MICROSCOPY RESEARCH AND TECHNIQUE 21:175-187 (1992)

Pinealocyte Synaptic Ribbons and Neuroendocrine Function JOHN A. McNULTY

AND

LINDA M. FOX

Department of Anatomy, Loyola University Stritch School of Medicine, Maywood, Illinois 60153

KEY WORDS

p-adrenergic receptor, Development, Isoproterenol, Melatonin, N-acetylserotonin, Ribbon fields

ABSTRACT A comparative study of pinealocyte synaptic ribbons (SR) revealed two predominant populations exhibiting either a rodhibbon shape (Sk) or a spherical/punctate shape (SR,,). Species-specific differences were found in the abundance of SR, the ratio of SR,JSRsp, and the occurrence of SR in ribbon fields. The close topographical relationship of SR to the plasma membrane and the numerical changes that occurred with changes in metabolism of the pinealocytes suggest that SR have important vesicle-mediated interactions with the cell membrane. Experiments designed to clarify the relationship between SR and pineal neuroendocrine function revealed a positive correlation between SR numbers and indole intermediates during pineal development in the rat, and increased SR frequency after denervation of the rat pineal gland or administration of the p-adrenergic agonist, isoproterenol. These data are consistent with the hypothesis that SR function is linked to receptor mechanisms regulating indoleamine production in the pineal gland. o 1992 Wiley-Liss, Inc.

INTRODUCTION The mammalian pineal gland is an ideal system to investigate structural-functional interrelationships in cell biology because the gland undergoes predictable, large amplitude metabolic changes over a 1ight:dark cycle. Moreover, pinealocyte synthesislsecretion can be easily altered by either changing the environmental lighting or by pharmacological means. One organelle, the synaptic ribbon (SR), has been of particular interest to pinealogists because it exhibits quantitative changes which in many instances parallel pineal gland production of the hormone melatonin. Based on the homology of SR to synaptic structures in photosensory pinealocytes and the close topographical relationship of SR to the plasma membrane, it has been suggested that the SR function in cell-to-cell communication and in regulating melatonin synthesis. This paper emphasizes structural-functional interrelationships of SR related to possible mechanisms of neuroendocrine transduction in the pineal gland. The first part discusses ultrastructural characteristics of SR and presents comparative data on SR populations, the formation and topography of SR, and species variations. Hypotheses of the functional role(s) of SR related to cell-to-cell signaling, melatonin production, and receptor mechanisms are addressed in the second part of the paper. ULTRASTRUCTURAL CHARACTERISTICS The SR typically comprises an elongated electrondense core associated with clear vesicles (Fig. l),although variations of this basic structural plan do occur and are discussed below. The central core, which is proteinaceous (Bunt, 1971; Krstic, 1976; McNulty, 19801,often exhibits laminae with thin arms projecting outward to the surrounding vesicles. The central core

0 1992 WILEY-LISS, INC.

of the SR has a consistent thickness of approximately 50-80 nm, but is highly variable in length extending several Fm.

Synaptic Ribbon Populations and Terminology The terms synaptic ribbon and, less commonly, uesicle-crowned rod were originally used to describe this organelle. Both terms refer to the most common shape of SR (ribbon or rod) and may or may not take into account other forms. In those studies which have not distinguished between various populations of SR the term synaptic ribbon presumably referred to all structural variations of this organelle. Other studies have used the term synaptic ribbon to designate a specific population of SR. In order to minimize confusion related to terminology, we propose utilizing subscripts to designate specific populations of SR. The most common profiles of SR (ribbon or rod) can be indistinguishable in thin-sectioned material. Occasionally, in fortuitous sections, SR also exhibit a platelike shape (Fig. 2). In this paper, ribbon-, rod-, and plate-like SR are designated S k . A second common population of SR is spherical or punctate in shape (Figs. 3,4). This population is designated SR, . In thinsectioned material, SR, can be confused wit[ rod-like SRr cut in cross section.kowever, the diam-eter of SRsp usually exceeds the width of SR, as demonstrated in Figure 5. SR, appear to be functionally different from SR, because tgese two populations exhibit phase differences over the 1ight:dark cycle (Matsushima et al., 1983a; Vollrath et al., 1983; Martinez-Soriano et al.,

Received October 10, 1989; accepted in revised form May 17, 1990. Updated December 4, 1991. Address reprint requests to Dr. John A. McNulty, Department of Anatomy, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153.

