The polyamine spermine potentiates the propagation of negatively charged molecules through the astrocytic syncytium

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium

“El club de lectura me ha hecho feliz”: Una investigación acción

This article presents the findings of an action research, which purpose was to explore how participating on a reading club positively contributes to the development of aesthetic reading (reading for pleasure) in elementary school students that showed challenges with reading and were participants on an RTI literacy development group. The techniques used to collect the information were a pre-test, a questionnaire, three journal entries, a post-test and the participation on a focus group. The research was based on the constructivist approach to education which is based on learning through personal construction of meaning. Reading is seen as an activity of transactional interaction between the reader and the text, as proposed by Rosenblatt (1995). Among the most significant findings, we saw that the integration of snacks, dramatizations, book loans and enriched activities surrounding the shared readings brought satisfaction, enjoyment and participation to the students.Este artículo presenta los hallazgos de una investigación-acción que tuvo como propósito explorar cómo contribuye la participación en un club de lectura al desarrollo de la lectura estética en niños de escuela elemental que presentan dificultad en la lectura. Las técnicas de recopilación consistieron de una preprueba, un cuestionario, tres entradas de diario, una posprueba y una entrevista de grupo focal. La investigación se fundamentó en la teoría constructivista del aprendizaje, la cual se enmarca en el aprendizaje a través de la construcción personal. Asimismo, se abordó el tema de la lectura desde la perspectiva transaccional, expuesta por Rosenblatt (1995). Entre los hallazgos más significativos, encontramos que actividades como la integración de merienda relacionada a los cuentos, la dramatización y los préstamos de libros evocaron, en los participantes, disfrute, motivación y participación

The Polyamine Spermine Potentiates the Propagation of Negatively Charged Molecules through the Astrocytic Syncytium

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium

Unidirectional photoreceptor-to-Müller glia coupling and unique K+ channel expression in Caiman retina.

Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Müller cells from the retina of crocodiles remain, hitherto, unknown.We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus), endowed with both diurnal and nocturnal vision, by (i) immunohistochemistry, (ii) whole-cell voltage-clamp, and (iii) fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications.Immunohistochemistry revealed that caiman Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100β, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling.Our data indicate that caiman Müller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K+ buffering

Localization of connexin 43 (Cx43) in the caiman retina.

<p>(<b>A</b>) Immunolocalization of the Müller cell-specific protein glutamine synthetase (GS, red) and staining of the nuclei by Hoechst 33258 (blue). The micrograph shows the overlay of the fluorescence image and the transmitted light with visible retinal layers. (<b>B</b>) Immunostaining of Cx43 (green), yellow arrowheads point to Müller cell-like structures. (<b>C</b>)-(<b>E</b>) Overlay of Cx43 and GS staining clearly demonstrates co-localization in Müller cell processes (yellow arrowheads). Moreover, cone outer segments and cone pedicles are stained by the lectin peanut agglutinin (PNA, purple). Localization of Cx43 in cone pedicles is demonstrated at higher magnification in (<b>E</b>) (white arrowheads). ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, OPL-outer plexiform layer, ONL-outer nuclear layer, OLM-outer limiting membrane, IS-inner segments of photoreceptors, OS-outer segments of photoreceptors.</p

Unidirectional flow of sulforhodamine-B dye from cone to caiman Müller cell.

<p>(<b>A</b>) Upper panel: whole-cell patch clamp technique using micropipette (MP) filled with sulforhodamine-B penetrating the inner segment of a cone attached to a Müller cell. The dye filled the cone and the Müller cell: tracing of the photoreceptor cell body and inner segment (IS) and spreading throughout the whole Müller cell from the soma (MS) to the endfeet. Lower panel: combined (DIC and fluorescent) image showing that sulforhodamine-B is not permeable to neighboring photoreceptors, but filled only the cone which was patched. Insert shows an enlarged image of fine attachments of three cones to a single glial cell where two cones are not showing fluorescent dye. (<b>B</b>) Upper panel: whole-cell patch clamp of a Müller cell resulted in the dye-tracing of the cell body and endfeet, while no spreading of the dye occurred to the photoreceptors attached to this Müller cell. Lower panel: patch of the Müller cell soma only traced the Müller cell; the dye did not spread to the attached photoreceptors.</p

Cellular tandem between Müller cells and photoreceptors: involvement of 2P domain K⁺ channels.

<p>(<b>A</b>) Müller cell soma (MS) with attached cones approached by a patch pipette (MP). In <b>A</b>, <b>B</b>, and <b>C</b>, white arrowheads and white dotted lines show the area of contact between the inner segment (IS) of photoreceptors and MS. S points to the soma of cones. (<b>B</b>) Rods with outer segments (black circle), inner segments (black diamond) and somata (open black square) attached to MS. In <b>B</b> and <b>C</b>, thick white arrow shows MS. (<b>C</b>) Isolated Müller cell without photoreceptors. Curved arrows show endfeet. Thin white arrow shows stalk. (<b>D</b>) Average currents recorded from MS of isolated Müller cells (solid line, n = 5) and recorded from MS of isolated Müller cells with photoreceptors attached (dotted line, n = 5). The I/V-curves were obtained in response to a linear voltage ramp from −150 mV to +150 mV in control ECS K⁺ = 2.5 mM. Müller cells have linear outward currents near K⁺-equilibrium potential. (<b>E</b>) Effect of 2-P domain channel blocker bupivacaine (BUPI 1 mM) on isolated Müller cells with (dotted line) and without photoreceptors attached (solid line). After BUPI, residual currents are reduced ∼15 times. (<b>F</b>) Adding the Kir channel blocker barium (Ba²⁺, 100 µM) to BUPI, caused a near complete block of residual current (from −100 mV to +50 mV). (<b>G</b>) Membrane potentials of cells in 2.5 and 10 mM K⁺ ECS. [K⁺]_o = 10 mM induces depolarization. (<b>H</b>) Application of BUPI 1 mM and BUPI 1 mM with barium 100 µM were used to test for 2P-domain and Kir channels respectively. Single Müller cells (solid black column, n = 8) and cells with photoreceptors attached (grey column, n = 10) show different sensitivity to BUPI. Addition of Ba²⁺ further depressed membrane potentials in both cells, but with no significant difference. Error bars represent standard errors of the mean (SEM), where p<0.05 was considered significant (*).</p