A number of adrenergic drugs were tested for their ability to induce luminescence in the extirpated lantern of the firefly larva. The drugs produce sigmoid doseresponse curves characteristic of drug-receptor interactions when drug concentration is plotted against either maximum intensity or maximum rate of intensity rise.
Amphetamine and saline of high potassium concentration induce intense luminescence in freshly extirpated lanterns but act only weakly or not at all in lanterns suffering from treatment by reserpine injection 48 hr. previously.
No significant difference in response to norepinephrine was observed in lanterns immersed in standard saline, 0.32 M sucrose, 0.16 M choline chloride or 0.16 M-NaCl2 but 0.107 M-CaCl2 considerably slowed the response.
Some generalizations concerning structural character and effectiveness in inducing luminescence were made by comparing the drugs tested. It was not possible to describe the mode of action of the drugs. The observation, however, that solutions lacking sodium and potassium did not significantly alter the response was felt to argue against the action of the adrenergic drugs in affecting ion movement across the photocyte membrane.
Smalley (1965) confirmed and extended the original observations of Kastle & McDermott (1910) that norepinephrine induces glowing in the lantern of the adult firefly. She further conclusively demonstrated that normal neural excitation involved an adrenergic rather than a cholinergic transmitter. Amphetamine apparently induced luminescence by causing the nerve endings to release their transmitter. Reserpine, a drug known to drain vertebrate nerve ends of adrenergic transmitters, abolished luminescence associated with neural stimulation. The ability of the lantern to produce a long-latency ‘slow flash’ or a short-latency ‘quick flash’, first reported by Buck, Case & Hanson (1963), was used to distinguish between the presence of transmitter in the nerve endings or its loss. Extracts of the lantern were tested for activity on rat uterus and colon and their effects were found to be intermediate between those of epinephrine and norepinephrine.
The paired lanterns of the larval firefly, Photuris sp., also respond to adrenergic agents. They represent a simpler structural system compared to the adult because electron micrographs show that the nerves terminate directly on the photocytes (M. Wetzel, unpublished). Further, the lantern can be extirpated, immersed in an oxygenated saline solution, tested with various drugs and its luminescent response can be monitored with some precision. This procedure has allowed us to study the pharmacological actions of various drugs and other chemical agents on this luminescent system in vitro.
MATERIALS AND METHODS
Larval fireflies of the genus Photuris were obtained from Iowa, Long Island and Maryland. They were maintained at 4°C. on moistened filter paper. They were occasionally fed with liver but otherwise required little care.
Drugs used were: DL- and L-norepinephrine hydrochloride, dopamine hydrochloride, DL-β-3,4-dihydroxy phenylalanine, eserine sulphate, 5-hydroxytryptamine and y-amino butyric acid (Sigma Chemical Co.); DL-epinephrine hydrochloride and tyramine hydrochloride (K and K Laboratories, Inc.); DL-isoproterenol hydrochloride (Aldrich Chemical Co.); D-amphetamine hydrochloride, acetylcholine chloride and atropine (Nutritional Biochemical Corp.); tyrosine (Eastman Organic Chemicals) and reserpine phosphate (supplied through the courtesy of Ciba Pharmaceutical Co.). All drugs except reserpine phosphate were dissolved in a saline containing 9 g./l. NaCl, 0·2 g./l. KCl, 0·2 g./l. CaCl2, 0·2 g./l. MgCl2, 4 g./l. glucose buffered with 0·04M Tris buffer to pH 7·3.
The tiny, paired larval lanterns lie on the ventral sides of the 8th abdominal segment and are composed of a ventral photogenic layer and a dorsal reflector layer. The lanterns were extirpated, their dorsal connexions such as nerves and tracheae were severed and the transparent ventral cuticle was left covering the organ. The lanterns were placed, ventral side down, on Plexiglass spatulas and held in place by fine Nylon cloth. The spatulas were placed in test-tubes, and these were positioned in front of a photomultiplier. The photomultiplier could be shifted to observe either lantern. Compressed oxygen, led through a flow meter, was bubbled into the test-tube via a small plastic tube. Solutions were introduced via a funnel which led through a stopcock into a narrow tube which extended to the bottom of the test-tube. The photomultiplier output was led into the top and bottom channels of a Grass two-channel Polygraph.
