Introduction•Chemoreception, magnetoreception, and water flow detection influence navigation and locomotive behaviors in the heterobranch sea slug Tritonia tetraquetra. •A factor that does not affect their navigation is vision. •However, Tritonia are still able to detect light – electrophysiological recordings from the optic nerve show activity of 5 photoreceptors in response to light [1, 2]. •In the similar species Hermissenda crassicornis, GABA and serotonin (5-HT) modulate photoreceptors [3] and the eyes synthesize and release acetylcholine (ACh) [4, 5]. •Research on Hermissenda’s photoreceptors has provided a molecular and neurobiological understanding of how locomotive learning takes place. •A current structural, compositional, and functional description of Tritonia’s eyes does not exist.•This project aims to explore the structural composition of Tritonia’s eyes and hopefully lead to finding a nexus between vision and locomotive behaviors.

Research MethodImmunohistochemistryThe eyes underwent a series of soaking and rinsing in chemical labels, as antibodies, and were mounted onto glass coverslips in mounting medium. The eyes were labeled with antibodies for serotonin (5-HT), GABA, 5-HT receptor, SCPb, FMRF-amide, and the enzyme that synthesizes ACh, choline acetyltransferase (ChAT). An antibody that labels β-tubulin, a scaffolding protein of neurons, was also used. To detect fluorescence under the microscope, the eyes were exposed to goat anti-rabbit and goat anti-mouse antiserum conjugated to fluorescent molecules that fluoresce under 488 nm and 555 nm lasers.

Confocal Microscopy The prepared eye tissue was visualized fluorescently with a Leica confocal laser scanning microscope (Leica TCS SP8) using 20X and 40X objectives.

3D images of the eyes were made through compiling z-stacks, the sum of scans through every layer in the z-plane.

AcknowledgmentsConfocal imaging was made possible by the W.M. Keck foundation. We would also like to thank Samantha Zacharias and Kelsey Wallace for assisting in tissue dissection.

ConclusionsOur results strongly suggest that Tritonia’s eyes contain ChAT. The presence of ChAT would indicate that the eyes synthesize ACh. The uneven distribution of ChAT inside the eye may denote that individual photoreceptors contain ChAT. We’ve tentatively identified four photoreceptors.

β-tubulin immunoreactivity reveals five axons adjacent to the eye. The optic nerve is established by the meeting of photoreceptor axons inside the eye.

Crystal A. Vardakis1 and James A. Murray1

1Biology Department; crystal.vardakis@csueastbay.edu, james.murray@csueastbay.edu

Literature cited1. Chase, R., The electrophysiology of

photoreceptors in the nudibranch mollusc, Tritonia diomedia. J Exp Biol, 1974. 60(3): p. 707-19.

2. Chase, R., The initiation and conduction of action potentials in the optic nerve of Tritonia. J Exp Biol, 1974. 60(3): p. 721-34.

3. Crow, T. and N.G. Jin, Chapter 19 - Multisite Cellular and Synaptic Mechanisms in Hermissenda Pavlovian Conditioning, in Handbook of Behavioral Neuroscience, M. Randolf and R.B. Paul, Editors. 2013, Elsevier. p. 236-250.

4. Heldman, E., et al., Cholinergic features of photoreceptor synapses in Hermissenda. J Neurophysiol, 1979. 42(1 Pt 1): p. 153-65.

5. Heldman, E. and D.L. Alkon, Neurotransmitter synthesis in the nervous system of the mollusc Hermissenda. Comp Biochem Physiol C, 1978. 59(2): p. 117-25.

Fig. 1. Laser scanning confocal mircoscopic 3D image of a vertically positioned eye immunoreactive for ChAT and β-tubulin.

Fig. 2. Laser scanning confocal mircoscopic 3D image of a horizontally positioned eye immunoreactive for ChAT and β-tubulin.

Fig. 1. The eye is immunoreactive for ChAT and β-tubulin. The sums of all z-stacks produced a 3D construct of the eye at 40X and 488 laser, 555 laser, and transmitted light. A) ChAT immunofluorescence was detected with the 488 laser and is presented as green. B) β-tubulin immunofluorescence was detected with the 555 laser and is presented as red. C) The overlay image of A, B and the eye under transmitted light (not shown) reveals that this eye was positioned vertically in the mounting medium.

Fig. 2. The eye is immunoreactive for ChAT and β-tubulin. The sums of all z-stacks produced a 3D construct of the eye at 40X using the 488 laser, 555 laser, and transmitted light. A) ChAT immunofluorescence was detected with the 488 laser and is presented as green. B) β-tubulin immunofluorescence was detected with the 555 laser and is presented as red. White arrows point to axons comprising the optic nerve. C) The overlay image of A, B and the eye under transmitted light (not shown) reveals that this eye was positioned horizontally in the mounting medium.

Fig. 3.

Fig. 3. Tritonia in its natural environment. Its eyes can’t be seen here because they are embedded inside of its body.

Fig. 4. Fig. 4. Tritonia’s brain and eyes during a dissection. The eyes are 250 μm in diameter [1] and look like small black dots. Its brain is between the blue brackets.

ChAT β-tubulin ChAT, β-tubulin, transmitted light

ChAT β-tubulin ChAT, β-tubulin, transmitted light

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