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Studies on visual experience-dependent plasticity can benefit tremendously from experimental protocols in which sensory stimulation is precisely controlled for extended periods over which neuronal, circuit, and behavioral plasticity occurs. Small vertebrates, such as Xenopus tadpoles and zebrafish, are excellent systems for studying brain plasticity. Here, we present a detailed protocol to perform controlled visual stimulation for extended time periods. These methods have been used to study structural plasticity induced by temporally controlled visual stimulation in Xenopus tadpoles. For further details on the use and execution of this protocol, please refer to Hiramoto and Cline (2014, 2020).
Figure 1. A chamber for visual stimulation
Top view photo (A) and schematic drawings of the chamber and components (B). The chamber holds six tadpoles between the two acrylic plates that are 2 mm thick × 3 cm wide. There is a gap between the two plates of 2.3–3 mm where the tadpoles are positioned. A strip of mesh is glued under the gap of the two plates with acrylic glue and forms a sling under the tadpoles. Six rectangular rods are glued under the acrylic plates. The animal is held snugly in the chamber as follows: the ventrolateral sides of the head are supported by 0.4 mm styrene rods or strips of Sylgard glued to the lower edge of the acrylic plates. The head is supported by a Sylgard block positioned in front of the head. It is held in place by the acrylic plates on either side and the coverslip laid over the animal. The tail is supported on both sides by two strips of Sylgard glued to the painted inner edge of the acrylic plates. LEDs are positioned on each side of the chamber at eye level for the tadpoles. LEDs are glued to the acrylic plates with epoxy glue. Before mounting the LEDs on the chamber, a slit is cut in the plastic dome on top of the LED which provides more even illumination to the eye. Wires are soldered to LEDs. Either the anodes or the cathodes are connected to a common line. The other terminals are connected to the power source independently through resistors. Tadpoles are inserted in the slit. The head supporter is positioned to immobilize the tadpoles. Cover slips are overlaid on the tadpoles and attached to the acrylic plates using utility wax.
Figure 2. Visual stimulation controller
(A) A diagram of the visual stimulation setup. A controller (lower) is connected to LEDs on the plates that holds the animals (upper).
(B) The visual stimulation controller in operation.
(C) The pattern of the circuit board. The parts are soldered to the board. The electric parts are described in the Key resources table. Jumper lines (red) need to be soldered to the custom-ordered circuit board by users.
Figure 3. Electrodes for electroporation
(A) Design of the platinum electrode for electroporation. The anode is cut to a point for focal contact with the back of the eye and the cathode is larger for greater contact area with the front of the eye. The platinum plates are soldered to wire that is thick enough to hold the electrodes.
(B) The wires are attached to a 3 mm rod with a heat-shrink tubing (gray).
Figure 4. Visual stimulation strategy
Retinal ganglion cells (RGCs) on a retina cannot be stimulated independently. (A) Cartoon showing an attempt to stimulate two RGCs on a retina. (B) Drawing of retinal ganglion cells in frog (Cajal, 1972). RGCs extend dendrite over wide area in the retina. This induces unintended interaction of the two visual inputs within the retina. (C and D) To control visually driven activity in convergent RGCs, animals in which one tectal lobe is ablated are used, so both eyes innervate the remaining optic tectum. Each eye can be stimulated independently. The green and pink traces are activity in the tectal cells evoked by the green and pink retinal ganglion cells, respectively.
Figure 5. Electroporating the eye
An incision (red line) is made in the skin to insert the anode to contact the back of the eye. The cathode is placed on the skin in front of the eye to make a large contact area. Electroporate immediately after injecting the DNA solution. Make sure that the DNA solution is retained between the retina and the lens before electroporation.
Figure 6. Strategy to stimulate one eye
(A) Unilateral visual stimulation activates both eyes. Electrophysiological recordings from the left and right optic nerves and whole cell recording from neurons in the left and right optic tecta show responses in the retinal ganglion cell axons and the tectal cells respectively following unilateral visual stimulation (red LED and lines indicate visual stimulus). In tadpoles each eye projects axons primarily to the contralateral optic tectum. Stimulus timing is indicated with arrowheads. The light presented to one eye stimulates both eyes due to scattering and reflection.
(B) Masking light presented to one eye (red) prevents the indirect activation of the eye receiving the masking light and results in unilateral responses to the eye receiving the direct visual stimulation (black LED and lines indicate OFF visual stimulation).
(C) For repeated presentation of OFF stimuli, the LED has to be turned on for the next OFF stimulation; however even a stepwise increase in light intensity can indirectly stimulate the other eye (red circle).
(D) Gradually increasing LED intensity prevents the unintended indirect stimulation of the eye. In this example, the ramp of increased intensity was 11 s long. The recordings were collected in voltage clamp mode at −70 mV.
Figure 7. In vivo images of retinotectal axon arbors collected over a 2-day interval
The lefteye was stimulated 15 ms earlier (A and B) or later (C and D) than the righteye. The cell body of the labeled RGC is in the lefteye. DIC images of the tectum are superimposed. Day 0: start of visual stimulation. Two RGCs are labeled in the left panels. Scale bar, 100 μm. From Hiromoto and Cline (2020). The diagrams show the intensity of the LEDs during the visual stimulation. One cycle of the stimulus is 11 s. The visual stimulation continued for 10 h per day. The RGCs that were stimulated earlier and later developed their arbors in the rostral and caudal direction, respectively.
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