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J.A. McNULTY AND L.M. FOX

Fig. 1. A typical SR,, in rat pinealocyte. The electron-dense core bordering the plasma membrane is associated with clear vesicles 4060 nm in diameter. Bars for Figs. 1-5 indicate 0.25 pm.

Fig. 4. Punctate SR,, in rat pinealocyte. Fig. 5. RF in rat pinealocyte comprising SR, and SR,,. Note the diameter of SR,, is greater than the thickness of the SR, rod.

Fig. 2. SR, with a plate-like shape. Rat pinealocyte. Fig. 3. SR,, in rat pinealocyte with typical spherical shape.

1984; Khaledpour and Vollrath, 1987; Diaz e t al., 1990), which is seasonally dependent in some species (Riemann et al., 1990), and respond differently to environmental lighting (Vollrath, 1986) as well as to exogenously applied indoles (Vollrath et al., 1985). Other less common forms of SR have been described, includ-

ing ring-shaped and irregular-shaped SR (Matsushima et al., 1983a,b; Banks et al., 1985; Khaledpour and Vollrath, 1987). It is our opinion that many of these intermediate forms represent SR, or SRFPcut in various planes of section, although the possibility of additional distinct populations of SR is not discounted.

SYNAPTIC RIBBONS

177

TABLE I . Average numbers of synaptic ribbons (SR) comprising ribbon fields (RF) in adults of representative species

Number Species

SRIRF

Rat Rhesus monkey Baboon Ground squirrel Guinea pig Eastern chipmunk Cat

1.1-1.7 1.3-4.8 3.0-16 1.0-1.6 1.6-3.4 3.1-3.5 1.2-1.9

Largest RF

5 22 >40

8 21 80 13

Reference King and Dougherty, 1980 McNulty et al., 1986a Theron et al., 1979, 1981 McNulty, unpublished Vollrath, 1973 Karasek et al., 1982d McNulty, unpublished

Formation of Synaptic Ribbons Turnover of SR is implied by numerical changes that occur over periods of time as short as 10-30 min (Maitra et al., 1986; Karasek et al., 1988b), but to date there is little information on the formation and degradation of this organelle. However, several hypotheses have been proposed. Karasek (1976) suggested that microtubular sheaves were precursors of SR (Fig. 6a). Although little evidence exists to support this hypothesis, examples of microtubular sheaves which may represent stages of maturation leading to the formation of SR are occasionally seen (Fig. 6b). A second hypothesis states that SR are formed by division of pre-existing SR (McNulty et al., 1986a).This suggestion was based primarily on the observation that the number of SR in a field changed in response to certain experimental conditions. Alternatively, numerical changes in SR could result from simply an increase in the size of the central core thereby increasing the probability of inclusion in the plane of section. An increase in the length of SR has been observed during the dark period when increased frequency of SR normally occurs (Matsushima et al., 1983a; McNulty, 1981). Our understanding of the new mechanism(s1of SR formation must await information on the identity of the protein(s) comprising this organelle. If de novo synthesis of SR occurs, mRNA expression should precede the nighttime rise in SR numbers. Using drugs that interrupt transcription andlor translation, Net0 et al. (1990) found that the nighttime rise in SR numbers could be inhibited if protein transcription was blocked during the first half of the light phase. Translation of proteins related to SR formation appeared to take place during the first part of the dark phase.

exhibited quantitative changes in response to steroid hormones (Saidapur et al., 1991a). Species differences also exist in the distribution of the organelle. In the rat and guinea pig, the majority of SR (50-93%) bordered the plasma membrane opposite pinealocytes (Vollrath and HUSS,1973; King and Dougherty, 1980). By contrast, only 18%of SR were situated along the plasma membrane opposite pinealocytes in the vole (Hewing, 1981).

