Light input to crustacean neurosecretory cells

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Brain Research, 265 (1983)307-311 Elsevier Biomedical Press

Light input to crustacean neurosecretory cells RAYMON M. GLANTZ, MARK D. KIRK and HUGO ARECHIGA*

Department of Biology, Rice University, Houston, TX 77001 (U.S.A.) and (H.A.) Departamento de Fisiologia y Biofisica, Centro de In vestigacibn y de Estudios A vanzados del IP N, Apdo. Postal 14- 740, M~xico D.F. 07000 (Mexico) (Accepted December 28th, 1982)

Key words: neurosecretion - crayfish - photoreception - neurosecretory cells

Electrical activity was recorded intracellularly from neurosecretory cells in the crayfish eyestalk identified by lucifer yellow injection. The activity is most commonly enhanced by illumination of retinal fields. Increments in spontaneous activity as well as bursts in otherwise silent cells were the most common type of response. Occasionally light-induced inhibitory responses were recorded. At neuropil level, light pulses result in EPSPs with amplitudes dependent on intensity of light and the previous adaptation to darkness.

The major neurosecretory system in crustaceans is the X-organ (XO), a cluster of 10(~150 neurons located in the medulla terminalis (MT) of the eyestalk 1.~5, with axonal projections ending in a neurohaemal organ, the sinus gland (SG) which is located more distally, between the medulla interna and the medulla externa (Fig. 1). A large number of physiological functions are influenced by the neurosecretory products released from this system (see ref. 19) and so far 4 neuropeptides have been isolated and purified from these cells. For two of them, the chemical structure is known. Such is the case of the erythrophore concentrating hormone (ECH) 1° and the distal pigment light-adapting hormone 9. For the other two, the approximate amino acid composition is known. The hyperglycaemic hormone (HGH) 17 and the neurodepressing hormone ( N D H ) 13.

These neurosecretory cells are electrically excitable, and action potentials have been intracellularly recorded at the cell bodies in the X-organ ~4 and from the endings in the sinus gland 7. The coupling of electrical activity to the secretory process has been documented for two peptides, ECH 8 and N D H 5. Light is a powerful stimulus for the secretion of several of these neurohormones. For instance, the release of DPLH is

known to be triggered by light6, and there is good indirect evidence for a similar phenomenon in connection with ECW 8, H G W 2 and N D H 4. The location of the photoreceptors and pathways of such neuroendocrine reflexes are as yet unknown. DPLH release appears to involve extra-retinal photoreceptors (see ref. 3). The main aim of this communication is to present direct evidence for light responsiveness in the X-organ-sinus gland (XO-SG) system along with some considerations regarding the location of the receptive structures. The experiments were conducted in adult crayfishes, Pacifastacus, of either sex using a previously documented procedure t6. The animals were pre-cooled in oxygenated iced water for 30 min and later transfered to cold saline solution 2° while maintaining the oxygenation. The dorsal carapace was removed to expose the heart and the preparation was left to equilibrate in the saline for another 30 min. The exoskeleton was partly removed from the eyestalks, which had been previously glued to the carapace. The X-organ, and part of the XO-SG tract, and the SG were then visible, depending on the rotation imposed to the eyestalk before glueing. The connective tissue surrounding the neurosecretory elements was then removed. The recording was made with glass

0006-8993/83/000(~0000/$0Y00 © 1983 Elsevier Science Publishers

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Fig. I. A: lucifer yellow fill of a neurosecretory cell in the eyestalk of Pacifastacus. The cell body is located superficially in the medulla terminalis (MT) and the axon, after branching in the neuropil of MT runs distally. MI, medulla interna: ON, optic nerve. B: detail of the same neuron, at higher magnification. Scale 60/~m for A and 24 #m for B.

micropipettes filled with 3% lucifer yellow m distilled water. The tip resistances were in a range of 50-100 M~2. Signals were led to a WPI preamplifier displayed on a Tektronix oscilloscope, and recorded on a Tandberg instrumentation tape recorder. Light pulses were delivered from an incandescent source with an approximate intensity of 100 lux. The duration was regulated with a shutter whose half-time of excursion was 0.5 ms. The light signal was monitored with a photocell. The impaled cells were stained with the procedures described by Glantz and Kirk

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As expected from histological data, the lucifer fills reveal a heterogeneous population of cell bodies. The most commonly found were pearshaped, as shown in Fig. 1. The diameters range from 25 to 40/~m, with an enlarged initial segment. A second common type was of rounded shape with smaller dimensions and the least common was a cylindrical or polyhedrical shape. Nuclei were clearly visible in various positions, most commonly near the center of the soma. The common feature of all of these neurons is that the cell body gives rise to a long axon

which runs from the medial to the external side of the eyestalk along the curvature of the medulla terminalis. Some axons were straight in their paths, others showed kinks of various dimensions. Once in the external rim of MT, the axons proceed distally until ending in the sinus gland. As reported by Andrew and Saleuddin 2, after the initial segment, the axons branch profusely in the neuropil of the medulla terminalis. The total volume of branches is greater than the combined volume of the rest of the cell, not considering the endings at the sinus gland. The branches are oriented in all planes in the depth of the neuropil. There are abundant varicosities, similar to those described in other secretory neurons. They are mostly in the branches, but also common in the main axon, chiefly in the proximal part. Stable impalements were obtained at various levels of the neurosecretory cells, at cell bodies, axons and endings. The cell bodies have resting potentials which vary from - 6 0 to - 8 0 mV, their input resistances are around 20 M~2, and overshooting action potentials could be recorded, confirming previous results H. However, quite often, the cell bodies were electrically si-

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dings are more prolonged (half-width of ~ 5 ms) than any other spike recorded from crayfish axons. Spikes at nerve endings also exhibited half-widths of ~ 10-20 ms. Some impalements were done at the branching region near the cell body, the activity was higher and spontaneous PSPs were apparent. Diffuse illumination quite commonly resulted in enhancement of the ongoing spiking activity, as shown in Fig. 3. The response to illumination was of a phasic-tonic nature, with an initial burst followed by a sustained discharge. The total firing rate in both phases was proportional to the intensity of illumination. Light-evoked EPSPs were observed with impalements at the branI ji''!

Fig. 2. Electrical activity recorded intracellularly at various points of a neurosecretory cell in the eyestalk of Pacifastacus. A: action potentials recorded at the cell body, evoked by current passed through the microelectrode. B: abortive spike recorded at the cell body after stimulating the XO-SG tract in the distal part of the axon. C: spontaneous spike recorded from the proximal axon, at the level of the branching region. Notice some PSPs. D: spontaneous activity in a distal axon of the XO-SG tract. Calibration: A, 40 mV, 100 ms; B, 10 mV, 5 ms; C, 20mV, 50ms; D, 20mY, 20ms.

lent even after inducing depolarizations beyond 20 mV. Spontaneous bursting was rarely recorded, and when stimulating the XO-SG tract antidromically with a suction electrode, abortive spikes were recorded (Fig. 2B). This suggests that in the whole animal, as described in other crustacean neurons, the spike initiating zone is distant from the cell body, which is only passively invaded. As shown in Fig. 2A however, some spikes could be elicited by depolarizing current passed from the recording electrode in the cell body. Such spikes are quite prolonged (halfwidth "" 10-20 ms) thus consistent with the notion of Ca 2÷ origin, proposed by Iwasaki and Sat o w ]4.

In contrast with the pattern found in cell bodies, spontaneous action potentials were commonly found in axons and nerve endings. As seen in Fig. 2C and D even the spikes recorded from axons, far away from the somas or the en-

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Fig. 3. Responses induced in a neurosecretory cell in the eyestalk of Pacifastacus by light applied on the corneal surface of the eyestalk. Lower traces recorded from the branching region of the axon. Left:enhancement of ongoing activity. Right: EPSP obtained from the same cell with extrinsic hyperpolarization. Top record: burst of spikes triggered in a distal axon by a light pulse. At bottom of every trace is the signal from a photocell, monitoring the illumination. Calibration: lower left, 20 mV, 100 ms; lower right, 5 mV, 200 ms; upper trace, 40 mV, 500 ms.

310 ching region if spiking was arrested by passing hyperpolarizing current. The EPSPs consisted of both discrete unitary events at low stimulus intensities and compound events associated with steady-state shifts in membrane potential at higher light intensities. The PSP amplitudes were proportional to the level of hyperpolarization. Only rarely were neurons observed to be inhibited by illumination. The latency of the responses to light was 20-40 ms, depending on the intensity used. In order to locate the source of the photosensitive structures, the light pipe was moved about the cornea and from the cornea, along the eyestalk in a proximal movement toward the X-organ. The response was only seen when light was focused on the ipsilateral cornea, thus suggesting that, at least for the observed responses, the responsible photoreceptors are located in the ipsilateral retina, and that the neurosecretory cells are not directly sensitive to light. These results indicate that, as might be antic-

ipated, light modulates the electrical activity of neurosecretory cells, by presumably acting on structures synaptically connected to these elements. Since the eyestalk contains visual neurons whose response to light in their retinal fields is of a tonic sustained natur~ j and their cell bodies have been located in the medulla externa ~6 they are obvious candidates to mediate the light-induced excitation of the neurosecretory cells. Correspondingly, there is a set of dimming detectors whose firing rate is inversely related to light intensity 21, and these could participate in the light-induced inhibition. Such possibilities, however, are presently open to further studies necessary to clarify the afferent input to this neuroendocrine system.

