Symmetrically Organized Dorsal Unpaired Median (Dum) Neurones and Flash Control in the Male Firefly, Photuris Versicolor by Thomas A

r exp. Biol. (1981), 93. I33"i47 133 Hbt/i 8 figures Printed in Great Britain

SYMMETRICALLY ORGANIZED DORSAL UNPAIRED MEDIAN (DUM) NEURONES AND FLASH CONTROL IN THE MALE FIREFLY, PHOTURIS VERSICOLOR BY THOMAS A. CHRISTENSEN AND ALBERT D. CARLSON Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York 11794 USA

(Received 1 October 1980)

SUMMARY 1. Male fireflieso f the species Photuris versicolor produce a species-typical triple-pulsed flash which is used as a courtship signal. The neural anatomy was examined to determine if this complex behaviour could be attributed to the organization within the central nervous system. 2. The lantern is innervated primarily by the two most posterior abdominal ganglia. Bilateral roots from these ganglia form a symmetrical pattern of innervation to both sides of the lantern tissue. With minor exceptions, this pattern is similar to that described for other firefly species. 3. The neural organization within the lantern ganglia was determined by back-filling the roots with cobalt or Lucifer Yellow CH, and then examining the ganglia in whole mount. Clusters of three or four large dorsal unpaired median (DUM) neurone somata, each sending bilateral processes out of the lantern roots, were found in both lantern ganglia. 4. The DUM neurone axons bifurcate several times and ramify throughout the dorsal surface of the lantern tissue. More than one DUM neurone may innervate a particular region of photogenic tissue. 5. When dye was back-filled into peripheral branches of the lantern roots that do not innervate photogenic tissue, no DUM somata were stained. Instead, the fibres that filled carried the dye anteriorly up the nerve cord through the ipsilateral connective. No fibreswer e observed to cross the ganglion midline or exit from the contralateral root, nor were any fibres stained in the contralateral connectives. 6. DUM neurones within the lantern ganglia have resting potentials between 30 and 45 mv and they exhibit multiple, as well as single-peaked spontaneous action potentials. The presence of multiple spikes might reflect the special bilateral morphology of these neurones. 7. The lantern nervous system is organized in an arrangement capable of synchronizing the excitation of all the lantern photocytes. This neural organization could aid in the control of the complex flash pattern displayed by male Photuris versicolor fireflies.

INTRODUCTION The firefly flash is a rapid burst of light used as a courtship signal, and each species firefly produces a unique flash exchange pattern (Lloyd, 1966). Male Photuris I 'eflies, in particular, produce complex flashes by rapid variations of flash intensity 134 T. A. CHRISTENSEN AND A. D. CARLSON

Fig. i (A). Relationship between electrical activity in the lantern tissue and the dynamics of a P. verticolor male spontaneous triple-pulsed flash. Upper trace: photomultiplier output of light emission. Lower trace: spike activity recorded via external electrodes in the anterior lantern segment. Temp: 22 °C. Time marker: 200 ms. (B) Gross innervation of the lantern (dorsal view). The two large lantern organs (shaded) completely cover the sternitea of the 6th and 7th abdominal segments. The lantern tissue is innervated by paired roots from the 5 th, 6th and 7th abdominal ganglia. These roots break up into a network of smaller fibres (not illustrated) that densely innervate all the photogenic tissue. Note also that intersegmental tracts link adjacent roots in the periphery. The four genital roots from A 7 are cut short in this diagram. Scale marker: 1 mm.

