Structure and Function of Pedal Neurons Controlling Muscle Contractions in Tritonia Diomedea

Page 1 of 16 Impulse: The Premier Undergraduate Neuroscience Journal 2012 Structure and function of pedal neurons controlling muscle contractions in Tritonia diomedea Reakweeda Zazay1,2,3, Josh B. Morrison2,4, Roger Redondo2,4, James Alan Murray1,2,4 1California State University East Bay, Hayward CA 94542; 2Friday Harbor Laboratories, Friday Harbor WA 98250; 3George Washington University, Washington DC 20052; 4University of Cen- tral Arkansas, Conway AR 72035 There are 16 pairs of “identified neurons” in the pedal ganglion of any sea slug of the species Tritonia diomedea that have published behavioral functions. Many of the pedal neurons cause flexion of the ipsilateral body wall and foot when activated, but they are not thought to innervate muscle directly. The goal of this study was to examine the motor functions of brain neurons with no previously-identified functions. We described the activity of two such cells and their motor effects, and further characterized the motor effects of a previously-identified neuron (Pedal 3). We stimulated the Pedal 3 flexion neuron and characterized where and how it contracted the foot. For each neuron, we described the latency to contraction, the time to relaxation, and the dis- tance and speed of movement. These neurons may be involved in turning during crawling, and these results will help us understand how the activity of specific neurons is translated into behavior (neuromechanics), and determine how fast the animal can respond to sensory feedback during locomotion. The relative simplicity of this brain allows us to understand how behavior is gener- ated on a cellular basis, and to generate neural network and neuromechanical models of naviga- tion that can be applied to robotics. Abbreviations: SFA – spike frequency adaptation; Pd – Pedal neuron (identified neuron); PdN – pedal nerve (identified nerve) Keywords: motor control; turning; identified neuron; neuroethology Introduction tion of muscles that may change the amount The marine gastropod Tritonia dio- of cilia contacting substrate on a given side medea (Figure 1A) is a model organism for of the animal (Murray et al., 2006). The neu- studying the neural control of behavior, as it rons that elicit movement are referred to as has giant, re-identifiable brain cells (neu- “flexion neurons” because they do not con- rons) that have been extensively studied for tact muscle directly, as would a true motor more than forty years (Willows, 1967). The neuron (Hoyle and Willows, 1973). Many of slug has a relatively simple and limited the larger neurons have been identified visu- amount of behavioral responses to environ- ally, physiologically, functionally, and are mental stimuli, such as predator, prey and morphologically mapped in the brain (Wil- conspecific odors, water flow and the geo- lows et al., 1973a; Hume et al., 1982; Mur- magnetic field (Willows, 1999, Wyeth et al., ray et al., 1992; Cain et al., 2006; Neuron- 2006; Murray et al. 2011). T. diomedea have Bank). Willows et al. (1973) published a a cilia-covered foot, and crawl by beating four-paper series describing structure and these cilia against the substrate; it is sus- function of the brain, nerves, and neurons of pected that no muscles are involved in this T. diomedea, and categorizing several “iden- process (Audesirk, 1978). However, we hy- tified neurons.” They identified 11 neurons pothesize that turning involves the contrac- in the pedal ganglion, and Page 2 of 16 Impulse: The Premier Undergraduate Neuroscience Journal 2012 Figure 1: Photographs and diagrams of the sea slug Tritonia diomedea, and its brain. A. T. diomedea is a nudibranch gastropod that crawls using ciliary beating and uses asymmetrical contractions of the muscular foot to turn during crawling. B. Experimental setup (Drawing from Willows AOD et al., The neuronal basis of behavior in Tritonia I. functional organization of the central nervous system. Journal of Neurobiology 4: 204-237, 1973. Used with permission, License Number 2707211025474). C. Dorsal view of the brain of Tritonia showing fused cerebral, pleural, and pedal ganglia, comprising ~7000 neurons. Each of the orange circles is the soma of a single brain cell, and the white nerves radiate out to all part of the body. D. The brain has six ganglia, and the left and right pedal ganglia contain ~1000 neurons each and most of them send axons out of Pedal Nerves (PdN 1-4) to relay motor commands to the foot muscle, cilia, and other targets. determined the motor functions of three (Pe- (Pd21 by Audesirk 1978; Pd5 by Popescu dal neurons 1, 3, and 8; a.k.a. Pd1, Pd3, and Willows, 1999), and the remaining two Pd8). Hoyle and Willows (1973) noted that (Pd6, Pd7) are presumed to be ciliary motor these pedal neurons likely do not directly neurons (Wang et al., 2003, 2004; Cain et innervate muscle fibers, but