Neural Excitation of the Larval Firefly Photocyte: Slow Depolarization Possibly Mediated by a Cyclic Nucleotide

J. exp. Biol. (1976), 65, 213-327 213 ~Vith 10 figures xrinUd in Great Britain NEURAL EXCITATION OF THE LARVAL FIREFLY PHOTOCYTE: SLOW DEPOLARIZATION POSSIBLY MEDIATED BY A CYCLIC NUCLEOTIDE BY DONATA OERTEL AND JAMES F. CASE Department of Biological Sciences University of California at Santa Barbara, Santa Barbara, California 93106 (Received 3 February 1976) SUMMARY 1. In firefly larvae, extracellular recordings from the light organ nerve show that a volley of action potentials elicits a glow similar to the glow of an intact animal. 2. Intracellular recordings from the photocytes show that they respond to nerve stimulation with a slow, graded depolarization which precedes light emission. The depolarization begins about 0-5 s after the nerve is stimulated; it peaks about 1 s after stimulation; and subsides about 2^5 s after the stimu- lus. The glow increases fastest when the photocyte depolarization is at its peak and lasts 5-15 s. 3. Photocyte depolarization is associated with a decrease in the input resistance of the cell. 4. Adrenergic receptors in the light organ are pharmacologically similar to vertebrate a-receptors. 5. Phosphodiesterase inhibitors, aminophylline and theophylline, cause the light organ to glow, suggesting that cyclic nucleotides may mediate the effect of the adrenergic nerve transmitter. INTRODUCTION Two aspects of neural control of bioluminescence in the firefly larva have been in- vestigated using electrophysiological and pharamacological methods: (1) synaptic mechanisms of the nerve-photocyte synapse; (2) intracellular mechanisms controlling luminescence. Although the biochemical reactions leading to light emission in the firefly have been extensively studied (McElroy & De Luca, 1973; Bowie, Horak & De Luca, 1973; Bowie et al. 1973), little is known about the physiological role played by nerve impulses. Several lines of evidence indicate that light emission in adult light organs is indeed controlled neurally (Buck & Case, 1961; Case & Buck, 1963; Buck, Case & Hanson, 1963). In addition it has been shown that the innervation of adult and larval light organs is probably adrenergic (Smalley, 1965; Carlson, 1968 a, b). The morphology of the light organ of the larva is simpler than that of the adult (Peterson, 1970; Oertel, Linberg & Case, 1975; Smith, 1963). A mass of about 2000 photocytes, well tracheated and innervated, is covered by dorsal layer cells. The cell 214 DONATA OERTEL AND J. F. CASE layers are clearly distinguishable in vivo because the photocyte layer is transparent id contrast to the opaque, white, dorsal cell layer. Each light organ receives one nerve branch containing only two axons which branch to innervate the photocytes directly. The synapse between nerve and photocyte has been identified as an electron-dense, irregularly shaped, inward projection from the nerve cell membrane which is sur- rounded by light core vesicles (Oertel et al. 1975). Several features of the light organ of the firefly larva make it a useful physiological preparation, namely: (1) Light emission is easily measured; (2) the photocyte layer is homogeneous, containing only photocytes, small nerve terminals and small tracheoles; (3) photocytes are easily exposed for experimentation by removing a small patch of dorsal layer cells; (4) the synapse between nerve and photocyte can be studied both presynaptically by recording extracellularly from the nerve, and postsynaptically by recording intracellularly from the photocyte. MATERIALS AND METHODS General Larvae of Photuris pennsylvanica DeGeer were collected in the fall near Bethesda, Maryland and flown to California where they were maintained at 11 °C in Petri dishes lined with moist filter paper with a light cycle of 16 h light/8 h dark. Periodi- cally they were brought to room temperature, fed and cleaned (McLean, Buck & Hanson, 1972). The preparations on which electrophysiological recordings and pharamacological tests were made were similar. Larvae were decapitated and pinned on to a clear, Sylgard resin-filled dish, ventral side down. All subsequent procedures were done at x 62-5 magnification. The tergites were removed from all segments to expose the gut and fat bodies, which were then excised leaving the sternites, ventral musculature, light organs, and central nervous system (Fig. 1). For intracellular recording and for pharamacological tests a small hole about 75 /tm in diameter was made in the white, opaque, dorsal cell layer with an electrolytically sharpened tungsten pin, to expose the dorsal surface of the transparent mass of photocytes. If this is done carefully there is no background glow, as is characteristic of injured firefly light organs, and neurally excited glows do not change in shape or amplitude after the dissection. Extracellular recordings from the light organ nerve were made from the small branch just proximal to the site where the nerve enters the dorsal cell layer, a point which is conspicuous as a break in the dorsal cell layer. The preparations were bathed in physiological