(Barber, 1951), and these modulated flashes are recognized by prospective mates of the same species during courtship (Nelson, Carlson & Copeland, 1975; Zorn & Carlson, 1978). The P. versicolor male typically emits a triple-pulsed flash which appears as a twinkle to the human observer (Fig. 1 A). The rhythmic flash patterns of many fireflies,includin g P. versicolor, are believed to be controlled by a neural 'pacemaker* within the animal's brain(Case & Buck, 1963; Bagnoli et al. 1976). Bursts of action potentials travel down the segmental nerve cord and by some means activate the photocytes (light-producing cells) of the lantern. These action potentials can be recorded by external electrodes in the lantern tissue (Case & Buck, 1963; Fig. 1 A). In P. versicolor males, the neural volley activates both lantern segments simultaneously, resulting in three rapid pulses of light from the photogenic tissue. Although Chang (1956), and later Buck & Case (1961) demonstrated that the adult firefly lantern behaves like a typical neuroeffector such as striated muscle, we still do not fully understand how neural activity initiates luminescence in the photocytes (Case & Strause, 1978), which are actually derived from fat cells (Hess, 1922). The gross neural anatomy of the lantern was originally described by Hanson (1962). The lantern, which occupies the sternites of the sixth and seventh abdominal segments, is innervated by nerve roots from the fifth, sixth and seventh abdominal ganglia (A 5, A 6 and A 7, respectively), the latter two ganglia lying dorsal to the anterior lantern segment (Fig. 1B). Hanson (1962) showed that it was possible to deganglionate the posterior lantern segment by severing the connexions with its more anterior ganglion, A 7. The adult lantern ultrastructure was first described for a Photinus species by Beaim & Anderson (1955), and later for Photuris by Kluss (1958) and Smith (1963). DUM neurones and flash control in the firefly 135 Prganized into a ventral photogenic layer and an overlying reflector layer. Trachea and nerves plunge ventrally into the photogenic tissue forming a uniform array of tracheal cylinders. Tracheal processes, enclosed in tracheolar cells, project horizontally away from the tracheal cylinders, through specialized, mitochondria-filled tracheal end cells, and eventually reach the photocytes which are situated in a rosette pattern around the tracheal cylinders. Nerve axons split away from their surrounding sheath cells and terminate in pad-like endings between the tracheal end cells and tracheolar cells. Synaptic specializations can be seen between the vesicle-filled nerve endings and tracheolar cells, and it is believed that these cells transmit the excitation to the photocytes (Case & Strause, 1978). Several lines of evidence indicate that the phenylethylamine octopamine could function as a neurotransmitter in the lantern. It has been shown that this amine is extremely effective in eliciting lantern luminescence (Carlson, 19686), and that substantial amounts are found in the lantern segments (Robertson & Carlson, 1976). More recently, octopamine has been found to activate an octopamine-sensitive adenylate cyclase in the lantern which catalyses the production of cyclic AMP (Nathanson, 1979). It is suggested that activation of the photocyte-end-cell complex could occur through the actions of this second messenger, but the details of activation remain unresolved. From this description it is clear that we understand a great deal about the neural and physiological control of firefly flashing, but the anatomical organization within the central nervous system, through which the triggering bursts from the brain pass to the lantern, remains unexplored. In this article we report the discovery of distinct populations of large dorsal unpaired median (DUM) neurones within the lantern ganglia that appear to perform a key function in conducting the neural bursts from the brain to the photocytes of the lantern. Moreover, these neurones are organized to ensure the synchronous activation of the thousands of photocytes in both lantern segments. To further define the functional significance of these neurones, we have monitored their spike activity and identified them anatomically using intracellular recording and dye- marking techniques. The results presented here match the expected results, given the geometry of the neurones and their proposed function. In short, these cells appear to be both physiologocally and anatomically suited to an important role in the production of light by the photocytes. A preliminary report of these results has appeared elsewhere (Christensen, 1980).

METHODS Organisms Adult male fireflies (Photuris versicolor) were obtained from a tree-lined grassy field near Stony Brook, Long Island, New York. Males displaying their species-specific triple-pulsed flash were lured out of the trees with a single flash from a flashlight pointed into the grass, which served to mimic the typical female response to the male's flash. After capture, the fireflies were kept in glass or plastic containers at room tem- perature with moist paper-towelling and grass. All were dissected within a few days after capture. 136 T. A. CHRISTENSEN AND A. D. CARLSON

Anatomical methods The gross morphology of the lantern nervous system was examined by two methods. To locate the destinations of the lantern ganglia roots, the abdominal dorsal cuticle was removed and the exposed ventral nerve cord and lantern were stained with methylene blue dye (1%, w/w, in saline; Hanson, 1962). The finer peripheral nerve processes were traced by back-filling one lantern ganglion root with cobaltous chloride (6%, w/w, in distilled water, containing o-i% bovine serum albumin), and allowing the cobalt to be carried into the ganglion and out again through the contralateral nerve root to the photogenic tissue. Subsequently, the preparation was rinsed with ammo- nium sulphide which reacts with the cobalt to form a black precipitate (Pitman, Tweedle & Cohen, 1972; Strausfeld & Obermayer, 1976). The organization within the lantern ganglia was determined by back-filling the ends of select peripheral branches of the lantern ganglia roots with cobaltous chloride as previously described, or with the highly fluorescent dye Lucifer Yellow CH (5%, w/w, in distilled water; Stewart, 1978). Cobalt back-fills of lantern nerves for 24 h at 5 °C and Lucifer Yellow CH back-fills for 24 or 48 h at 5 °C were required to elucidate the neural organization within the lantern ganglia. Whole mounts of cobalt-filled nerve cords were prepared by fixing in Carnoy's fluid, dehydrating in ethanol, clearing in xylene, and mounting on a slide with Per- mount. Whole mounts of Lucifer Yellow CH-filled nerve cords were prepared by fixing with 4% formaldehyde in o-i M sodium phosphate buffer (pH 7-4; Stewart, 1978), dehydrating in ethanol, clearing in xylene, and mounting on a slide with Fluormount (Edward Gurr, Ltd). Camera lucida drawings were made of slide-mounted specimens. Measurements were made using an eyepiece grid in conjunction with a stage micrometer. To test for the presence of monoamine-containing neurones within the lantern ganglia, excised nerve cords were stained for 24-48 h at 5 °C with a o-1 % (w/w) solution of the vital dye, Neutral Red (Sigma Chemical Company; Stuart, Hudspeth & Hall, 1974), in firefly saline (Carlson, 1968a). Whole mounts were prepared by fixing the stained nerve cords in 10% formalin, destaining in ethanol, clearing in xylene, and mounting on a slide with Permount, Unless otherwise specified, all chemicals used in this study were obtained from Fisher Scientific Company. Lucifer Yellow CH was the generous gift of Walter W. Stewart.