Topography A characteristic feature of SR is its close topographical relationship to the plasma membrane, an arrangement that has important functional implications which are discussed in detail below. Between 75% and 100% of SR border the plasma membrane in such species as the rat, vole, and guinea pig (Vollrath and Huss, 1973; Hewing, 1981; Saidapur et al., 1991a). By comparison, SR in the monkey (Macaca mullata) pineal gland more frequently occur in large fields away from the plasma membrane (McNulty et al., 1986a). In the rat, SR located along the plasma membrane appear to be functionally different from those located at a distance from the cell membrane because only the former population

Species Variations Table 2 lists some of the species in which pinealocyte SR were counted in a way that allowed comparisons. From this table, it is clear that considerable variability exists among species with regard t o the frequency of SR irrespective of phylogenetic status. Some of this variability is probably due to the method of counting. As mentioned above, it is not always clear whether authors have counted all SR or only specific populations of SR (e.g., SR,). Differences in the method of quantification could account for the more than tenfold discrepancy between SR frequency in unstimulated cat pinealocytes in the present study and previous reports. Variability in SR counts reported by different labora-

Ribbon Fields A distinctive feature of SR in many species is their tendency to occur in groups or ribbon fields (RF) comprising either regularly aligned SRf (Fig. 71, randomly oriented SR, (Figs. 8, 91, or mixtures of SR, and SR,, (Figs. 5, 9, 10). Large RF such as those depicted in Figures 7-9 have never been observed in contact with the plasma membrane, indicating that these “supraorganelles” may function as storage sites. A functional heterogeneity is implied by the predominance of SR, with plate-like shapes when they occur in large RF (Theron et al., 1981; McNulty et al., 1986a). From Table 1,it is evident that large RF are more prevalent in some species than in others. A possible inverse relationship between size of RF and degree of sympathetic innervation has been suggested (Karasek et al., 1983). In this regard, it is noteworthy that large RF are more prevalent in neonatal rats (Fig. 11)prior to full development of the sympathetic innervation. RF comprising more than 5 SR (SR, + SR,,) were present in pinealocytes of 5-day-old rats, but were never observed in 20day-old rats. The average number of SWRF declined as follows: 5 day = 1.48; 10 day = 1.45; 20 day = 1.24 (McNulty, unpublished data).

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J.A. McNULTY AND L.M. FOX

Figs. 6 and 7.

SYNAPTIC RIBBONS

Fig. 8. An RF in pinealocyte of a cat. Bars for Figs. 8-11 indicate 0.5 pm. Fig. 9. An RF in pinealocyte of the rhesus monkey.

Fig. 6. a: A microtubular sheave in rat pinealocyte. Bars for Figs. 6-7 indicate 0.5 pm. b: A microtubular sheave associated with severa1 clear vesicles. Fig. 7. An RF in the rhesus monkey comprising SR, reconstructed from thin serial sections. Ten of the SR, seen in a are numbered in b. The plane of section is depicted by a n arrow in a. The section b was

179

Fig. 10. An RF in pinealocyte of adult rat that had been superior cervical ganglionectomized for 7 days. Fig. 11. An RF in pinealocyte of a 5-day-old rat sacrificed during the middle of the light period.

tilted 45" by a goniometer stage in order to visualize the plate-like structure of SRs 1-6. In c, the section was rotated 90" and tilted 45" in order to visualize the plate-like structure of SRs 7-10. The two SR, in the upper left of b and c are not shown in the 3-dimensional view. Reprinted from McNulty et al. (1986a) with permission from Springer-Verlag.

J.A. McNULTY AND L.M. FOX

180

TABLE 2. Synaptic ribbon (SR) frequency (numberl20,OOO min') in pinealocytes in adults of various species' Species Rat Sprague-Dawley

SR,

SR,,

Number Total SR2

11-38

40-72

6-12

14-77

0.6-13

Long-Evans 22-180 34 19-24 12-22 30 32 40 23-44 12-49

2-20 0.8 5-1 .1-.2 0.8 0.8 1.7 1-3 0.1-8

2-15 29-50 32-97 40-95 47-84 15-31 16-43 10-30 30-50 22-34 52-99 50-128 15-153 25-201 35 19-24 13-22 31 34 42 27-51 13-73

Cotton rat

9 5-14

Kangaroo rat

Rare

Vole Mouse ICR C57B116J BALBIC Microphthalmic White-footed mouse Brush mouse Guinea pig