instead work al., 2006), but definitive evidence is not yet through a peripheral nerve net that includes published. local motor neurons, and this results in de- In some of these experiments we lays in contraction. were looking to identify un-mapped neurons Six of the others (e.g. Pd5, Pd6, Pd7, that were involved in movement of the slug. Pd20, Pd21, Pd22) elicited no obvious motor Identified neurons in the pedal ganglia con- effect, though two of those neurons were tract the ipsilateral side of the foot and body later determined to be ciliary motor neurons wall when active (Willows et al., 1973a), Page 3 of 16 Impulse: The Premier Undergraduate Neuroscience Journal 2012 contributing to turning (Redondo and Mur- Brain cell recording ray, 2005). Pedal neuron 3 (Pd3; Figure 2A) Figure 1C shows a picture of the is a previously-identified unipolar neuron brain underneath a microscope with the out- that sends an axon into Pedal Nerve 3 er sheath removed from all ganglia and the (PdN3), which projects to the foot and lat- epineurium intact. The orange circles are eral body wall (Figure 1). This neuron has single brain cells, some of which can be several neurites that extend into the neuropil identified visually in any individual (by col- of the ganglion, and are presumed to be or, size, and position in the ganglion). We dendritic inputs since no neurons have been identified the Pd3 neuron in five slugs by shown to receive synaptic input from this position in the ganglion, size, color, sponta- neuron in the isolated brain. In this study, neous activity and motor effect (Murray et we characterized the location, extent, and al., 1992), and we characterized two new speed of contractions caused by activation of cells in one slug each when the cells did not an identified neuron (Pd3), and two newly- match published criteria. We visually- identified neurons. guided sharp glass microelectrodes to pene- trate the epineurium above a haphazardly- Material and Methods selected neuron. We characterized only one neuron per pedal ganglion. We made elec- Animals & surgery trodes with a Sutter P-97 puller and thin- We collected T. diomedea via SCU- walled glass (A-M Systems, 1.0 OD, 0.75 BA near Dash Point, Federal Way, Wash- ID) to have a resistance of 5-20 MΩ when ington. We kept the slugs at the Friday Har- filled with potassium acetate. We used elec- bor Laboratories in sea tables that had flow- trodes with the tip backfilled with the nega- ing sea water. We used seven slugs in this tively-charged fluorescent tracer AlexaFluor study. We exposed the brain for recording hydrazide 488 or 568 nm (Invitrogen, Grand while it was intact and connected to the Island, NY), or Lucifer Yellow (Vector body (a “semi-intact” preparation, see Fig- Labs, Burlingame, CA), to impale a cell, and ure 1B) by its many nerves (Figure 1C, D). the resistance was then between 20-50 MΩ. We made a one-inch incision on the dorsal We first inhibited the cells from firing by body wall above the brain. We placed the injecting them with negative DC current. animal in an acrylic tank and kept open the Once the cell stopped firing, we injected it incision with non-magnetic hooks. We made with positive current via a bridge amplifier an incision in the next layer of connective (A-M Instruments, Sequim, WA, Neuro- tissue posterior to the brain, and inserted a probe model 1600) to induce and record its non-magnetic wax-covered platform ventral firing activity. The number of trials varied to the brain. We pinned the surrounding from 10-50 for each neuron. connective tissue onto the wax to immobi- lize the brain. We cut the connective tissue Behavioral recording & analysis above the pedal ganglia (“outer sheath”) to We simultaneously recorded video of expose the tight-fitting epineurium (“inner the resulting movements (spike data were sheath”), which is the last layer of connec- recorded with a PowerLab 26T, Colorado tive tissue above the nerve cells in the pedal Springs, CO), and video was recorded with a ganglia of the brain (Figure 1D). Panasonic firewire camcorder and synchro- nized using LabChart v.6 (ADInstruments, Colorado Springs, CO), displayed on a com- puter monitor and recorded to a hard drive. Page 4 of 16 Impulse: The Premier Undergraduate Neuroscience Journal 2012 Movement of the body was monitored in 0.1M PBS twice for 30 min intervals, then real-time with a photovoltaic sensor (MTI dehydrated with 50%, 70%, 90%, 95%, Fotonic, Albany, NY, model 1000) placed 1- 100%, 100%, and 100% EtOH for 30 min 3 cm from the skin. This device measured each. The brain was then put in methyl sa- movement by detecting decreases in light licylate to clear the tissue for microscopy, reflection as the slug moved away from the and mounted between two cover slips using probe and put out a voltage proportional to paper clips as spacers for the ~1 mm thick reflected brightness.

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