Physiological techniques Extracellular The spontaneous neuronal potentials were recorded with paired electrodes of insulated copper transformer wire (80 //.m diam.) pushed through the ventral cuticle into the photogenic tissue of the anterior lantern segment. The electrodes were con- nected to a Grass P15 a.c. pre-amplifier, the output of which was fed into one channel of a Tektronix D 10 storage oscilloscope and also into one channel of a Hewlett- Packard instrumentation tape recorder. The animal was standing upright attached by its dorsal surface to a wax tether and restrained by wires around its body. Its legs wem positioned on another wax platform which allowed its posterior lantern to project fr DUM neurones and flash control in the firefly 137 ILantern flashes were conducted via light pipe to a photomultiplier, and its output was monitored on a second oscilloscope channel and recorded on an FM tape recorder channel. Oscilloscope displays were photographed with a Grass C4 oscilloscope camera.

Intracellular Glass pipettes used for monitoring spike activity were one of two types: either Omega Dot (1 o mm O.D., Glass Company of America) or Ultra-Tip (10 mm O.D., Frederich Haer Co.). The micropipettes were pulled on either an M-i Micropipette Puller (Industrial Science Associates, Inc.) or a Brown-Flaming Type (Sutter Instrument Co.). They were filled with either a solution of 27 M-KC1 and 150-200 mM-CoCl2 or a 4% (w/w) solution of Lucifer Yellow CH, with resistances of 25-50 MCI (Omega Dot) or 5-15 M fi (Ultra-Tip). For some recordings, due to the tough glial sheath surrounding the firefly ganglion, the micropipettes were bevelled to between 85 and 95% °f their original resistances using a K. T. Brown Type Model BV-10 Micro- electrode Beveler (Sutter Instrument Co.). Intact (non-decapitated) male fireflies were pinned dorsal side up to a Sylgard- filled dish (Dow Corning). The dorsal cuticle over the lantern was removed to expose the underlying ventral nerve cord, and the preparation was immersed in firefly saline. A small plastic spatula was guided with a micromanipulator underneath the ganglion to be studied. Once in position, a fibre-optics light pipe from a Narishige MEI-i Micro Electrode Illumination System was guided to the ganglion with another micromanipulator. Under the proper conditions, and by virtue of their large size (30-75 fim), the DUM somata could easily be seen under a dissecting stereomicroscope (Zeiss). Electrical activity from the DUM somata was amplified through a W.P.I. Model M-707 Micro-Probe System electrometer equipped with capacity compensation. Spikes were monitored on a Tektronix D 13 storage oscilloscope and simultaneously recorded on a Hewlett-Packard 7404 A chart recorder or a Hewlett-Packard four- channel FM tape recorder. To positively identify the cells after recording, dye was injected iontophoretically into the DUM somata using the same recording electrode with the aid of a bridge circuit built into the electrometer. Typical stimulating para- meters used were depolarizing pulses of 50 nA, 0-5 s duration at 1 Hz for 1-2 h for CoCl2 (Pitman, Tweedle & Cohen, 1972), and hyperpolarizing pulses of 20 nA, 1.0 s duration at 0-5 Hz for 10-30 min for Lucifer Yellow CH (Stewart, 1978).