L;BB;cold L;Culture;OVX L

10

L

4-15 24-35 28-70 10-21

17-25 30-65 35-86 12-122 25-160 27 26 25 31 10-38 7 4-9

14-29 0 28-55

0 23-55 8 Rare 7-116

1-20 1-3 2-24

Hamster Djungarian Syrian 7-36

7-23 3-16

Gerbil

34-53 0 7

6-55

7-41

130 125-200 15-84

Karasek et al., 198313 Karasek et al., 1983c

L

Hewing, 1981

LID L;Pharm L;Pharm L L

McNulty et al., 1987 McNulty et al., 1989 Satoh and Vollrath, 1988 McNulty, unpublished Karasek et al., 1983b Karasek et al., 1983a Vollrath, 1973 Vollrath and Huss, 1973 Vollrath and Howe, 1976 Vollrath, 1986 Vollrath et al., 1983 Khaledpour and Vollrath, 1987

LID L;LL;DD L1D;Pharm LL LID LID

L 28-61 25 24 33-86 8-17 4 -28

60

Ground squirrel Richardson's

L L;SCGX

L 2-47 2-52 2-37 2-6

60-125 42-108 43 50

LID LL;DD;Pharm SCGX;Pharm L;SCGX L L L;Pharm L L L LID LID

L1D;Culture LID L1D;BB LID LID LID L;Pharm L:Stim LiD:Culture:Dharm

Karasek et al., 1982a

40-83 11-16 0

Reference Cos et al., 1989 Karasek, 1976 Karasek et al., 1982e Karasek e t al., 1983b Karasek and Vollrath, 1982 Kurumado and Mori, 1977 Kurumado and Mori, 1980 Maitra et al., 1986 McNulty et al., 1985 McNulty et al., 1987 McNulty et al., 1989 Reuss et al., 1989 Vollrath et al., 1985 Vollrath and Maitra, 1986 Vollrath and Welker, 1984 Net0 et al., 1990 Saidapur et al., 1991b King and Dougherty, 1980 King and Dougherty, 1982a King and Dougherty, 1982b McNulty, this study Saidapur et al., 1991b Kosaras et al., 1983a Kosaras et al., 1983b Saidapur et al., 1991b Saidapur et al., 1991b Saidapur et al., 1991b Riemann et al., 1990 Seidel et al., 1990a

12-32 9-24

39

3 -25 7-20 1-20

13-lined

12-42 1-20 6-20 . -.

Lewis DA Brattleboro Roman

Experimental conditions3

18-80 35-75 14

Wistar

RF

65 65-85 13-56

L

Fechner, 1986 Karasek et al., 1982c Karasek et al., 1983b Hewing, 1979 Hewing, 1980 Vollrath and Maitra, 1986 Diaz et al., 1990 Heinzeller, 1985

L LID L

Karasek et al., 1983b Karasek et al., 1982d McNulty, unpublished

L;SCGX L 1, L E;DD L;DD L;LL LID

fcontinued)

181

SYNAPTIC RIBBONS

TABLE 2. Synaptic ribbon (SRI frequency (number/20,000 m i d ) in pinealocytes in adults of various species' (Continued) Number Species Chipmunk

SR,

SR,,

Cat

Experimental

Total SR2 247 250-450 260

RF 80-130 85

Reference Karasek et al., 1982b Karasek et al., 1982d Karasek et al., 19831,

12-68 12-58 14 175

8 107

L L

Gonzalez and Alvarez-Uria, 1986 Gonzalez and Alvarez-Uria, 1987 Karasek et al., 1983b McNulty, unpublished

Fox

11

7

L

Karasek et al., 1983b

Pig

Rare

140

Rabbit

7-33

35

14-27

Rhesus monkey Owl monkey

30-106

11

Baboon

65

79 16-21

9-53

conditions3 L LID L L;Stim L;SCGX

Karasek and Wyrzykowski, 1980 21-60

LID

Martinez-Soriano et al., 1984

14-48

L;Pharm

McNulty et al., 1986a

54

L

McNulty, unpublished

LID

Theron et al., 1979

1-7

'All published values are rounded. 'Total numbers are given where authors did not specify the population(s) of SR. 3BB, bilateral optic enucleation; Cold, subjected to cold; Culture, glands in culture; DD, continuous darkness; L, counts taken during the light period WD, range of counts taken over the 1ight:dark cycle; LL, continuous lighting; OVX, ovariectomized; Pharm, pharmacological study; RF, ribbon field; SR,, rod type SR SR,,, spherule type SR, Stim, electrical stimulation of nerve.

