex of these animals. This study showed that the Otx1 gene has a more complex role than previously reported, as it is involved in the maturation and differentiation of various cerebral cortices, and, specifically, in the development of CaBP cells.

The basilar pontine nuclei and the nucleus reticularis tegmenti pontis subserve distinct cerebrocerebellar pathways.

Previous studies often considered the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) as relays of a single cerebro-(ponto)-cerebellar pathway. Conversely, the different cortical afferences to the BPN and the NRTP, as well as the anatomical and functional features of the cerebellopetal projections from these pontine nuclei, support the different, and for some aspect, complementary arrangement of the cerebrocerebellar pathways relayed by the BPN or NRTP. Both the BPN and the NRTP are innervated from the cerebral cortex, but with regional prevalence. The NRTP is principally innervated from motor or sensori-motor areas while the BPN are principally innervated from sensory, mainly teloceptive, and associative area. Projections from sensory-motor areas were also traced to the BPN. The BPN and NRTP project to all parts of the cerebellar cortex with a similar pattern. In fact, from single areas of them projections were traced to set of sagittal stripes of the cerebellar cortex. In variance to such analogies, the projections to the cerebellar nuclei differed between those traced from the NRTP and from BPN. In fact, BPN and NRTP have private terminal areas in the cerebellar nuclei with relatively little overlaps. The BPN innervated the lateroventral part of the nucleus lateralis and the caudoventral aspect of the nucleus interpositalis posterioris. The NRTP principally innervated the mediodorsal part of the nucleus lateralis, the nucleus interpositalis anterioris, the nucleus medialis. Since the single cerebellar nuclei have their specific targets in the extracerebellar brain areas, it follows that the BPN and the NRTP, passing through their cerebellar nuclei relays, are devoted to control different brain areas and thus likely to play different functional roles. From single pontine regions (of both BPN and NRTP) projections were traced to the cerebellar cortex and to the cerebellar nuclei. In some cases these projections reached areas which are likely anatomically connected (by Purkinje axons). This pattern of the pontine projections was termed as coupled projection. In some other cases, the projections reached areas of the cerebellar cortex but not the nuclear regions innervated by them. We termed this as uncoupled projection. The existence of both coupled and uncoupled projections, open new vistas on the functional architecture of the pontocerebellar pathway. More in detail, this study showed the different quantitative and topographic distribution of the coupled and uncoupled projections visualized in the cerebellar projections from BPN and NRTP. All these evidences strongly support the anatomical and the functional differences that characterise the cerebrocerebellar pathways relayed by the BPN and the NRTP.

Role of carbon monoxide in vascular diseases.

During the degradation of heme by the enzyme heme oxygenase (HO), Carbon monoxide (CO) is generated. Although it is considered as a non-significant and potentially toxic waste gas of heme catabolism, CO is a key signaling molecule used to regulate different cardiovascular functions. In this review, we focus the protective roles of CO in vascular injury/disease, which may be important to explore the overall protective roles of HO-1/CO system in the pathogenesis of human vascular disease.