RESULTS 1. Gross neural anatomy of the lantern nervous system The male firefly lantern occupies the ventral sclerites of abdominal segments 6 and 7 (Fig. 1 B) and its neural organization is similar to that described by Hanson (1962). Abdominal ganglia 5, 6 and 7 provide motor nerves which innervate the lantern tissue. While A 6 and A 7 supply the bulk of the motor innervation, A 5 innervates only a small area of the anterior lantern segment (Hanson, 1962; Fig. 1B). Each of the lantern ganglia sends out a single root on either side which ramifies T. A. CHRISTENSEN AND A. D. CARLSON

Fig. 2. Camera lucida drawings of six cobalt-filled preparations showing the relative sizes and positions of the large DUM cells within A 7. Letter within each ganglion refers to which root was filled. R = right root; L = left root. Scale bars: 100 /im. into a dense network of branches that spreads out over the photogenic tissue. Further- more, there is a similar pattern of innervation to both the right and left sides of the lantern (Hanson, 1962; Fig. 1 B).

2. Neural organization of the lantern ganglia Seventh abdominal ganglion (A 7)

Cobaltous chloride (CoCl8) back-fills of all branches of the two roots from A 7, except those branches not innervating the lantern, reveal a cluster of four large dorsal unpaired median (DUM) somata ranging in size from 30 to 75 fim in diameter (Fig. 3). All four lie apposed in a diamond-shaped array on the dorso-medial surface of the ganglion. Fig. 2 is a set of camera lucida drawings of six CoCl2 back-filled preparations of A 7 showing the relative sizes and positions of these cells. Back-fills of a given root not only fill the axons and somata of these large neurones, but cobalt diffuses into their contralateral axons as well (Fig. 3). Each of the four large DUM somata sends out a single neurite anteriorly, and all four neurites bifurcate in the same region to form a T-shaped juncture. From here, each neurite gives rise to two bilaterally symmetrical axons which exit to the periphery through their respective roots. Before exiting the ganglion, the four bilateral axon pairs take different p Journal of Experimental Biology, Vol. 93 Fig-3

Fig. 3. Dorsal view of A 7 which was back-filled with CoClj through the left lantern root. Four large dorsal unpaired median (DUM) somata are filled, each giving rise to a bifurcating neurite. Bilateral axons from each neurite exit to the periphery through both roots and innervate the posterior lantern segment. Scale bar: 100 /im.

A. CHRISTENSEN AND A. D. CARLSON (Facing p. 138) Journal of Experimental Bio fogy, Vol. 93

Fig. 4. Two views of A 6 in which the right root (white arrows) was back-filled with Lucifer Yellow CH. Three large DUM somata filled, each giving rise to a neurite which bifurcates forming a T-shaped juncture (black arrows). From here, bilaterally symmetrical axons leave the ganglion through both roots. (A) Dorsal view of A 6 showing the somata clustered toward the posterior edge of the ganglion. (B) Dorso-lateral view of A 6 showing the somata situated in a row on the midline. The auto-fluorescent strands on either, side of the ganglion are trachea. Scale bars: ioo fim.

T. A. CHRISTENSEN AND A. D. CARLSON DUM neurones and flash control in the firefly 139 Pirough the neuropile. Usually two of these pairs run directly toward the roots, while the other two pairs proceed anteriorly before exiting the ganglion. Although the significance of this divergence is not immediately apparent, it is certain that each of the DUM neurones innervates both the right and left sides of the seventh abdominal segment Back-fills of a single root also reveal smaller somata, 10-15 Z"11 m diameter, which cluster around the region where the root arises from the ganglion. These cells are certainly not bilateral, nor are they situated on or near the dorsal midline. In addition to demonstrating the neuronal structure within A 7, CoCl2 back-filled into the roots of A 7 stains fibres that direct the dye anteriorly through the interganglionic connectives (Fig. 3). These fibres and their destinations are currently under investigation. Sixth abdominal ganglion (A 6)

Although back-fills of lantern ganglia roots with CoCl2 or Lucifer Yellow CH in A 6 do not reveal results as consistent as those of A 7, a number of striking similarities are found within the two ganglia. Back-fills of all nerve root branches, except those that do not innervate photogenic tissue, reveal three (unlike A 7) closely clustered DUM somata, each ranging in size from 40 to 70 /on in diameter. As found in A 7, each of the large neurones sends bilateral axons out both sides of the ganglion (Fig. 4). Each soma sends out a single neurite anteriorly which bifurcates into two bilaterally symmetrical axons. All three neurites bifurcate at a common point in the neuropile, which is always marked by an aggregation of dye. Unlike A 7, however, the positions of the DUM somata within A 6 vary slightly from one preparation to the next. The somata either form a cluster toward the posterior-medial edge of the ganglion (Fig. 4 A), or they form a linear array on the midline (Fig. 4B). Although these small positional differences occur, the three DUM somata within A 6 are by far the most easily recognized cells in the ganglion. Back-filling lantern roots with either cobalt or Lucifer Yellow CH reveals several other morphological similarities between A 6 and A 7. When either root is back-filled, smaller somata, 10-15 fim in diameter, appear on the lateral edges of A 6 where the roots leave the ganglion. These cells are certainly not DUM neurones, but their possible function in lantern luminescence will be discussed later. Also, dye back- filled into either lantern root travels anteriorly through fibres in the interganglionic connectives. We have observed no fibres in the posterior interganglionic connectives when dye is back-filled into a lantern root of A 6.