tories may also be related to the time of year that the experiments were conducted. It was recently reported that frequency of SR, and SR,, displays differences over the circannual cycle (Karasek e t al., 1988a; McNulty et al., 1990; Riemann et al., 1990; Seidel e t al., 1990a). The functional significance of species variability in frequency of SR was studied by Karasek et al. (1983b). These authors reported that numbers of pinealocyte SR were inversely related to the density of noradrenergic nerve endings and the concentration of noradrenalin in different mammals. The authors proposed that the increased density of SR reflected enhanced sensitivity of pinealocytes, which was compensatory for reduced innervation. It is noteworthy that the pigmented LongEvans strain of rat, which tends to have a higher density of SR than the albino Sprague-Dawley strain (Table 2), is also more sensitive to the effect of light on melatonin synthesis (Lynch et al., 1984). The frequency of SR, and SR,, in pinealocytes of different strains of rats was recently compared and found not to vary. However, samples in this study were collected during the photophase of the 1ight:dark cycle when the circadian rhythm in SR number is a t its nadir (Saidapur e t al., 1991b).

linked to endocytotic processes involving turnover of membrane components (e.g., receptors).

Relationship of SR to Melatonin Production Increased frequency of SR during the dark, when pineal melatonin production is normally elevated, has been reported in several species (rat, guinea pig, ground squirrel, chipmunk, rabbit, hamster, and baboon) and has led to speculation that the SR is a morphological substrate for indoleamine synthesis. Furthermore, when rats were exposed to unexpected light a t night there was a rapid and significant decline in SR numbers which paralleled the rapid and significant decline in activity of the N-acetyltransferase enzyme (Maitra et al., 1986). To test the hypothesis that SR are related to indoleamine production, developmental changes in SR frequency in neonatal rat pinealocytes were correlated with ontogeny of the circadian rhythm in indole biochemistry. For the present study, pups entrained to a 1ight:dark cycle of 12 hours light were sacrificed during mid-light and mid-dark at 5, 10, and 20 days of age. Pineal glands were collected and processed for either electron microscopy or high performance liquid chromatography for analysis of pineal indoles. Counts of SR and RF were greater at night in all age groups with peak numbers for both day and night FUNCTION OF SYNAPTIC RIBBONS occurring at 10 days of age (Fig. 12a). Two-way analThe SR evolved a s a specialization for synaptic trans- ysis of variance (ANOVA) revealed a significant effect mission in special sensory organs. It seems logical to of age and day vs. night sampling on SR numbers. The assume that a modification of this synaptic function nocturnal increase in SR at day 10 was significantly has been retained with the phylogenetic transition of greater than at 5 and 20 days of age as determined pinealocytes from a photoreceptive to a n endocrine by the Tukey-Kramer multiple comparisons test. Alfunction. The close juxtaposition of SR to the plasma though SR were elevated during night at 5 days of age, membrane suggests that SR are involved in cell-to-cell day vs. night differences were not significant (P > signaling where SR-associated vesicles release trans- 0.05). Pineal levels of melatonin and its precursor, Nmitter-like compounds into the extracellular compart- acetylserotonin (NAS), were elevated at night in all ment. Alternatively, SR and associated vesicles are age groups with a significant effect of day vs. night-

J.A. McNULTY AND L.M. FOX

182

0.12-

70 60

-

-Y

-

=L

c

? 0.08-

4 0-

%

30-

9

5 0.049

C

0

-0.06

5aJ

-

In

x

Y

a,

U

40

J C

1

.4

% .

v)

10 -

G C

2 50-

c)

20-

d

2

-

E

0.06-

-Y v

a

H

\

% *

-0.08

a,

s .+

2

60 L4

v

C

0 0

-0.10

0.10-

\

2 50:

70 7

u

-

-0.04

2 0.02-

5 Day 10 Day 20 Day

5 D a y 10Day 20Day

AGE

AGE

@

5Day 10Day20Day

AGE

Fig. 12. Means ( ? SEM) of day vs. night measurements of SR numbers (a)and N-acetylserotonin (b) during pineal development in the rat. The high correlation between SR and N-acetylserotonin is emphasized when nighttime values are compared (c).