When a new solution was to be introduced into a test-tube the stopcock was set so that the old solution could be sucked into a refuse bottle. The new solution was poured into the funnel. The stopcock was rotated 90°, allowing entrance of the solution to the test-tube and closing a switch which caused the signal pen to vibrate rapidly, thereby providing an automatic marker.
Oxygen concentrations were varied by changing the flow rates of oxygen and nitrogen through calibrated flow meters. Denervation experiments were carried out on animals by transecting the nerves via a slit through the intersegmental membrane between the 6th and 7th or 7th and 8th abdominal segments. The animals were subsequently maintained at 20°C. for 48 hr. before testing. In order to study the effects of reserpine, larvae were injected with 5−15 μg. of reserpine phosphate in 3 μl. of distilled water. They were maintained for 48 hr. in a 20°C. box prior to testing.
Expirtated larval lanterns immersed in saline normally remain dark or maintain a very low level of luminescence. They can be maintained in the extinguished state in a wide variety of solutions such as 0·32 M sucrose, 0·107 M-CaCl2, 0·16 M-choline chloride, and 0·16 M-NaCl. De-ionized water, however, induces bright luminescence within a few minutes, evidently due to its osmotic effect. Lanterns which glow brightly in saline are usually damaged, but occasionally this occurs in apparently undamaged lanterns as well.
Introduction of saline containing 0·5 mM norepinephrine results in the characteristic response shown in Fig. 1. Following a lag period of a few seconds in most cases the luminescence builds up rapidly, eventually reaching a maximum which can be maintained for periods of many minutes. Removal of the norepinephrine solution results in the slow extinction of luminescence. If the lantern is glowing, removal of solution from the test-tube results in an immediate fall in intensity followed by restoration of the original level when new solution is added. This fall in intensity stems from loss of light reaching the phototube due to reflexion on the test-tube walls; it is a useful means of showing whether the lantern is still glowing or not.
As is demonstrated in Fig. 2, oxygen becomes a limiting factor between 15 % and 20%, above which no further increase in intensity can be obtained when organs were treated with 1 mM norepinephrine. Since the solutions are bubbled with 100 % oxygen it was felt that oxygen concentration was not a limiting factor in the luminescent response.
Pairs of dose-response curves obtained from one larva relating the percentage maximum intensity and drug concentration for DL-norepinephrine and DL-epinephrine are shown in Fig. 3. Note that while the concentration range for the lanterns differ for DL-norepinephrine, DL-epinephrine is more potent in each case. A similar relationship occurs when maximum rate of intensity rise is plotted against concentration as shown in Fig. 4. Both types of dose—response curve are of sigmoidal character. A threshold near 0·1 mM for both drugs is the same as that found in the adult by Smalley (1965).
The relative effectiveness of some adrenergic drugs in inducing luminescence in the larval lantern is shown in Table 1. The following drugs were found to have no effect at concentrations of 10mM: acetylcholine, 5-hydroxytryptamine, tyrosine, atropine, eserine, γ-amino butyric acid and DL-β-3,4-dihydroxyphenylalanine.
No significant difference in maximum intensity or maximum rate of intensity rise was observed in lanterns tested with 0·5 mM DL-norepinephrine in the following solutions: standard saline, 0·32 M sucrose, 0·16 M choline chloride and 0·16 M-NaCl. The response was considerably slower and of lower intensity to a solution of 0·107 M-CaCl2. Standard saline with the NaCl replaced by 0·16 M-KCI (high K+ saline) induced brilliant, rapid luminescence without norepinephrine. This luminescence could not be maintained, however, The response to high K+ saline in amphetamine-treated or reserpinized lanterns was usually reduced or abolished.
1 mM amphetamine induces intense luminescence which requires a long period in standard saline to extinguish. Confirming Smalley’s (1965) observations on the adult lantern, larval organs continue to respond to norepinephrine after their response to amphetamine has failed. This is true in organs previously treated in reserpine phosphate and it is true of lanterns which have suffered denervation for 48 hr. by prior nerve transection posterior to the last abdominal ganglion.