Fifth abdominal ganglion (A 5) Only a small region of the anterior lantern is innervated by A 5. This ganglion as well as every other abdominal ganglion in the firefly contains DUM cells, but they are no larger than the smallest DUM somata found in A 6 and A 7, there are fewer of them in a given ganglion and the positions of the somata vary considerably from one preparation to the next. For now we will direct our attention to the two ganglia providing the bulk of the lantern innervation, A 6 and A 7. A thorough investigation of A 5 and other ganglia will be covered at another time. 140 T. A. CHRISTENSEN AND A. D. CARLSON

Fig. 5. Camera lucida drawing of the two lantern segments (dorsal view) illustrating the bilateral pattern of innervation from the last two abdominal ganglia, A 6 and A 7. CoClt was back-filled into the right root of A 6 and the left root of A 7. Double broken lines represent a peripheral branch of the left root of A 6 which contains no DUM cell axon and which corresponds to the Lucifer Yellow-filled root shown in Fig. 6. Scale marker: 500 fim. Inset 1. Enlargement of the area at (1) highlighting the extensive branching of the DUM axons innervating the anterior lantern segment. Some axons exhibit marked cytoplasmic swel- ling at their points of bifurcation. Scale bar: 50 /im. Inset 2. Enlargement of the area at (2) highlighting three DUM axons from A 7 innervating the posterior lantern segment. A fourth axon separates from the others to innervate the upper right portions of the segment. The three larger axons travel together within the root until they reach a point where each splits into two branches. Branches to the left innervate more medial regions while those trailing off to the bottom right travel to the most posterior edges of the segment. Scale bar: 50 /im.

3. Large DUM neurones innervate the photogenic tissue

If the ventral nerve cord is left attached to the photogenic tissue, CoCl2 back-filled into either root in each lantern ganglion reveals the pattern of DUM cell innervation of the lantern (Fig. 5). Three axons in the case of A 6, and four in the case of A 7 travel out to the periphery and branch several times before penetrating the dorsal reflector layer of cells overlying the photocyte layer. The axons may branch together to innervate the same area of the lantern, or they may diverge to innervate different areas. In either case the outcome is the same: the large bilaterally symmetrical neurones which arise in A 6 and A 7 branch profusely to innervate their respective lantern segments. Within both lantern segments we have observed peripheral branches of the lantern ganglia nerve roots which do not contain axons from DUM neurones (Fig. 5, dotted outline). At the point where these branches split off toward the lateral edges of the body wall, the DUM axons do not send processes into the peripheral branches, but turn away in a posterior direction to innervate photogenic tissue. If CoCla or Lucif:iieer Yellow CH is used to back-fill one of the long peripheral branches which trave DUM neurones and flash control in the firefly 141

Fig. 6. Spontaneous intra-somatic recording made from DUM soma in A 7. Note the presence of multiple- as well as single-component spikes. Physiologically, these DUM neurones resemble those of other insect species (see text). Vertical: 10 mV; horizontal: 100 ms. toward the lateral body wall in the vicinity of the spiracle, axons carry the dye back to the ganglion but the large DUM somata do not fill (Fig. 7). Furthermore, there are no stained fibres found in the contralateral root. Instead, the dye travels through fibres in the anterior ipailateral connective to other regions of the nerve cord. The DUM neurones therefore confine their innervation to the lantern tissue in these two segments. 4. A sample of spike activity from a large firefly DUM neurone Firefly DUM somata positively identified after dye-injection display negative resting potentials from 30 to 45 mV and small action potentials with amplitudes from 10 to 15 mV (Fig. 6). These neurones typically display multiple- as well as single- component spikes. In many of the recordings, a small potential appears as a shoulder on the rising phase of the larger potential. The two may occur at varying degrees of summation or, in other records, one or more smaller potentials may be completely separated from the larger. The different types of spikes observed might reflect the characteristic morphology and special function of these bilateral neurones. Compared with the spike frequencies recorded intracellularly from other insects (Hoyle & Dagan, 1978; Heitler & Goodman, 1978), firefly DUM neurones fire spontaneously at rather high frequencies while maintaining a steady resting potential. After firing at over 50 Hz for several minutes immediately after penetration, frequencies typically drop to a level of about 10 Hz for 30 min or more, which is an order of magnitude greater than the typical frequencies reported in other DUM neurones.