sampling (ANOVA) on both indoles. Analysis for the effect of age was significant for NAS, but not melatonin. Multiple pair-wise comparisons indicated that this effect of age on NAS was the result of elevated nighttime levels in 10-day-old animals (Fig. 12b) reflecting the dramatic increase in the pineal N-acetyltransferase enzyme which has been reported to occur at 10 days of age (Altar et al., 1983). The results of the present developmental study are in close agreement with the observations of King and Dougherty (1980), who demonstrated that the day vs. night rhythm in SR numbers appears during the second postnatal week with a peak a t 10 days. The present study extends these observations revealing that the nocturnal increase in SR numbers at 10 days of age is closely correlated with elevation of NAS a t this age, particularly when nighttime values are compared (Fig. 12c). These findings are consistent with the hypothesis that SR formation is either a prerequisite or a consequence of activation of the melatonin biosynthetic pathway. Several other studies dispute this hypothesis. First, exposure to continual lighting, which suppresses melatonin production, leads to increased SR numbers in most species (Lues, 1971; Vollrath and HUSS,1973; Romijn, 1975; Roux et al., 1977; Vollrath and Maitra, 1986). Second, SR exhibited day vs. night differences in mouse pineal glands incapable of producing significant amounts of melatonin (McNulty et al., 1987). Third, increased N-acetyltransferase activity after stimulation of the superior cervical ganglia was not accompanied by increased numbers of SR in the rat (Reuss et al., 1989). Finally, as opposed to pooled samples, there was no correlation between SR numbers and melatonin levels in single rat pineal glands (Vollrath and Welker, 1984). With regard to the latter observation, a strong

correlation between SR and melatonin might not be expected in individual animals because melatonin exhibits few correlations with intermediate products and enzymes due to melatonin’s pulsatile production and/or release under normal 1ight:dark cycles (Champney e t al., 1984; McNulty et al., 1986b). More recently, Net0 e t al. (1990) have demonstrated that transcription of proteins related to melatonin production and SR formation are not temporally synchronized over the light: dark cycle.

Role in Intercellular Communication The hypothesis that SR function in cell-to-cell signaling is supported by three important observations: 1) specializations resembling pre- and post-synaptic densities are present where SR contact the plasma membrane (Figs. 13-17); 2) coated pits are found along the plasma membrane of pinealocytes opposite SR (Hewing, 1981; McNulty et al., 1987; McNulty et al., 1989); and 3) SR are specifically oriented along the plasma membrane bordering cell processes that resemble neurites (Figs. 17-19). Conventional synapses are also found between neuron-like processes and pinealocytes (Figs. 17,20). Although direct cell-to-cell synaptic-like signaling may occur in some instances, i t is more likely that SR participation in cell-cell interactions is of a more diffuse nature (Hewing, 1981). This possibility is based on the observation t h a t SR are topographically related to various other cellular components and compartments including the perivascular space (Fig. 16). In the rat and the vole, between 20% and 25% of all SR bordered the perivascular spaces (King and Dougherty, 1980; Hewing, 19811,whereas in the Malaysian rat SR were

SYNAPTIC RIBBONS

183

Fig. 13. SR,, with thickening of plasma membrane. Rat pinealocyte. Bars for Figs. 13-16 indicate 0.5 pm.

Fig. 15. Plasma membrane thickenings (arrow) adjacent to an SR,, in rat pinealocyte.

Fig. 14. SR in pinealocyte of rat that had been superior cervical ganglionectomEed. The SR, parallels the plasma membrane which exhibits several thickenings (arrows).

Fig. 16. SR, with thickening of the plasma membrane adjoining the basal lamina of the pericapillary space. Rat pinealocyte.

184

J.A. McNULTY AND L.M. FOX

Figs. 17-20.

SYNAPTIC RIBBONS TABLE 3. Effects of bilateral superior cervical ganglionectomy on counts of synaptic ribbon rods (SRJ and synaptic ribbon spherules (SR,,) (number120,0001mm2)'

