Journal of Pineal Research 1: 139-147 (1984)

Responses of Synaptic Ribbons in Pineal Photoreceptors Under Normal and Experimental Lighting Conditions

John A. McNulty Department of Anatomy, Loyola University Stritch School of Medicine,

Maywood, Illinois

The size of synaptic ribbons (SR) in photoreceptor cells of the goldfish pineal organ was quantified over 24-h 1ight:dark cycles of long (16:8) and short (10:14) photoperiods during summer and winter months, respectively. The amplitude of both rhythms was similar with peak values occurring toward the latter part of the photophase or early dark. When fish were entrained to the long photoperiod and exposed to continual light, SR size continued to increase during the expected dark time. The effect of extending the photoperiod into the expected dark time was diminished when fish were entrained to a short photoperiod and presented with 6 h of darkness at the end of the 24-h period. The size increase in response to environmental lighting is believed to reflect a greater demand for either vesicle attachment sites or neurotransmitter storage sites since vesicles (neurotransmitter) have been hypothesized to accumulate in the synaptic pedicles during inhibition by light. From a comparative standpoint it is noteworthy that synaptic ribbons (vesicle-crowned rods) in mammals react in a similar manner to both normal and experimental lighting conditions.

Key words: synaptic ribbons, pineal, photoreceptors, circadian cycle

INTRODUCTION

The synaptic ribbon (SR) is an organelle found in pineal photoreceptors of lower vertebrates as well as the secretory pinealocytes of mammals. Their common occurrence in both cell types is consistent with the concept that

Received December 30, 1983; revision accepted February 28, 1084

Address reprint requests to Dr. John A. McNulty, Department of Anatomy, Loyola Univ. Medical Center, 2160 S. First Avenue, Maywood, IL 60153.

@ 1984 Alan R. Liss. Inc.

140 McNulty

the mammalian pinealocyte is phylogenetically derived from neurosensory cells. Synaptic ribbons typically consist of numerous clear vesicles surround- ing an electron-dense core which has been described as rodlike, platelike, or spherical in shape [Karasek and Vollrath, 1982; King and Dougherty, 1982a; McNulty, 1980; Theron et al., 19811. Recently, there have been numerous studies demonstrating that SRs exhibit numerical changes over 24-h 1ight:dark cycles with the greatest number occurring during the dark period [see Pevet, 1983; Vollrath, 19811. This response of SRs to environmental lighting and the observation that SR in the mammalian pinealocyte is inversely related to the level of adrenergic innervation [Karasek et al, 1982a; Karasek et al., 1983; King and Dougherty, 1982bI have implicated participation of SR in the neuroendocrine activities of the pineal gland.

The SR in pineal photoreceptors of lower vertebrates is usually found in basal cytoplasmic processes bordering the plasma membrane adjacent to dendrites from pineal ganglion cells and presumably plays a role in synaptic transmission. Quantitative morphological studies have suggested that SR are functionally related to photoreception since their frequency and size in both the pineal organ and retina vary over a 1ight:dark cycle and are affected by continual lighting conditions [McNulty, 1981, 1982; Omura and Ali, 1980; Spadaro et al., 1978; Wagner, 1975; Wagner and Ali, 19771. A working hypothesis proposed for both pineal and retinal photoreceptors states that these fluctuations in size and number of SR are directly related to the storage and depletion of neurotransmitter(s) that occur during light and dark adap- tation, respectively [Spadaro et al., 1978; Wagner and Ali, 1977; McNulty, 19811.

As part of a series of investigations on the structure and function of SR, the present study sought to determine the effects of seasonal differences in day-length on the circadian cycle in SR morphology in the pineal organ of the goldfish. A second purpose was to test the hypothesis that inhibition of pineal photoreceptors by light causes increased size of SR. In these experi- ments, the response of SR to environmental lighting was measured in animals where the expected time of lights off was delayed over 24-h periods.