5. Correlations made using the vital dye Neutral Red Because the neurones innervating the lantern are believed to be octopaminergic, we immersed the lantern ganglia in a o-i % solution of Neutral Red dye in saline (Stuart, Hudspeth & Hall, 1974), and obtained the result shown in Fig. 8. A cluster of large somata, each 30-60 /im in diameter, are deeply stained on the anterior dorsal surface of A 7 (Fig. 8 A). Some of these cells are located in the same position as the large dorso- medial somata found using cobalt back-fills (Fig. 8B, dotted outlines; compare Fig. 2). Similarly, select cells are stained in A 6 (Fig. 8 C, D; compare Fig. 4). In addition to the ^ dorsal somata characteristically found using dye back-filling, there is a pair of T. A. CHRISTENSEN AND A. D. CARLSON more intensely stained dorso-medial cells (Fig. 8 A, C: arrows; B, D: solid outlines)! each about 20 fim in diameter, that do not appear in any of the cobalt or Lucifer Yellow CH back-fills of the ganglionic roots of either A 6 or A 7. These same two cells appear in relatively the same position in every other abdominal ganglion as well.

DISCUSSION Morphological and electrophysiological examination of the male P. versicolor lantern apparatus reveals an organization within the central nervous system which may help explain the physiology of flash control in this insect.

1. Gross morphology of the lantern nervous system The organization of nerves innervating the lantern of male P. versicolor firefliesi s similar to that described for Photuris and Photinus fireflies by Hanson (1962), but differs in one conspicuous detail: axons exiting from a lantern ganglion leave through single (not paired) lateral nerve roots, only later splitting into branches that innervate the photogenic tissue (Fig. 1 B). It is not known whether these differences in gross neural anatomy have any significant physiological consequences. Unlike the roots arising from the more anterior abdominal ganglia, the lantern ganglia roots branch profusely and spread out over the entire surfaces of the two lantern segments (Fig. 1 B). Extensive branching helps to ensure that when excitation passes from the brain and reaches the lantern all regions of the lantern luminesce simultaneously. This photogenic synchrony is essential for the production of the courtship flash, which rapidly alternates between brilliant luminescence and near total extinction three times within one half-second.

2. Neural organization of the lantern ganglia Hoyle et al. (1974) assigned the term DUM (for dorsal unpaired median) to a cluster of large somata located along the dorsal midlines of several insect thoracic and abdominal ganglia. These cells, first discovered in Locusta migratoria by Plotnikova (1969), have since been described in other insect species (Crossman et al. 1971; Hoyle, 1975; Heitler & Goodman, 1978). They are given the designation 'unpaired' because they have been found to give rise to processes which extend out on both sides of a given ganglion rather than occurring in bilaterally symmetrical pairs. Using whole-mount preparations of CoCls and. Lucifer Yellow CH back-fills of all the branches of the lantern ganglia roots, except those that do not innervate the lantern, we have discovered a symmetrical array of large neurones whose somata are situated on the dorso-medial surfaces of the two lantern ganglia A 6 and A 7. Further- more, each of these large neurones bifurcates and sends bilateral axons to both sides of the photogenic tissue. These neurones therefore fit the three criteria for being termed 'DUM'.

3. Similarities between firefly and other insect DUM neurones Intracellular records from firefly DUM neurones closely resemble those from DUM neurones found in other insects, including the cockroach (Crossman et al. 1971) ani Journal of Experimental Biology, Vol. 93 Fig-7

Fig. 7. Photomicrograph and corresponding camera lucida drawing of A 6 in which a peripheral branch of the left root, which innervates lateral body-wall musculature (but not lantern tissue) was back-filled with Lucifer Yellow CH (see Fig. 5 and text for further explanation). Broken lines within the drawing represent the DUM somata and contralateral fibres that appear in typical back-fills of branches that innervate lantern tissue, but which are absent in this preparation. Scale marker: 150 /im.

T. A. CHRISTENSEN AND A. D. CARLSON (Facing p. 142) Journal of Experimental Biology, Vol. 93 Fig. I

Fig. 8. Photomicrographs and corresponding camera lucida drawings of A 6 and A 7 stained with Neutral Red dye. (A) A 7 in dorsal view, showing a cluster of large somata in the anterior portion of the ganglion. Note the pair of smaller darkly stained somata (arrows) just anterior to the larger ones. (B) Camera lucida drawing of the preparation in A, outlining the larger somata (broken lines) and the smaller pair (solid lines). Compare this drawing with Figs. 2 and 3. (C) A 6 in dorsal view, showing a pair of somata (arrows) very darkly stained, along with a cluster of larger somata which are out of the focal plane of the photograph, and are therefore blurred. (D) Camera lucida drawing of the preparation in C, outlining the larger somata (broken lines) and the smaller pair (solid lines). Compare this drawing with Fig. 4. Scale bars: B, D : 100 /im.