185

sulted in the formation of SR,, which are normally absent in this species, and a proliferation of SR,, (Masson-Pevet and Pevet, 1990). SR frequency was also SRSR.. elevated in denervated pineal gland transplants into Control (n = 6) 31.7 2 2.08 5.73 2 234 the cerebral cortex (McNulty et al., 1991). King and SCGX (n = 6) 132.8 ? 19.49 11.03 -+ 286 Dougherty (1982b) examined more closely the temporal effects of SCGX on SR numbers in the rat and re'Figures represent means ? SEM ported an initial decline 12-24 hours after SCGX followed by a gradual increase in SR numbers which peaked between 3-7 days after the surgery. predominantly located along the plasmalemma borderOther investigations have used pharmacological ing the pericapillary spaces (Pevet and Yadav, 1980). techniques to test the hypothesis that SR are functionally related to p-adrenergic receptors. The p-adrenRole in Endocytosis and Receptor Mechanisms ergk agonist, isoproterenol, elevated SR numbers Because coated pits and coated vesicles are found in when administered during the day in rats, but not in the vicinity of SR (Fig. 14, 18) (King and Dougherty, mice (McNulty et al., 1989). These effects of isoprotere1982a1,it is conceivable that endocytotic processes and no1 on rat SR were observed 3 hours after injection. SR membrane turnover are linked to SR function(s). King numbers were not elevated above saline-injected conand Dougherty (1982a) proposed that SR participation trols when rats were administered isoproterenol (i.p.) in membrane internalization could involve the p- during the middle of the photoperiod and sacrificed afadrenergic receptor which activates, via second mes- ter either 20 minutes or 1hour (McNulty, unpublished sengers, the N-acetyltransferase enzyme leading to the data). This pharmacologically induced gradual information of melatonin. The evidence supporting this crease in SR numbers over an extended period of time hypothesis is indirect. mimics the gradual increase in SR numbers that occurs In one experiment, male Long-Evans rats (3-4 during the scotophase of the 1ight:dark cycle and may months) were superior cervical ganglionectomized help explain the recent finding that significant (SCGX) bilaterally, a procedure that sympathetically changes in SR frequency did not occur in rat pineal denervates the pineal gland and up-regulates p-adren- glands 1-2 hours after isoproterenol administration ergic receptor sites (Craft et al., 1985). Control animals (Seidel et al., 1990b). The discrepancies in reported efunderwent the same surgical procedures, but the gan- fects of isoproterenol on SR numbers appear also to glia were only exposed and not removed. Each of the be age related (Vollrath, personal communication). animals which had undergone SCGX experienced pto- Vollrath and Howe (1976) reported a nearly twofold sis confirming successful denervation. After 5-7 days, increase in SR numbers after norepinephrine treatpineal glands from both groups were collected for elec- ment of guinea pigs during the day. However, the effect tron microscopic analysis. Both SR, and SR, showed a of norepinephrine was not statistically significant due statistically significant increase in the SZGX group perhaps to the small sample size and the fact that an(Table 3). Furthermore, RF comprising 4 or more SR imals were sacrificed 4-5 hours post-injection when were observed in denervated pinealocytes (Fig. lo), but the effects of the drug are diminished. If rat pineal not in control animals. These results suggest a positive glands are supersensitized, an isoproterenol-induced correlation between p-adrenergic receptor density and increase in SR numbers was seen after a much shorter SR numbers. Similar effects of denervation on fre- time period of 15-20 minutes (King and Dougherty, quency of SR have been reported in the pineal gland of 1982a,b). Propranolol, a p-adrenergic blocker, prethe rabbit (Romijn, 1975), the gerbil (Welsh et al., vented the normal nocturnal increase in SR numbers 1979), the cotton rat (Karasek et al., 1983c), the Djun- in guinea pigs (Vollrath and Howe, 1976), but had no garian hamster (Karasek et al., 1982c), and the cat effect on SR in rats (Seidel et al., 1990b). Propanolol (Gonzalez and Alvarez-Uria, 1987). Sympathetic den- did, however, reverse the increased frequency of SR in ervation of the European hamster pineal gland re- rats after exposure to continual darkness (King and Dougherty, 1982a). Studies in which the superior cervical ganglia have been electrophysiologically stimulated to release enFig. 17. SR, in pinealocytes of the rhesus monkey bordering a dogenous pools of norepinephrine have yielded results neuron-like process with specializations resembling pre- and post- which are inconsistent. Gonzalez and Alvarez-Uria synaptic densities along the cytoplasmic membranes (arrowheads). A (1986) were able to demonstrate a significant increase second unidentified cell is forming a conventional synapse with this process (arrow). Bar indicates 0.5 pm. Reprinted from McNulty et al. in SR number after 15 minutes stimulation of the SCG in the cat. In contrast, Reuss et al. (1989) could not (1986a) with permission of Springer-Verlag. detect a significant increase in SR after SCG stimulaFig. 18. Several SR, oriented along the plasma membrane oppo- tion for either 15 minute or 1hour in rats. These dissite neuron-like processes in the 13-lined ground squirrel. Coated pita crepancies may be related to species differences in the (arrows) are in the vicinity. Bar indicates 0.5 pm. innervation of the gland because norepinephrine levels Fig. 19. Pinealocyte SR, abutting a neuron-like process in the in the cat pineal gland are about threefold greater than American opossum. Bar indicates 0.5 pm. in the rat (Kus et al., 1989). It should also be noted that Fig. 20. A conventional synapse between a neuron-like process the lack of effect of 1hour SCG stimulation in the rat is consistent with our findings that 1 hour treatment and a pinealocyte in the oppossum. Bar indicates 0.25 pm. ~

