Transgenic Overexpression of Myocilin Leads to Variable Ocular Anterior Segment and Retinal Alterations Associated with Extracellular Matrix Abnormalities in Adult Zebrafish.

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.

Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABA-activated GIRK channels in Purkinje cells.

Activation of G protein-gated inwardly-rectifying K(+) (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA(B)) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA(B) receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA(B) receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA(B) receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA(B) receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA(B) receptors. The association of GIRK channels and GABA(B) receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.

Cell type-specific subunit composition of G protein-gated potassium channels in the cerebellum.

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels regulate cellular excitability and neurotransmission. In this study, we used biochemical and morphological techniques to analyze the cellular and subcellular distributions of GIRK channel subunits, as well as their interactions, in the mouse cerebellum. We found that GIRK1, GIRK2, and GIRK3 subunits co-precipitated with one another in the cerebellum and that GIRK subunit ablation was correlated with reduced expression levels of residual subunits. Using quantitative RT-PCR and immunohistochemical approaches, we found that GIRK subunits exhibit overlapping but distinct expression patterns in various cerebellar neuron subtypes. GIRK1 and GIRK2 exhibited the most widespread and robust labeling in the cerebellum, with labeling particularly prominent in granule cells. A high degree of molecular diversity in the cerebellar GIRK channel repertoire is suggested by labeling seen in less abundant neuron populations, including Purkinje neurons (GIRK1/GIRK2/GIRK3), basket cells (GIRK1/GIRK3), Golgi cells (GIRK2/GIRK4), stellate cells (GIRK3), and unipolar brush cells (GIRK2/GIRK3). Double-labeling immunofluorescence and electron microscopies showed that GIRK subunits were mainly found at post-synaptic sites. Altogether, our data support the existence of rich GIRK molecular and cellular diversity, and provide a necessary framework for functional studies aimed at delineating the contribution of GIRK channels to synaptic inhibition in the cerebellum.

Differential maturation of GIRK2-expressing neurons in the mouse cerebellum.

The cerebellar cortex is among the brain regions showing the highest expression levels of G-protein-gated inwardly-rectifying potassium (GIRK/Kir3) channels. Despite their critical contribution in modulating neuronal excitability during development and adult, the spatiotemporal expression of specific GIRK subunits in identified cerebellar neuron populations is unresolved. To characterize this onset of expression, we examined the GIRK2 protein expression in mouse cerebellum by western blot, light microscopy immunohistochemistry and immunofluorescence during perinatal development. Using western blots, GIRK2 expression was low at birth but reach its maximum at P5 before decreasing gradually to adult levels. Immunohistochemical localization indicated that GIRK2 is expressed in granule cells from early stages of development. At the embryonic stage, immunofluorescence techniques for the transcription factor Pax6 allowed to demonstrate that GIRK2 is expressed in granule cell precursors. This GIRK2 expression in granule cells continued throughout postnatal development and adulthood. In addition, the expression of Pax2-GFP allowed selective visualization of Golgi cells during pre- and postnatal development. We could not detect co-expression of Pax2-GFP and GIRK2 during prenatal and early postnatal development, but only at post-migratory stages of Golgi cells, once they are morphologically differentiated and located at the granule cell layer. In the adult cerebellum, we performed a detailed characterization on the expression of GIRK2 in different subpopulations of Golgi cells, using metabotropic glutamate receptor 2 (mGlu(2)) and neurogranin as markers, in GlyT2-GFP and GAD67-GFP mice, and showed that GIRK2 is present in at least four morphological and neurochemical non-overlapping populations of Golgi cells. Altogether, these findings shed new light on the developmental regulation of GIRK channels in the cerebellum, and the main expression in granule cells during perinatal development support the idea that GIRK2 may provide a significant route for modulating different aspects of cerebellar development.

Evolución de los signos y síntomas gastroinstestinales durante el embarazo y el puerperio / Gatrointestinal signs ans symptoms evolution guring pregnancy and puerperium

En los manuales de obstetricia se encuentran numerosos signos y síntomas gastrointestinales asociados con el embarazo normal que aparecen en determinados momentos del mismo. Los porcentajes de mujeres que los presentan no son registrados en estos manuales y, cuando lo hacen, las cifras son muy variables de uno a otro. En las revisiones de la bibliografía, los signos o síntomas no se toman en conjunto, sino que los trabajos suelen referirse a las náuseas y a los vómitos principalmente. Por ello, el objetivo de este estudio es describir el comportamiento de los signos y síntomas gastrointestinales más frecuentes a lo largo del embarazo y en el puerperio mediato.Material y métodos: 963 mujeres embarazadas, elegidas de modo accidental, entrevistadas entre las 8 y 12 semanas de gestación (n = 198), entre las 18 y 22 (n = 255), entre las 37 y 42 (n = 292) y dos semanas después del parto (n = 218) y procedentes de centros de salud de la ciudad de Esmeraldas (Ecuador). El cuestionario, contestado de forma voluntaria, contenía un listado sobre los signos y síntomas gastrointestinales más frecuentes durante el embarazo, y se preguntaba sobre la presencia o no de los mismos en las dos semanas anteriores al momento de la entrevista.Resultados: El porcentaje de náuseas y vómitos, aunque más elevado en el primer trimestre, se mantiene alrededor del 35-40 por ciento en el tercer trimestre de gestación. La pirosis se presenta en un 40,4 por ciento de las gestantes ya en el primer trimestre. El estreñimiento se mantiene constante a lo largo de todo el embarazo. La prevalencia de hemorroides es muy baja durante la gestación (2,3 por ciento).Conclusiones: La descripción de la evolución de los signos y síntomas del embarazo descrita en los manuales de obstetricia no se corresponde con datos obtenidos en este estudio. Algunos de los síntomas clásicos atribuidos a la gestación aparecen en proporciones muy bajas en la población estudiada.