MATERIALS AND METHODS Experimental Groups

Goldfish (Curassius auratus, 50-80-mm standard length) were kept in 20-gal aquaria illuminated by two 15-w incandescent lamps connected to automatic 24-h timers. The experiments were conducted during the months of June and February since the day-length for these months coincided most closely with the photoperiods of interest. For the first experiment in June, two groups of animals were entrained to a long photoperiod of 16-h light:% h dark (lights on at 0500 h) for 3 wk. The fish were fed daily between 1500 and 1700 h. One group (controls) was sacrificed over this 24-h L:D cycle at six time points (1300, 2000, 2200, 0100, 0400, and 0600 h). These time points allowed sampling at midlight, middark, and either side of the L:D interphases. Fish in the experimental group were sacrificed at the same time points over the same 24-h period, but in this case the lights remained on

Synaptic Ribbons in Pineal Photoreceptors 141

during the expected dark time. A minimum of five animals from each group were sacrificed at each sample time for this and the following experiment. Some tissue was lost during processing, resulting in an n < 5 at some time points.

In the second experiment conducted in February the goldfish were entrained to a short photoperiod of 10: 14 (lights on at 0700 h) and the same feeding schedule as above. Control animals were again sacrificed over the entrained L:D cycle at six time points (1200, 1600, 2000, 2300, 0200, and 0400 h). Experimental fish were taken at the same time points over the same 24-h period, but lights off was delayed by 8 h. The time points in this experiment were selected in order to sample at midlight, middark, and either side of the L:D interphase for both the control and experimental groups.

Microscopy

Fish in both experiments were sacrificed by decapitation and the dorsal part of the cranium with pineal organ attached was quickly removed using a razor blade. The tissue was placed in 4% glutaraldehyde in monobasic phosphate buffer (340 m0sm; pH 7.4) for 1 h. A photographic safelight (Kodak No. 1 filter) was used to collect samples in the dark and this tissue was kept in light-tight containers for the first hour of fixation. Following a brief wash in buffer, the tissue was postfixed in 1% osmium tetroxide in phosphate buffer, dehydrated in a graded series of acetone, and embedded in Epon. The pineal organ was carefully dissected from the cranium prior to embedding. All organs were sectioned in a sagittal plane and sections col- lected from the middle two thirds of the pineal end-vesicle. Sections were mounted on one-hole copper grids that had been Formvar coated and carbon backed. Contrast of the sections was enhanced with both uranyl acetate and lead citrate. The electron microscopes used to record the data included a RCA EMU-3F and Hitachi H-6000 equipped with 35-mm cameras.

Morphometrics The first section encountered from each grid was scanned and every SR

in the plane of section photographed. A minimum of 30 SRs were recorded from each organ up to a maximum of 122 from a single section in one organ. This sampling procedure resulted in a total of 4,830 SR recorded and mea- sured in this study. The micrographs were enlarged to a final magnification of X54,000 and every SR from each animal traced onto a single sheet of paper. The length of these tracings was subsequently measured using a Zeiss Videoplan image analyzer, Each roll of film was coded and the tracings and measurements made by an individual unfamiliar with the codes.

Statistics

The mean length of SR was calculated for each specimen. Means and standard errors of the grouped means were calculated and plotted according to experiment and time of sacrifice. The effects of time, lighting condition, and the interaction between time and lighting condition were tested by two- way analysis of variance. A Duncan’s Multiple Range test was subsequently used to test for differences between groups at individual time points.

142 McNulty

RESULTS

The ultrastructure of SR in this species was variable, with the central electron-dense core appearing in thin-sectioned materials as a circular, rod- like, or platelike structure (Figs. 1, 2). For this study, circular profiles were not measured since it was not possible to distinguish in two dimensions whether these SRs were spherical or were rods cut in cross section. All of the SRs were associated with clear vesicles measuring 40-60 mm in diameter. Occasionally, larger dense-core vesicles were in the vicinity of the SR. The SRs were typically located in basal synaptic pedicles although they were found in other regions of the cell (e.g., perikarya, inner segment) usually during the dark period. Sometimes SRs bordered the plasma membrane opposite supportive cells or other photoreceptors (Fig. 3).

Animals sacrificed over a L:D cycle of long photoperiod @me) exhibited significant changes in the size of SR over the 24-h period. In these control animals, SR length was greatest during the latter part of the photophase or early dark and tended to diminish during the scotophase (Fig. 4). When fish entrained to this long photoperiod were subjected to constant light on the day of the experiment there was a continued increase in SR length during the expected dark time (Fig. 4). The F test for the effect of lighting gave a P < 0.001, supporting the hypothesis that the light pattern plays a role in determining SR size. Subjecting the data to Duncan’s Multiple Range test showed that the significant differences between control and experimental groups occurred toward the end of the 24-h period (0400 and 0600 h).