T. A. CHRISTENSEN AND A. D. CARLSON DUM neurones and flash control in the firefly 143 Be locust (Hoyle & Dagan, 1978; Heitler & Goodman, 1978). Firefly DUM neurones spike spontaneously (Fig. 6) and exhibit both excitatory and inhibitory postsynaptic potentials. Presumably these spontaneous potentials are synaptically driven at least in part by axons which descend from the oscillator in the insect's brain. Heitler & Goodman (1978) have shown that each of four regions of a locust DUM neurone (the soma, the neurite and the two bilateral axons) has associated with it a different action potential type. Presumably, this condition is due to the presence of four separate spike-initiation zones on this neurone. The firefly DUM neurone spikes resemble these locust potentials so closely that, considering their morphological similarity to the locust neurones, one might expect to find multiple spike-initiation zones on firefly DUM neurones as well. Morphological and physiological experiments that could help determine this are currently being conducted. Although the spike types recorded from the firefly are similar in appearance to those of orthopteran insects, there are also obvious differences between them. Firefly DUM neurones positively identified after dye injection exhibit negative resting potentials of 30-45 mV and spontaneous multiple-component potentials. These potentials are usually double, consisting of a larger primary component and a smaller secondary component. Primary components range in amplitude from 10 to 15 mV, while the secondary components fall between 2 and 4 mV. In the locust, however, typical amplitudes for soma-initiated spikes range from 86 to 89 mV, while those for axon- initiated spikes are closer to 15 mV (Hoyle & Dagan, 1978; Heitler & Goodman, 1978)1 Another property of firefly DUM neurones helps identify them easily upon microelectrode penetration. Unlike other neurones within the lantern ganglia, the DUM neurones typically fire spontaneously at frequencies as high as 56 Hz for several minutes before slowing down to a steady level of around 10 Hz for another 30 min or more without any change in resting potential. In sharp contrast, typical spontaneous firing rates for locust DUM neurones are reported at less than 1 Hz (Hoyle & Dagan, 1978; Heitler & Goodman, 1978). Likely explanations for the low resting potentials and high spike frequencies observed in firefly DUM neurones are injury to the soma due to microelectrode penetration or depolarization of the neurone by elevated po- tassium in the saline. Precluding these possibilities, another explanation for the small spike amplitudes is that the potentials have propagated over some distance from their site(s) of initiation and invade the soma electrotonically. In order to answer these questions we must expand upon these preliminary findings and conduct more extensive physiological tests on these neurones. Another similarity of note is that firefly DUM neurones, like the DUM neurones found within the locust (Evans & O'Shea, 1977), the cockroach (Evans, 1978; Dymond & Evans, 1979), and the grasshopper (Goodman et al. 1979), stain deeply with the vital dye Neutral Red (Fig. 8) which stains monoamines. This might be an indication that firefly DUM neurones, like these other insect neurones, are octopaminergic. The results of a radio-enzymic assay for octopamine on the isolated cells would be very interesting in light of the fact that octopamine is a suggested transmitter operating in the lantern (Carlson, 19686; Robertson & Carlson, 1976; Oertel & Case, 1976; Nathanson, 1979). ^Finally, perhaps the most startling difference between firefly DUM neurones and 144 T. A. CHRISTENSEN AND A. D. CARLSON - other insect DUM neurones concerns the tissues they innervate. Until now, typicaP insect DUM neurones such as DUMETi, which innervates the extensor tibiae (ETi) muscles used in jumping (Hoyle et al. 1974), and DUMDL, which innervates the dorsal longitudinal (DL) flight muscles (Hoyle, 1978) of the locust and grasshopper, have all had a common effector - peripheral skeletal muscle. Firefly DUM neurones innervate photogenic tissue which, although physiologically similar to muscle (Chang, 1956; Buck & Case, 1961), is derived from fat cells (Hess, 1922). The means by which excitation is passed from the nerve terminals to the photocytes is still unknown, but the answer undoubtedly lies in a better understanding of the lantern DUM cells and their connexions between the lantern and the rest of the central nervous system.

