The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in “circadian” light detectors of higher vertebrates. Because in amphioxus melanopsin activates a Gq/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na+. In addition to Na+, Ca2+ is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at Vm > ENa; (b) in Na-free solution a small residual inward photocurrent persists at Vm near resting level, provided that Ca is present; and (c) Vrev exhibits a modest shift with [Ca]o manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K+, as [K]o only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La3+ and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates

Melanopsin-expressing amphioxus photoreceptors transduce light via a phospholipase C signaling cascade

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e29813, doi:10.1371/journal.pone.0029813.Melanopsin, the receptor molecule that underlies light sensitivity in mammalian ‘circadian’ receptors, is homologous to invertebrate rhodopsins and has been proposed to operate via a similar signaling pathway. Its downstream effectors, however, remain elusive. Melanopsin also expresses in two distinct light-sensitive cell types in the neural tube of amphioxus. This organism is the most basal extant chordate and can help outline the evolutionary history of different photoreceptor lineages and their transduction mechanisms; moreover, isolated amphioxus photoreceptors offer unique advantages, because they are unambiguously identifiable and amenable to single-cell physiological assays. In the present study whole-cell patch clamp recording, pharmacological manipulations, and immunodetection were utilized to investigate light transduction in amphioxus photoreceptors. A Gq was identified and selectively localized to the photosensitive microvillar membrane, while the pivotal role of phospholipase C was established pharmacologically. The photocurrent was profoundly depressed by IP3 receptor antagonists, highlighting the importance of IP3 receptors in light signaling. By contrast, surrogates of diacylglycerol (DAG), as well as poly-unsaturated fatty acids failed to activate a membrane conductance or to alter the light response. The results strengthen the notion that calcium released from the ER via IP3-sensitive channels may fulfill a key role in conveying - directly or indirectly - the melanopsin-initiated light signal to the photoconductance; moreover, they challenge the dogma that microvillar photoreceptors and phoshoinositide-based light transduction are a prerogative of invertebrate eyes.This work was supported by the National Science Foundation of the USA (grant 0918930)

La extinción facilita la nueva adquisición de la memoria espacial en ratas en el laberinto de Barnes

Para observar los efectos de la extinción sobre la adquisición de un nuevo aprendizaje espacial, se entrenaron dos grupos de 11 ratas en el laberinto circular de Barnes. Un grupo recibió, tras el entrenamiento, un procedimiento de extinción y enseguida un segundo entrenamiento, mientras que el otro grupo recibió el segundo entrenamiento a continuación del primero. El procedimiento de extinción fue exitoso para suprimir la preferencia espacial previamente adquirida. A pesar de que ambos grupos adquirieron de la misma forma una asociación espacial durante el primer entrenamiento, en el segundo entrenamiento las ratas sometidas a extinción cometieron menos errores, recorrieron menor distancia y desarrollaron una preferencia espacial más fuerte (p < 0,05). Estas diferencias fueron debidas a que las ratas que no recibieron el procedimiento de extinción continuaron expresando el primer comportamiento aprendido. La extinción produjo un efecto inhibitorio sobre la expresión de la primera preferencia espacial, facilitando la nueva adquisición. El paradigma aquí propuesto constituye un nuevo enfoque para el estudio de la extinción. Adicionalmente, en este estudio se desarrolló y se puso a prueba un nuevo método para modelar y evaluar las trayectorias de las ratas en el laberinto

G_q expresses in the microvillar membrane of Hesse cells.

<p>(<i>A</i>) The sequence of the immunogenic peptide used to raise anti-G_q antibodies was aligned with the predicted C-terminal region of G_q of amphioxus, and those of other organisms. (<i>B</i>) Western blot of neural tube using anti G_q, showing the detection of a single band of ≈42 kDa. (<i>C</i>) Morphological characteristics of the Hesse cell: the accessory pigmented cell engulfs the microvilli-covered region of the clear sensory cell; the position of the villous membrane within the occluded region is drawn in red. (<i>D</i>) <i>Left</i>: Nomarski micrograph of a 10 µm section of fixed neural tube containing two Hesse cells. <i>Right</i>: fluorescence image of the same section incubated with anti-G_q antibodies and Alexa Fluo 488-conjugated secondary antibodies. The immunostaining is confined to the region of the microvilli.</p

Lack of effect of diacylglycerol analogs on membrane conductance and light-evoked current.

<p>(A) Control Hesse cell dialyzed with standard internal solution. (<i>Left</i>) Recording of membrane current in the dark immediately after attaining the whole-cell configuration; (<i>right</i>) currents evoked by flashes of increasing intensity. (B,C) Similar experiments conducted in Hesse cells dialyzed with SAG (1-Stearoyl-2-arachidonoyl-<i>sn</i>-glycerol; 10 µM) and OAG (1-Oleoyl-2-acetyl-sn-glycerol; 10 µM), respectively. Neither the holding current nor the photocurrents were affected. Membrane potential was clamped at −50 mV throughout.</p

Amphioxus melanopsin groups with G_q-coupled rhodopsins.

<p>(<i>A</i>) The translated sequence of <i>Branchiostoma</i> was subjected to a BLAST search, and subsequently aligned with the highest-ranking hits among <i>non</i>-melanopsin photopigments. All belong to microvillar photoreceptors from eyes of invertebrates. The Clustal W alignment shows the core region of the proteins (omitting the carboxy-terminus, and part of the amino-terminus, which are generally divergent); black shading indicates identity, whereas grey shading indicates conservative aminoacid substitutions. Accession numbers: amphioxus (<i>Branchiostoma</i>) Q4R1I4; scallop (<i>Mizuhopecten</i>), O15973.1, octopus (<i>Octopus</i>). P09241.1; horseshoe crab (<i>Limulus</i>), ACO05013.1; and squid (<i>Loligo</i>) P24603.1. (<i>B</i>) Western blot with anti-melanopsin antibodies. A single band of ≈71 kDa was detected, closely approaching the size expected from the predicted aminoacid sequence. This confirms that the melanopsin protein expresses in the neural tube of <i>B. floridae</i>. (<i>C</i>) Phylogenetic tree illustrating the grouping of the main classes of photopigments found in the animal kingdom. Amphioxus melanopsin is closely related to the G_q-coupled rhodopsin of mollusks and arthropods.</p