The SR from animals sacrificed over the short photoperiod in February exhibited a pattern similar to that seen in the previous experiment with SR size being greatest toward the latter part of the photophase and early dark (Fig. 5) . Delaying the onset of darkness by 8 h resulted in a tendency for SR length to remain elevated during the expected dark time. There was a subsequent delay in the decline in size of SR that normally would have occurred toward the end of the scotophase (Fig. 5) . The F test for the effect of lighting gave P < 0.01, further supporting the hypothesis that SR size is determined by photoperiod. However, the effect was diminished by expo- sure to some darkness over the 24-h period. This is supported by the Dun- can’s Multiple Range test, which showed that a significant difference occurred between the two groups at only a single time point (0600 h).

DISCUSSION

The results of this study support earlier observations that SR in both pineal and retinal photoreceptors are dynamic organelles exhibiting daily changes in morphology [McNulty, 1981; Omura and Ali, 1980; Spadaro et al., 1978; Wagner and Ali, 19771. However, some variability exists in the rhythmic oscillations depending upon the photoreceptor-cell type and the species. SR in the pineal organ of the goldfish and brook trout [Omura and Ali, 19801 tend to peak during the latter part of the photophase or early dark. This pattern was consistent with that reported for retinal SR of the rat [Spadaro et al., 19781, a species with a predominantly rod retina [Cicerone

Synaptic Ribbons in Pineal Photoreceptors 143

Fig. 1. Some SRs are spherical in shape (arrows). Bar 1 = I 0.5 pm.

Fig. 2 . dark time in June. Bar I = I 0.5 pm.

Fig. 3. photoreceptor cell. Bar I = I 0.5 pm.

Several SRs bordering a dendrite (D) of a pineal ganglion cell from a control animal.

A platelike SR in the pineal organ from an animal exposed to light during the expected

A cluster of SRs from a control animal bordering the plasma membrane of another

et al., 19791. Wagner and Ali [1977] were able to detect a circadian rhythm in the frequency of SR only in retinal cones of the brook trout. In this case, the SR peaked at midlight and gradually declined during the latter part of the photophase and early dark.

Neurophysiological studies on pineal photoreceptors have demon- strated that a light stimulus causes hyperpolarization of the cells with a corresponding decrease in the spontaneous spike activity of ganglion cells [see Meissl and Dodt, 19811. Daily oscillations in the morphology of SR may be related to the inhibition of neurotransmitter (vesicle) release by light, as appears to be the case in retinal photoreceptors [Schaeffer and Raviola, 19781. Assuming an accumulation of vesicles during light adaptation, there would be a greater demand for either vesicle attachment sites or neurotrans- mitter storage sites, resulting in increased size and frequency of SR. This

144 McNulty

Long Photoperiod (June)

n

E Y

5 m C

E 0 n g K

600-

460-

MODEL

GROUP TI M E ~ G R O U P

400-

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 n t m m ~ n t n t m m ~ , r r r n n n o o 0 o r

ANALYSIS OF V A R I A N C E

SUM OF -- SQUARES D.F. PR,F 197385 11 4.82 0.0001 82347 5 4.42 0.0024 93300 1 25.07 0.0001 21737 5 1,17 0.3405

Clock Hours

Fig. 4. Mean values (+SEM) of SR length over 24-h L:D cycle of long photoperiod. Controls (solid line) were sacrificed over the entrained L:D cycle (labeled #l). The photoperiod (labeled #2) for the experimental group (dashed line) was extended into the dark period. The number of animals in each group are given at the top and bottom of the standard error bars. The statistics from the two-way analysis of variance are given in the table to the right. Asterisks indicate significant differences (P < .05) between control and experimental groups at individ- ual time points according to the Duncan’s Multiple Range test.

hypothesis has been proposed previously [Spadaro et al., 1978; Wagner and Ali, 19771 and is supported by the present results demonstrating that when animals are subjected to continual light the size of SR continues to increase over the expected dark time. This effect of extending the photoperiod into the expected dark time is diminished if animals are exposed to some darkness over the 24-h period. Wagner and Ali [1977] also found that the frequency of SR in retinal cones was greater in animals subjected to continual light compared to those kept in constant darkness.