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with isoproterenol had no effect on SR numbers in this species. The plasticity of SR function(s) is further evidenced by data implicating involvement of SR in steroid hormone regulation of pineal melatonin production. Most recently, Saidapur et al. (1991a) demonstrated a significant decline in SR frequency when pineal glands of rats were incubated in the presence of either progesterone or estrogen (estradiol-17P). Interestingly, only those SR adjacent to the plasma membrane were affected, a finding consistent with the hypothesis that SR play an important role in plasma membrane functionb). The response of SR to steroids also emphasizes the complexity of the cell biology of these organelles because estradiol and progesterone either enhance or have no effect on melatonin production and CAMPlevels in the pineal gland (Cardinali et al., 1986, 1987).

CONCLUSION In summary, it is apparent from the data presented in this study that SR in mammalian pinealocytes are complex organelles that may be linked to cellular mechanisms regulating indoleamine production by the gland. Although specific mechanisms regulating temporal changes in SR have still to be elucidated, most of the available evidence suggests that the mechanisms involve interactions with the plasma membrane and receptor functions. REFERENCES Altar, A,, Motroni, T.P., and Lytle, L.D. (1983) Functional synaptogenesis and the rat pineal gland: A pharmacological investigation. J. Neural Transm., 58:231-244. Banks, J.C., Dalgleish, A.E., and Vollrath, L. (1985) Postnatal development of “synaptic” ribbons and spherules in the guinea pig pineal gland. Am. J. Anat., 173:43-53. Bunt, A.H. (1971) Enzymatic digestion of synaptic ribbons in amphibian retinal photoreceptors. Brain Res., 25571-577. Cardinali, D.P., Vacas, M.I., Sarmiento, M.I.K., Etchegoyen, G.S., Pereyra, E.N., and Chuluyan, H.E. (1987) Neuroendocrine integrative mechanisms in mammalian pineal gland: Effects of steroid and adenohypophyseal hormones on melatonin synthesis in vitro. J. Steroid Biochem., 27:565-571. Cardinali, D.P., Vacas, M.I., Solveyra, C.G., Sarmiento, M.I.K., and Vollrath, L. (1986) In vitro effects of estradiol, testosterone, and progesterone on 5-methoxyindole content, cyclic adenosine 3’,5’monophosphate synthesis and norepinephrine release in different parts of the female guinea pig pineal complex. J . Pineal Res., 3: 351-363. Champney, T.H., Holtorf, A.P., Steger, R.W., and Reiter, R.J. (1984) Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland. J. Neurosci. Res., 1159-66. Cos, S., Bardasano, J.L., Mediavilla, M.D., and Sanchez Barcel, E.J. (1989) Synaptische Bander in den Pinealocyten bulbektomierter Ratten unter experimentellen Bedingungen. J. Hirnforsch., 30:9198. Craft, C.M., Morgan, W.W., Jones, D.J., and Reiter, R.J. (1985) Hamster and rat pineal gland B-adrenoreceptor characterization with iodocyanopindolol and the effect of decreased catecholamine synthesis on the receptor. J . Pineal Res., 251-66. Diaz, C., Alvarezuria, M., Tolivia, J., and Lopez, J.M. (1990) Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus aurutus). Cell Tissue Res., 262:165169. Fechner, J . (1986) Influence of photoperiod on dense-cored vesicles and synaptic ribbons of pinealocytes of the Djungarian hamster (Phdopus sungorus).J. Neural Trans., 67:139-145. Gonzalez, G., and Alvarez-Uria, M. (1986) Morphometric analysis of the synaptic ribbons and nerve vesicles of the cat pineal gland after

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