saline containing 90 mM-NaCl, 7 mM-KCl, 7 mM-CaCl2, 66 mM glucose, 1 mM Tris, pH 7-4. The proper concentrations of Na and K were determined by flame photometry of larval haemolymph. Blood was drawn into haematocrit capillary tubes and centrifuged. The serum was diluted in 116 mM Tris and the Na and K concentrations were then measured on an Eppendorf Flame Photometer. The optimal concentration of Ca2"1" was determined empirically and glucose was added to increase the osmolarity to 275 m-osmoles which was found to be optimal in electron microscopical fixation (Oertel et al. 1975). Neural excitation of the larval firefly photocyte 215 Int. Ext. VIII abd. seg. Tail Fig. i. A diagram of the dissected firefly larva illustrating the intracellular recording arrangement from photocytes on the left and extracellular recording arrangement from the light organ nerve on the right. The light organs lie on the sternite of the eighth abdominal segment just anterior to the tail. The last of the chain of ganglia has a pair of nerves which leave the ganglion ventrolaterally, recognizable by their association with tracheae, and run posterior to the mid line of the eighth abdominal segment. One branch of this pair of nerves inner- vates each light organ. Its point of entry into the light organ can be seen as a break in the dorsal cell layer. To record intracellularly from photocytes, a patch of dorsal layer cells was cleared from the underlying photocyte layer so that the electrode approached the mass of photocytes dorsally, as shown on the left part of the diagram. A suction electrode was used to record from the light organ nerve just proximally to its entry into the light organ as shown on the right. In either recording condition, suction electrodes were used to stimulate the light organ through the ipsilateral segmental nerve (S). 216 DONATA OERTEL AND J. F. CASE Photometric measurements Light emission from the preparation was monitored with an EMI Type ' S' photo- multiplier tube powered by a Fluke model 409 A high voltage d.c. power supply. Electrophysiology Extracellular recordings were made from the light organ nerve using glass suction electrodes of about 20 fim tip diameter, and a Grass P 5 a.c. coupled preamplifier. A second suction electrode was used for stimulation. Eserine, IO~3M, made up in physiological saline and applied to the eighth abdominal ganglion for short periods, was used in some experiments to cause the nerve to fire spontaneously (Case & Buck, 1963; Smalley, 1965). Intracellular recordings from photocytes were made using standard techniques with a WP Instrument, model M-4 A Electrometer. Microelectrodes were filled with 3 M-KC1 and had resistances of 60 MD. or more. Pharmacology For pharmacological studies the eighth abdominal segment was cut at the mid line so that only one light organ of each pair was used, to avoid recording amgibuities. The dorsal cell layer was pierced and a few photocytes were exposed in order to reduce complications due to diffusion problems. Drugs were dissolved in larval firefly saline and were delivered to the light organ through fine polyethylene tubes with syringes. The chamber had a volume of 0-3 ml. To test relative effectiveness of agonists, the sensitivity of a single light organ to two or three drugs at equal concentrations was compared. Saline containing the dissolved drugs was added to the preparation in the dark. The solution was removed completely and the preparation was rinsed one or more times before the following test. The system allowed solution changes to be completed in 10 s. Since the sensitivity of the light organ changed with time, tests and controls were repeated several times during an experiment. To test a putative blocking agent, its effectiveness in reducing a neurally stimulated glow was measured using a preparation similar to that just described, but with the segmental nerve severed from the ganglion. The distal nerve stump was stimulated through a suction electrode with pulses of 5-15 V, 50 ms duration, at constant voltage and at regular intervals of about 45 s. The effectiveness of some agonists was tested by local application to the dorsal surface of the mass of photocytes. One microlitre of saline containing a drug was delivered over about 10 s to the exposed photocytes through a glass pipette with a tip diameter of 15-25/an, positioned with a micromanipulator. A microinjection apparatus was used to infuse the test solution slowly to avoid mechanical disturbance. The following chemicals were used in this study: adenosine 3':5'-cyclic mono- phosphoric acid, L-epinephrine, DL-epinephrine, guanosine 3':5'-cyclic mono- phosphoric acid, DL-isopropylnorepinephrine (DL-isoproterenol), L-norepinephrine (L-arterenol), DL-norepinephrine HC1 (DL-arterenol HC1), DL-octopamine, L-phenyl- ephrine, DL-synephrine (Sigma); aminophylline (Schwarz/Mann); D-amphetamine Neural excitation of the larval firefly photocyte 217 B 5s Fig. 2. Extracellular recordings from light organ nerve showing that several impulses in close succession precede glowing with a constant latency.

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