Immunoreactivity to calbindin in purkinje cells in the cerebellum of mice is not affected by rabies virus infection

Introducción: La calbindina (CB) es una proteína reguladora del calcio intracelular y la célula de Purkinje del cerebelo es la neurona con más alta concentración de CB. Se ha demostrado pérdida de inmunorreactividad a CB en diferentes áreas del sistema nervioso en ratones inoculados con virus de la rabia, pero faltaba estudiar este fenómeno en el cerebelo. Objetivo: Determinar el efecto de la inoculación con virus de la rabia sobre la expresión de CB en células de Purkinje del cerebelo de ratones. Metodología: Se inocularon ratones con el virus por vía intramuscular. Se sacrificaron los animales cuando alcanzaron la fase avanzada de la enfermedad y se fijaron mediante perfusión intracardiaca con paraformaldehído al 4%. Se les extrajo el cerebelo y se hicieron cortes sagitales de 50 micrómetros de espesor en un vibrátomo. Estos se procesaron mediante inmunohistoquímica para revelar la presencia de CB o de antígenos del virus de la rabia. El mismo procedimiento se realizó con animales no infectados (controles). Resultados: Las células de Purkinje fueron masivamente infectadas con el virus de la rabia. En las imágenes panorámicas observadas en el microscopio se comprobó que sólo estas células fueron inmunorreactivas a CB. No se hallaron diferencias significativas en la inmunorreactividad a CB, evaluada por densitometría óptica, entre los animales infectados y los controles. Conclusión: La expresión de CB en las células de Purkinje del cerebelo parece no afectarse por la infección con rabia, a diferencia de lo que se ha demostrado en otras áreas del sistema nervioso del ratón.Introduction: Calbindin (CB) is a regulatory protein of intracellular calcium, and the cerebellar Purkinje cell is the neuron with the highest concentration of CB. Loss of CB immunoreactivity has been demonstrated in different areas of the nervous system in rabies virus-infected mice, but the study of this phenomena in the cerebellum lacked. Objective: To determine the effect of inoculation with rabies virus on the expression of CB in Purkinje cells of the cerebellum of mice. Methodology: Mice were intramuscularly inoculated with rabies virus. Animals were sacrificed when they reached an advanced stage of the disease and then they were fixed by intracardiac perfusion with 4% paraformaldehyde. Cerebellums were extracted and sagittal sections 50 microns thick were obtained in a vibratome. These were processed by immunohistochemistry to reveal the presence of CB protein or rabies virus antigens. The same procedure was performed with uninfected animals (controls). Results: Purkinje cells were massively infected with rabies virus. In the microscopic panoramic images observed was found that only these cells are immunoreactive to CB. No significant difference in CB immunoreactivity evaluated by optical densitometry was found between infected animals and controls. Conclusion: The expression of CB in Purkinje cells of the cerebellum appears not to be affected by infection with rabies unlike what has been shown in other areas of the mouse nervous system

Effect of rabies virus infection on the expression of calbindin and parvalbumin on mouse cerebellum: raising awareness to integrate neuroscience and virology

La rabia es transmitida por la mordedura de un animal infectado, generalmente mamíferos carnívoros o quirópteros. Los signos clínicos y la evidencia experimental en modelos in vitro e in vivo sugieren una posible alteración de la neurotransmisión gabaérgica durante la infección con rabia. La Calbindina (CB) y la Parvoalbúmina (PV) son proteínas de enlace de calcio que regulan el efecto de este ion en el metabolismo intracelular. Son marcadores de células gabaérgicas y la infección con rabia causa la pérdida de la inmunorreactividad a CB, pero el incremento de PV, en las diferentes regiones del sistema nervioso central de ratón. Sin embargo, en un estudio reciente en el cerebelo de ratones infectados no se evidenció pérdida de inmunorreactividad a CB, apartándose de la tendencia observada. En este estudio se evaluó a mayor profundidad el efecto de la infección con el virus de la rabia en el cerebelo de ratones estableciendo su influencia sobre la expresión de CB y PV a nivel de transcritos y de proteínas, dada la importancia de estas proteínas en la fisiología de las poblaciones neuronales del cerebelo. Se inocularon ratones de 4 semanas, por vía intramuscular, en las extremidades posteriores, con virus fijo CVS (Challenge Virus Standard) y ratones controles con solución vehículo. Cuando los animales alcanzaron la fase final de la enfermedad, se dividieron en tres grupos de acuerdo con los procedimientos a llevar a cabo: Inmunohistoquímica (IHQ), western blot y RT-qPCR. La IHQ mostró marcación positiva para PV en las células de Purkinje y células estrelladas/en cesta y CB exclusivamente para las células de Purkinje. No hubo diferencias evidentes en la distribución e inmunorreactividad entre ratones infectados y control. Se observó una disminución en el nivel de transcritos para CB y PV, así como la disminución del nivel de proteínas para CB y PV en ratones infectados, aunque para este último no fue significativo. Estos resultados sugieren que la infección por el virus de la rabia promueve una alteración en la homeostasis del calcio en el cerebelo lo cual, además, puede afectar la neurotransmisión del GABA.Rabies virus (RABV) is frequently transmitted from a bite or scratch of an infected animal, mostly dogs. Clinical signs of rabies, in vitro and in vivo models suggest a possible involvement of the gabaergic system during rabies virus infection. Calbindin (CB) and Parvalbumin (PV) are calcium binding proteins which regulate the effect of calcium ions on intracellular metabolism, both are markers of gabaergic cells and rabies infection caused loss of immunostaining for CB but increase of inmunoreactivity of PV in different regions from central nervous system (CNS) in mice. However, in a recent study in cerebellum from infected mice, there were not changes in CB immunostaining compared with controls, this result disagree with previous studies. Here, we evaluated deeply the effect of rabies virus infection in mice cerebellum stablishing its influence in CB and PV expression at transcript and protein levels, due to the importance of both in cerebellum physiology. Four-week-old mice were inoculated intramuscularly in the hindlimb with rabies fixed virus CVS (Challenge Virus Strain) and other mice only with vehicle. When the mice reached the terminal state of illness, the groups were divided according to the procedures: immunohistochemistry (IHC), western blot, RT-qPCR. The IHC showed clear PV-stain for Purkinje cells, and stellate/basket cells and CB stain only in Purkinje cells. No evidence of differences between CB and PV distribution in infected and control mice was found. CB and PV transcript levels were decreased and CB and PV protein levels were decreased in infected mice as well, however non-significant for PV protein levels. These results suggest that RABV infection promotes alterations in calcium ion homeostasis which could affect GABA neurotransmission.COLCIENCIASMagister en Neurociencias. Línea de Investigación: Vulnerabilidad selectiva neuronalMaestrí

Morphological and Molecular Changes in the Cortex and Cerebellum of Immunocompetent Mice Infected with Zika Virus

Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications