Brain Research, 105 (1976) 583-587 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Photosensitivity of a bursting pacemaker neuron in

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Aplysia californica

THOMAS H, NELSON, YONG I. KIM AND M. KIM Bioelectric Systems Laboratory, School of Electrical Engineering and Section of Neurobiology and Behavior, Cornell University, Ithaca, New York, N.Y. I4853 (U.S.A.)

(Accepted December 23rd, 1975)

Several neurons in the abdominal ganglion of Aplysia californica contain photopigments and are photosensitive with respect to their electrical behavior2, s. Unlike visual receptor cells, these neurons are not exposed to light in the adult organism and the significance of this photosensitivity in their normal functioning is obscure. However, due to their large neuronal size and the relative simplicity of the neuronal interconnections, the neurons have recently been studied as a model photoreceptor~,6,TA0,11. Such studies were performed mainly on the Aplysia giant neuron, R2 (ref. 8), and have revealed that the release of Ca z+ from the cytoplasmic lipochondria granules, which contain photopigments, causes the increase of K + conductance responsible for the light-induced membrane hyperpolarizationS, 11. To date, the structure or photochemical mechanism of the pigments present in these neurons have not been determined. While these recent investigations are concentrated on the 'silent' R2 neuron, we have attempted to characterize the photoresponse of the neurosecretory neuron, R 15 (ref. 8), of Aplysia and have measured the neuron's spectral sensitivity by electrophysiological methods. When compared to R2, R15 presents two particularly interesting aspects: (1) it is endogenously activO, 16 and exhibits slow membrane oscillation that gives rise to the unique bursting pacemaker activity12,14, and (2) it produces a circadian rhythm of impulse activity which is entrainable by photoperiodic exposure of the animals 13,16. Aplysia californica utilized in the experiments were of moderate size, 150-250 g each, and used within a month of capture. To prevent possible alteration in the photoresponse of R15 by a circadian rhythm, we uniformly entrained the animals with white fluorescent light to a 12-12 h light-dark cycle. Further precautions were taken by performing the experiments at the same time of day. The artificial seawater used as a bathing medium of the excised abdominal ganglion had a similar saline composition to the extracellular fluid of the animal 3. Its p H was adjusted to 7.4 and its temperature was maintained at 14 °C which was the physiological temperature of an intact animal. The neurons were penetrated by 0.6 M K2SO4-filled micropipettes having a typical resistance of 20 Mr2. Electrical activity of the neurons was measured with the conventional electronic instruments employed for intracellular

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Fig. 1. A: response of R15 to m o n o c h r o m a t i c illumination at 485 n m and m e a s u r e m e n t s of the slope m e m b r a n e c o n d u c t a n c e (AI/AV) before a n d after illumination. Double electrode penetration was performed on the neuron to inject the hyperpolarizing current pulses (AI, amplitude: 1.5 nA) seen in the lower trace a n d simultaneously to m e a s u r e the m e m b r a n e voltage displacement (AV) seen in the upper trace. A r r o w indicates the onset o f illumination (intensity: 18 m W / s q , cm), B: typical responses o f R15 to m o n o c h r o m a t i c illumination at various wavelengths. Illumination at each wavelength was m a d e in a r a n d o m sequence a n d the neuron was dark-adapted between each illumination until its bursting pattern returned to n o r m a l for a dark-adapted neuron. A r r o w s indicate the onset o f illumination (intensity: 18 m W / s q , cm).

585 measurements. The source of light stimuli was a high-intensity tungsten-filament lamp having a broad spectral emissivity ranging from 250 to 2600 nm. Monochromatic light was provided by projecting the lamp through a narrow band interference filter (Baird-Atomic B-10). Its intensity was calibrated with a photocell (Clairex CLT2010) to an equal energy output level by varying the DC voltage across the lamp. In the calibration, we additionally normalized the voltage output of the photocell on the basis of its known spectral characteristics. Step-wise illumination of R15 with white light produced a slow membrane hyperpolarization which apparently caused a temporary inhibition of the bursting activity. This inhibitory process was chiefly characterized by a lengthened interburst period. The hyperpolarization was only a transient effect, however, permitting the neuron to recover its normal bursting pattern with sustained illumination. The hyperpolarization was found to be a function of both light intensity and wavelength. Illumination during the middle of the interburst period most effectivelyevoked the hyperpolarization. While the reason for this is not clear, according to a model of the cyclic variation of the K ÷ conductance (Gk) in bursting neurons 12,14, Gk reaches a maximum near this point in a bursting cycle. The transient bursting pattern of the neuron under illumination also showed a smaller post-burst hyperpolarization and a smaller repolarization of the individual spikes superimposed on the slow waves. In addition, the number of spikes produced per burst of activity considerably decreased and their amplitude was slightly diminished. In order to assure the reproducibility of the photoresponse, a minimum dark adaptation of approximately 15 rain was required after a 10 min illumination with an intensity of 18 mW/sq.cm. This was also found to be a function of both intensity and wavelength of the incident light. Monochromatic illumination of the neuron at the proper wavelengths produced essentially the same effects as illumination with white light. Fig. 1A illustrates the neuron's photoresponse to monochromatic illumination at 485 nm. The corresponding slope membrane conductance measurements were performed prior to and subsequent to the illumination. In this particular data, the hyperpolarizing potential change reached its maximum, 8 mV, 26 sec after the onset of illumination. The hyperpolarization is shown to be associated with a membrane conductance increase of about 20 ~ as measured by injecting constant current pulses into the neuron. We did not examine the ionic basis of the hyperpolarization, and it is yet to be determined whether the conductance increase is exclusively due to an increase in K ÷ permeability as in R2 studied by Brown and Brown6,L Fig. 1B presents one of the recordings used for spectral sensitivity measurements of R 15. The measurements were based on the transient time delay between the onset of illumination and the reappearance of a burst of activity. These showed good repeatability in subsequent trials, while the measurements of the hyperpolarizing potential change varied, of the order of a few millivolts when illumination was made at different positions within a single bursting cycle. However, when illumination was made at the same point in a bursting cycle (for example, at the center of the interburst period), the resulting potential change was virtually constant and the sensitivity data was similar in form to that of the time delay. The most sensitive wavelength of

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Fig. 2. Spectral sensitivity of R15. The curve is plotted as the ratio of the first interburst period after illumination to the average interburst period prior to illumination. The ratio at 485 nm was normalized to 100 ~ . Wavelengths appearing in the curve are (from left to right): 400, 440, 470, 485, 500, 515, 530, 560, 590, 620, 650, 680, and 710 nm. Data was averaged for 3 experiments.

R I 5 in terms of either the transient time delay or the membrane hyperpolarization was 485 nm. The least sensitive wavelengths lie near the infrared region, for which there appeared to be no perceptible photoresponse. The neuron was highly responsive to 470 nm light, occasionally producing a response comparable to that of 485 nm light. Illumination at two ultraviolet wavelengths, 250 nm and 350 nm, and at an infrared wavelength, 1100 nm, caused no change in the electrical activity of the neuron. The sensitivity curve plotted is shown in Fig. 2. In general, the curve consists of a relatively narrow band, peaking at 485 nm, falling steeply toward the red and more gradually toward the violet. There appears no secondary peak in the curve. These results are comparable to those of Brown and Brown 7 on R2 which shows a peak sensitivity at 490 nm. We also have observed a distinct effect of the circadian rhythm on the photoresponse of R15. Experiments carried out on dark-adapted neurons, with no parameters varying except the time of day, indicate that the photoresponse varies as a function of the time of day. The period of least sensitivity was commonly coincident with the onset of dawn, namely the time the aquarium light was turned on each morning during the course of circadian entrainment. The period of maximum sensitivity was a few hours prior to dawn. It seems, in addition, possible that there may exist a seasonal variation in the photosensitivity since we have observed that apparently healthy neurons were almost totally insensitive to light during a certain period of the year. Moreover, the color of the surface membrane in R15 seemed to change at different times of the year; being yellowish-white at some times and at others yellowish-orange. The results reported here present that (1) the bursting pacemaker neuron, R I5, responds to light with a slow membrane hyperpolarization which is associated with an increase in the slope membrane conductance and an inhibition of the bursting activity, and (2) its spectral sensitivity curve contains a single peak at 485 nm. The

587 photoresponse, as well as the spectral characteristics of R15 appears to be similar to those of R2 as reported by Brown and Brown 7. The single sensitivity peaks found from R2 and R15 of Aplysia suggest that a single photopigment is involved in generation of their electrophysiological tahotoresponses. This view supports the recent observations that fl-carotene may be the only pigment acting on R2 (refs. 4,15). It also appears that the pigments present in the abdominal neurons of Aplysia californica differ from those of Aplysia depilans studied by Arvanitaki and Chalazonitis 2, who attributed those neurons' photosensitivity to the presence of both heme and carotene proteins. This study was supported in part by Grant GB-35498 from the National Science Foundation.

1 ALVING,B. O., Spontaneous activity in isolated somata of Aplysia neurons, J. gen. PhysioL, 51 0968) 29-45. 2 ARVANITAKI,A., AND CHALAZONITIS,N., Excitatory and inhibitory processes initiated by light and infrared radiations in single identifiable nerve cells. In E. FLOREY (Ed.), Nervous Inhibition, Pergamon, New York, 1961, pp. 194-231. 3 AUSTIN, G., YAL H., AND SATO, M., Calcium ion effects on Aplysia membrane potentials. In C. A. G. WmRSMA(Ed.), Invertebrate Nervous System, The University of Chicago Press, Chicago, Ill., 1967, pp. 39-53. 4 BROWN, A. M., personal communication. 5 BROWN, A. M., BAUR, P. S., AND TULEY, F. H., Phototransduction in Aplysia neurons: calcium release from pigmented granules is essential, Science, 188 (1975) 157-160. 6 BROWN, H. M., AND BROWN, A. M., Ionic basis of the photoresponse of Aplysia giant neuron: K + permeability increase, Science, 178 (1972) 755-756. 7 BROWN, H. M., AND BROWN, A. M., Light response of a giant Aplysia neuron, J. gen. PhysioL, 62 (1973) 239-254. 8 FRAZIER,W. T., KANDEL,E. R., KUPFERMANN,I., WAZIRI, R., AND COGGESHALL,R. E., Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia californica, J. Neurophysiol., 30 (1967) 1288-1351. 9 GORMAN,A. L. F., AND MCREYNOLDS,J. S., Control of membrane K + permeability in a hyperpolarizing photoreceptor: similar effects of light and metabolic inhibitors, Science, 185 (1974) 620-621. 10 HENKART,i . , Structural changes associated with illumination in the Aplysia giant neuron, Program Abstr. Soc. Neurosci., 1973, p. 358. 11 HENKART,i . , Light-induced changes in the structure of pigmented granules in Aplysia neurons, Science, 188 (1975) 155-157. 12 JUNGE,1:)., ANDSTEPHENS,C. L., Cyclic variation of potassium conductance in a burst-generating neurone in Aplysia, Amer. J. PhysioL, 235 (1973) 155-181. 13 LICKEY,i . E., ZACH, S., AND BIRRELL, P., Circadian rhythm in neuron R15 of Aplysia: circannual modulation and extraoptic entrainment, Fed. Proc., 29 (1970) 325. 14 PLANT, R. E., AND KIM, M., On the mechanism underlying bursting in the Aplysia abdominal ganglion R15 cell, Math. Biosci., 26 (1975) 357-375. 15 SMITH, C. D., Identification of Carotene in Aplysia Ganglion Cells using Laser Resonance Raman Spectroscopy, Master Thesis, Cornell University, Ithaca, New York, 1975, pp. 76-114. 16 STRUMWASSER,V., Neural and humoral factors in the temporal organization of behavior, Physiologist, 16 (1973) 9-42. 17 STRUMWASSER,F., JACKLET,J. W., AND ALVAREZ,R. B., A seasonal rhythm in the neural extract induction of behavioral egg-laying in Aplysia, Comp. Biochem. Physiol., 29 (1969) 197-206.