1 Andrew, R. D., Orchard, 1. and Saleuddin, A. S. M.. Structural reevaluation of the neurosecretory system in the crayfish eyestalk, Cell. Tiss. Res., 190 (1978) 235 246. 2 Andrew, R. D. and Saleuddin, A. S. M., Structure and innervation of a crustacean neurosecretory cell, Canad. J. Zool., 56 (1978) 423-430. 3 Ar6chiga, H., Modulation of visual input in the crayfish, In G. Hoyle (Ed.), Identified Neurons and Behavior of A rthropods, Plenum Press, 1977, pp. 387 403. 4 Ar6chiga, H. and Huberman, A., Hormonal modulation of circadian rhythmicity in crustaceans. In C. Valverde and H. Ar~chiga (Eds.), Comparative Aspects of Neuroendocrine Control of Behavior, S. Karger, 1980, pp. 16-34. 5 Ar6chiga, H., Huberman, A. and Martinez-Palomo, A.. Release of a neuro-depressing hormone from the crustacean sinus gland, Brain Research, 128 (1977) 93 108. 6 Ar6chiga, H. and Mena, F., Circadian variations of hormonal content in the nervous system of the crayfish, Comp. Biochem. Physiol., 52A (1975) 581-584. 7 Cooke, I. M., Electrical activity of neurosecretory terminals and control of peptide hormonal release. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Press, 1977. pp. 345 374. 8 Cooke, I. M., Haylett, B. A. and Weatherby, T. M., Electrically elicited neurosecretory and electrical responses of the isolated crab sinus gland in normal and reduced calcium salines, J. exp. Biol., 70(1977) 125 149. 9 Fernlund, P., Structure of a light-adapting hormone from the shrimp, Pandalus borealis, Biochim. biophys. A cta, 439 (1976) 17 25.

10 Fernluncl, P., Structure of the red-pigment-concentrating hormone of the shrimp, Pandalus borealis, Biochim biophys. A cta, 371 (1974) 304- 311. 11 Glantz, R. M. and Kirk, M. D., Intercellular dye migration and electrotonic coupling within neuronal networks of the crayfish brain, J. comp, Physiol., 140 (1980) 121 133. 12 Hamman, A., Die neuroendokrine Steuerung tagesrhythmischer blutzukershwankungen durch die Sinudsrusse bein Flusskrebs, J. comp. Physiol., 89 (1974) 197-214. 13 Huberman, A., Ar6chiga, H., Cimet, A., de la Rosa, J. and Aramburu, C., Isolation and purification ofa neurodepressing hormone from the eyestalk of Procambarus bouvieri (Ortmann), Europ. J. Biochem., 99 (1979) 203 208. 14 lwasaki, S. and Satow, Y., Sodium- and calcium-dependent spike potentials in the secretory neuron soma of the X-organ of the crayfish, J. gen. Physiol., 57 (1971) 216 238. 15 Jaros, P. P., Tracing of neurosecretory neurons in crayfish optic ganglia by cobalt iontophoresis, Cell Tiss. Res., 194 (1978) 297- 302. 16 Kirk, M., Waldrop, B. and Glantz, R. M., The crayfish sustaining fibers, l.Morphological representation of visual receptive fields in the second optic ganglion, J. comp. Physiol., 146(1982) 175 179. 17 KleinholL L. H., Purified hormones from the crustacean eyestalk and their physiological specificity. Nature (LondL 258 (1975) 256 25~/.

This work was partly supported by Grant NSF BNS 7910335 to R. M. G. Predoctoral Fellowship NIH Training Grant EY07024-03 to M. D. K., and a Guggenheim Fellowship to H. A.

311 18 Kleinholz, L. H., Pigmentary effectors. In T. H. Waterman (Ed.), The Physiologv ofCrustacea, Vol. 11, Academic Press, New York, 1961, pp. 133-169. 19 Kleinholz, L. H. and Keller, R., Endocrine regulation m crustacea. In Barrington, E. J. W. (Ed.), Comparative Endocrinology, Academic Press, New York, 1979, pp. 159213.

20 Van Harreveld, A., A physiological solution for freshwater crustaceans, Proc. Soc. exp. Biol. N. Y., 34 (1936) 428432. 21 Wiersma, C. A. G. and Yamaguchi, T., The neuronal components of the optic nerve of the crayfish, as studied by single unit analysis, J. comp. Neurol., 128 (1966) 333358.