Measurements of the size of SR as done in this study do not take into account some populations such as the spherical SR. Although this population comprises a small percentage of the total number of SR, it would be impor- tant to determine if the spherical SRs also respond to environmental lighting. The common method of expressing numerical changes in SR per unit area of tissue cannot be easily applied to the pineal gland of this species since the majority of these organelles occur only in specific areas of the tissue (ix., the neuropil). These neuropil formations are not distributed evenly through- out the parenchyma of the organ [Wake, 19731. Therefore, the results could be biased depending on the area from which the sections were taken.

Synaptic Ribbons in Pineal Photoreceptors 145

Short Photoperiod (February) n

4 ANALYSIS OF VARIANCE t Y

c 0 n n L 0

n Q c v)

.- c

400 -

8@0 -

800 -

@ SOURCE z r

TIME GROUP TIME*GROUP -

4 6

i I , 112 # # W I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 N W O ~ O N w N t O O O L - - C N N N 0 0 0 0 -

SUM OF SQUARES D& 102468 11 3.23 0.0026 58468 5 4.05 0.0040 23242 1 8.05 0,0068 20758 5 1.44 0.2290

Clock Hours

Fig. 5 . Mean values (+SEM) of SR length over 24-h L:D cycle of short photoperiod. Controls (solid line) were sacrificed over the entrained L:D cycle (labeled #l). The photoperiod (labeled #2) for the experimental group (dashed line) was extended 8 h into the dark period. The number of animals in each group are given at the top and bottom of the standard error bars. The statistics from the two-way analysis of variance test are given in the table to the right. The asterisk indicates a significant difference (P < .05) between control and experimental groups according to the Duncan's Multiple Range test.

The observation that SRs occur in regions of the cell far removed from the neuropil and sometimes face other photoreceptor or supportive cells suggests that these organelles may also play a role in communication between these respective cell types. Although SRs approach the plasma membrane adjacent to photoreceptor and/or supportive cells, there are no specializa- tions such as pre- and postsynaptic thickenings characteristic of the axoden- dritic contacts. In this regard they resemble the vesicle-crowned rods (VCR) present in the mammalian pinealocyte. With loss of the neurosensory func- tion evolutionarily, the mammalian VCR no longer participates in the for- mation of true synaptic contacts. However, morphological responses of VCR to various physiological and experimental conditions have suggested that they nevertheless are functionally important structures, possibly involved in intercellular communication between adjacent pinealocytes [see Pevet, 1983; Vollrath, 19811.

The functional significance of morphological changes in SRs may be gleaned from studies on VCRs since these structures are considered to be homologs and have similar responses to environmental lighting. First, there are significant changes in the frequency of VCR over L:D cycles with peak numbers occurring during the dark period [Karasek et al., 1982b; Kurumado and Mori, 1977; Theron et al., 1979; Vollrath, 19731. Second, rodents kept

146 McNulty

under the inhibitory condition of continual light showed large increases in the number of VCR [Lues, 1971; VoIlrath and Huss, 1973; Welsh, 19771. This treatment on rabbits resulted in greatly expanded size of VCRs [Romijn, 19751. The VCRs are frequently found in closely asociated “ribbon fields” (RFs) which also increase in number under continual illumination. It is conceivable that RFs represent expanded VCRs that are folded and sectioned along various planes. Theron et al., [19Sl] were able to demonstrate by tilting their sections with a goniometer stage that single VCRs were tubular whereas the RFs contained VCRs in the form of plates. This relationship of increased size and frequency of VCRs with inhibition of pineal gland function is further supported by studies demonstrating that similar responses occur several days after animals have undergone superior cervical ganglionectomy [Karasek et al., 1982a,b; King and Dougherty, 1982b; Romijn, 1975; Welsh, 19771.

ACKNOWLEDGMENTS

The author thanks Mrs. Dwan Taylor for her excellent technical assist- ance and Mr. John Corliss for statistical and computer assistance.

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