The lanterns of larvae injected 40−48 hr. previously with 5−15 μg. of reserpine phosphate gave somewhat variable responses when immersed in amphetamine while their response to DL-norepinephrine was unimpaired. These reserpinized lanterns which failed to give any response in high K+ saline gave feeble responses to amphetamine. The amphetamine-induced luminescence built up slowly, reacheda low intensity maximum and then declined to extinction. Following this the luminescence could no longer be revived in amphetamine. In these lanterns the response to norepinephrine was enormously stimulated when tested immediately following amphetamine treatment. The post-amphetamine responses of eight lanterns tested with norepinephrine averaged 426 % of the initial responses prior to amphetamine treatment.
The amphetamine response of lanterns suffering from denervation was highly variable and although dramatic reduction was obtained in some cases, none could be found in others. This result is probably due to the difficulty involved in ensuring denervation and further experiments are necessary.
DISCUSSION AND CONCLUSIONS
As in the adult, so in the larva, the lantern responds to various treatments in a manner closely resembling vertebrate adrenergic systems. Injections of amphetamine and reserpine result in failure of response to amphetamine. This loss does not affect the responsiveness of the photocyte to adrenergic drugs which in the vertebrate operate on the receptor side of the neuroeffector junction. Further, norepinephrine and its analogues induce a relatively rapid build-up of luminescence which reaches a plateau, whereas luminescence induced with amphetamine slowly increases, reaching a high intensity level.
The adrenergic drugs examined in Table 1 show many properties typical of drug-receptor interactions. The relationship of response to concentration is sigmoidal, showing saturation at the higher concentrations. Intermediate concentrations produce steady plateau responses which can be maintained without appreciable diminution. No attempt was made to determine whether the drugs varied in effectiveness due to affinity for a receptor or to activity once associated with a receptor.
The larval firefly system differs from typical vertebrate adrenergic systems in the relatively high drug concentrations required. The most effective drugs, norepinephrine and epinephrine, had thresholds between 0.1 and 1 mM. Vertebrate neuroeffectors are considerably more sensitive. This may either mean that the luminescent effector is physiologically less sensitive or, more likely, that the true transmitter is not represented in the drugs tested.
On the basis of relative effectiveness some tentative generalizations concerning structural character can be made. It appears that the hydroxyl group on the β carbon, as in norepinephrine and epinephrine, enhances the drug’s effectiveness because dopamine which lacks this group is considerably less effective. The stereoisomerism of the β carbon is not crucial, however, because DL-norepinephrine and L-norepine-phrine did not differ significantly in potency. While the terminal methyl group of epinephrine adds effectiveness, changing this to an isopropyl group drastically reduces activity.
The mode of action of adrenergic drugs in triggering luminescence cannot be outlined with any precision. Again turning to the well-documented vertebrate systems two mechanisms predominate. The transmitter may cause the photocyte membrane to become permeable to an ion, typically Na+, which depolarizes the cell, thereby initiating luminescence (Bulbring, 1962). The other possible mechanism, suggested by Smalley (1965) for the adult, is that the transmitter may in some way interact with ATP or its production. The former mechanism is strengthened by the observation of Buck et al. (1963) that in the larval lantern under stimulation at high intensities the latency of the response can be reduced to 1 msec., which suggests electrical coupling. The observations in this study do not support a mechanism such as depolarization via ion movements. Since no significant difference could be found between 0·32 M sucrose, 0·16 M choline chloride or 0·16 M-NaCl it would suggest that neither the sodium nor the chloride flux through the membrane is mediated by adrenergic drugs. Further, although high-K+ saline might be expected to depolarize the photocytes, it apparently induced luminescence by causing release of transmitter from the nerve endings.
This investigation was supported by National Science Foundation Grants GB3597 and GB6385 to the author. The author wishes to thank Dr John B. Buck for his criticisms of the manuscript. He also wishes to thank Mr and Mrs Baron Crane for supplying the bulk of the larvae. The technical help of Mr Gerald Pollack and Miss Janet Christian in this research is greatly appreciated.