4. Possible role of the DUM neurones We are now attempting more extensive electrophysiological and electron micro- scopic studies of the lantern nervous system. Experiments such as locating DUM cell synaptic contacts within the lantern and determing the connexions between the DUM neurones and the rest of the central nervous system could tell us more about the physiology of flash control. However, certain problems, unique to the firefly, make it very difficult to complete some interesting and important experiments. For example, artificial depolarization of a lantern motoneurone should cause a flash response. But direct exposure of the photogenic tissue of the adult firefly lantern to air or oxygenated saline causes the tissue to glow and can drastically reduce its flash response. Unfortu- nately, in order to stimulate a single cell within the nervous system, it is necessary to expose the dorsal reflector layer of the lantern and perfuse it and the overlying ventral nerve cord with saline. Because of this, it is difficult to demonstrate flash responses to intra-somatically stimulated DUM neurones in the adult firefly. However, larval lanterns do not glow spontaneously in oxygenated saline, but strongly luminesce in response to stimulation of the lantern root (Carlson, 1969; Oertel & Case, 1976). The paired larval lanterns are innervated by the 8th abdominal ganglion which contains four large DUM neurones (unpublished observations). It should be less difficult, therefore, to induce luminescence in the larval lantern by intracellular stimulation of these neurones. Up to the present, DUM neurones described in several insect species have all been found to innervate peripheral skeletal muscle. For example, the locust neurone com- monly referred to as DUMETi is known to inhibit an intrinsic myogenic rhythm of the extensor muscles in the leg (Hoyle, 1975). More recently, Evans & O'Shea (1977) have shown that DUMETi can modulate the activity of the slow motoneurone to the extensor muscles (SETi), and that this action is mediated through the effects of octopamine. On the basis of these roles assigned to this DUM neurone, one might suspect that firefly DUM neurones could be operating in a similar fashion by modulating the activity of lantern motoneurones. In this report we have described two populations of neurones within the lantern ganglia that send axons out of the lantern roots. One group of neurones has small laterally situated somata which cluster around the region where the root emerges from the ganglion. The other group includes the large bilateral DUM neurones. On basis of evidence presented here, we can propose two models that could help DUM neurones and flash control in the firefly 145 lie mechanism of lantern regulation. Although there is no direct evidence, the small unilateral neurones could be the lantern motoneurones, the activity of which is modulated by the DUM neurones. Another explanation is that the large DUM neurones could directly innervate the lantern end-cell complex, without performing a modulatory function. In point of fact, Smith (1963) and Case & Strause (1978) have demonstrated that nerve terminals in the lantern, interposed between the tracheal end cell and tracheolar cell, characteristically contain two types of synaptic vesicles: small electron-lucent vesicles and larger dense-core vesicles. Furthermore, the only synapses found in the lantern are of this neurosecretory type, and they are indistinguishable from the terminals of DUMETi in the locust (Hoyle et al. 1974). Until we can identify the origin of these terminals in the lantern, we cannot be sure which of these two models is correct. Many Photurid male fireflies are capable of producing intensity-modulated flashes (Barber, 1951; Fig. 1 A). The flash of P. versicolor males, in particular, is a very rapidly modulated example, composed of three flash pulses within approximately 500 ms (Fig. 1 A). In order to accomplish this behaviour, the onset of luminescence in the lantern photocytes must be closely synchronized, followed shortly thereafter by the simultaneous extinction of all the photocytes. From the results presented above, we can speculate on the possible processes involved in the neural control of this complex behaviour. Three short neural bursts, produced by a pattern generator in the male's brain, travel down the ventral nerve cord to the lantern ganglia, where the excitation impinges upon the DUM neurones. As previously outlined, each of the DUM somata gives rise to a medial neurite which travels anteriorly through the neuropile for a variable distance and then bifurcates into two bilaterally symmetrical axons. Each axon branches again and again sending fine processes out over the dorsal surface of the lantern. The DUM axons confine their processes to the lantern tissue, whereas other axons that run within the lantern roots continue farther out to the periphery to innervate spiracular and body wall musculature. Whether the DUM neurones act directly on the lantern or as modulators of the lantern motoneurones, it is clear that the organization of these neurones ensures the bilateral and simultaneous passage of excitation to the entire lantern. This, in turn, through still unsolved mechanisms, facilitates the production of the brilliant, triple-pulsed courtship flash of the P. versicolor male firefly.

The authors wish to thank Dr Kent T. Keyser, Steve Moorman and Thomas DiFilippo for their technical assistance, and Drs John B. Buck, James F. Case and Frank E. Hanson, Jr, for valuable comments on the manuscript. This research was supported by NSF grant BNS 761832701 awarded to A. D. Carlson and is in partial fulfilment of the requirements for the Ph.D. degree at SUNY, Stony Brook by T. A. Christensen. 146 T. A. CHRISTENSEN AND A. D. CARLSON

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