optogenetic-perturbation-neural-activity-with-laser-illumination-semi

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact <em>Drosophila</em> Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

The University of Tokyo

the university of tokyo

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibire(ts), namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1-10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.

Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin.

Halorhodopsin (NpHR), une pompe de chlorure microbienne ax&eacute;e sur la lumi&egrave;re, permet de faire taire de fonction neuronale avec superbe r&eacute;solution temporelle et spatiale. Ici, nous avons g&eacute;n&eacute;r&eacute; une lign&eacute;e transg&eacute;nique de drosophile qui anime l&#39;expression de NpHR sous le contr&ocirc;le du syst&egrave;me de Gal4/UAS. Ensuite, nous avons utilis&eacute; pour diss&eacute;quer les propri&eacute;t&eacute;s fonctionnelles des circuits neuraux qui r&eacute;gulent le p&eacute;ristaltisme larvaire, une onde entretenue de la contraction musculaire de segments post&eacute;rieurs &agrave; la partie ant&eacute;rieure. Nous d&eacute;montrons d&#39;abord l&#39;efficacit&eacute; de NpHR en montrant cette inhibition optique globale et continue par NpHR des neurones moteurs ou neurones r&eacute;troaction sensorielle induisent les m&ecirc;mes r&eacute;ponses comportementales chez les larves rampants &agrave; ces stimul&eacute;s lorsque la fonction de ces neurones sont inhib&eacute;es par la Shibire(ts), &agrave; savoir toutes les paralysies ou ralentissent la locomotion, respectivement. Nous avons ensuite appliqu&eacute; transitoire et/ou ax&eacute; sur des stimuli lumineuses inhibe l&#39;activit&eacute; des neurones moteurs dans un plus dans le temps et dans l&#39;espace restreint de mani&egrave;re et a &eacute;tudi&eacute; les effets de l&#39;inhibition optique sur le p&eacute;ristaltisme. Lorsqu&#39;un stimulus lumineux bref (1 &agrave; 10 sec) a &eacute;t&eacute; appliqu&eacute; &agrave; une larve rampante, la vague de la contraction musculaire s&#39;est arr&ecirc;t&eacute; de fa&ccedil;on transitoire mais a repris depuis la position arr&ecirc;t&eacute;e lorsque l&#39;&eacute;clairage est &eacute;teint. De m&ecirc;me, lorsqu&#39;un stimulus lumineux concentr&eacute; a &eacute;t&eacute; appliqu&eacute; pour inhiber les neurones moteurs dans un ou plusieurs segments qui &eacute;taient sur le point d&#39;&ecirc;tre activ&eacute; dans une larve diss&eacute;qu&eacute;e en cours de locomotion fictive, la propagation de constriction musculaire en pause pendant le stimulus lumineux mais a repris depuis la position arr&ecirc;t&eacute;e lorsque l&#39;inhibition (&gt; 5sec) a &eacute;t&eacute; supprim&eacute;. Ces r&eacute;sultats sugg&egrave;rent que (1) cuisson des neurones moteurs &agrave; la pointe de la vague est requis pour la vague de proc&eacute;der &agrave; plusieurs segments ant&eacute;rieurs et (2) les informations sur la phase de l&#39;onde, &agrave; savoir quel segment est active &agrave; un moment donn&eacute;, peuvent &ecirc;tre m&eacute;moris&eacute;s dans les circuits neurones pendant plusieurs secondes.

Optique dissection de circuits neuronaux responsables de locomotion larve de drosophile avec halorhodopsin.

Halorhodopsin (NpHR), una pompa di cloruro microbica basati sulla luce, consente di silenziamento della funzione neuronale con eccellente risoluzione spaziale e temporale. Qui, abbiamo generato una linea transgenica di Drosophila che spinge l&#39;espressione di NpHR sotto il controllo del sistema Gal4/UAS. Poi, abbiamo utilizzato per sezionare le propriet&agrave; funzionali dei circuiti neurali che regolano la peristalsi larvale, un&#39;onda continua di contrazione muscolare da segmenti posteriori all&#39;anteriore. Abbiamo prima dimostrare l&#39;efficacia del NpHR mostrando che globale e continua NpHR-ottico inibizione mediata dei motoneuroni o neuroni sensoriali feedback inducono le stesse risposte comportamentali in larve striscianti a quelli indotti quando la funzione di questi neuroni sono inibiti da Shibire(ts), vale a dire completano paralisi o rallentarono locomozione, rispettivamente. Abbiamo poi applicato transitori e/o concentrato leggeri stimoli per inibire l&#39;attivit&agrave; dei neuroni motori in maniera pi&ugrave; temporaneamente e spazialmente limitato modo e ha studiato gli effetti dell&#39;inibizione ottico sulla peristalsi. Quando uno stimolo luminoso breve (1-10 sec) &egrave; stato applicato a una larva strisciante, l&#39;onda di contrazione muscolare si ferm&ograve; transitoriamente ma ripreso dalla posizione interrotta quando la luce &egrave; stata spenta. Allo stesso modo, quando uno stimolo luminoso concentrato &egrave; stato applicato per inibire i motoneuroni in uno o pochi segmenti che stavano per essere attivato in una larva sezionata in fase di locomozione fittizia, la propagazione della costrizione muscolare in pausa durante lo stimolo luminoso ma ripreso dalla posizione interrotta quando l&#39;inibizione (&amp;gt; 5 sec) &egrave; stato rimosso. Questi risultati suggeriscono che (1) Firing dei neuroni motori all&#39;avanguardia dell&#39;onda &egrave; necessaria per l&#39;ondata di procedere a pi&ugrave; segmenti anteriore e (2) le informazioni sulla fase dell&#39;onda, vale a dire quale segmento &egrave; attivo in un dato momento, pu&ograve; essere memorizzato nei circuiti neurali per alcuni secondi.

Teruyuki Matsunaga

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