Journal of Pineal Research 3:15-23 (1986)

Morphometric Analysis of the Synaptic Ribbons and Nerve Vesicles of the Cat Pineal

Gland After Electrical Stimulation of the Superior Cervical Ganglia

Gabino Gonzalez and Manuel Alvarez-Uria

Consejo Superior de Investigaciones Cientificas, Madrid (G.G.), and Department of Microscopic Morphology, University of Oviedo, Oviedo (G.G., M.A.-U.)

The nerve vesicles and synaptic ribbons were quantified in thc cat pineal gl:ind after electric:ll stimulation o f the pineal sympathetic nerve fibers. I t has heen shown that the bilateral electrical stimulation o f the preganglionic fibers innervating the superior cervical Kanglia ( S C G ) m:irkedly reduces the number o f dense cores o f small dark vesicles (SDV) and. o n the other hand. modifies the number and shape o f the large dark vesicles (LDV). A n increase in the nuniher o f the synaptic ribbons after stimulation o f the SCG supports the hypothesis that the numerical reduction o f dense cores o f nerve vesicles in the c:it pineal gl;ind influences the level o f synaptic ribbon formation.

Key words: pineal gland, cat , noradrenerg ic innervat ion, synapt ic rib- bons, electrical stimulation, ul t ras t ructure . m o r p h o m e t r y

INTRODUCTION

The pineal gland has been shown t o receive an autonomic innervation in all mammals studied so far; with special regard to the rabbit pineal gland, a detailed description of the penetration and terminal arborization o f sym- pathetic nerve fibers in this organ was given by Ram6n y Cajal [1895], who

Received March 4, 1985; accepted July 31, 1985.

Address reprint request to G. Gonzalez, Department of Microscopic Morphology, University of Oviedo, 33006 Oviedo, Spain.

0 1986 Alan R. Liss, Inc.

16 Gonzalez and AlvareZ-Uria

stated that these fibers originated from the superior cervical ganglia (SCG). It is now generally accepted that the mammalian pineal gland receives a rich nervous supply from nerve cell perikarya in the SCG [Bowers and Zigmond, 19821.

Superior cervical ganglionectomy in several mammalian species has furnished convincing evidence of the sympathetic origin on the intrapineal nerve fibers [Kappers, 1960; King and Dougherty, 19824. Furthermore, fluorescence microscopy [Mdller and Van Veen, 198 1 ; Hess, 198 1 ; Hwang, 1982; Matsuura and Sano, 19831, cytochemical techniques [Romijn, 19753, immunocytochemical studies [Matsuura et al., 19831, and autoradiography [Taxi and Droz, 19661 have demonstrated the noradrenergic nature of these fibers.

The functional activity of the pineal gland is believed to be controlled by norepinephrine release from sympathetic nerves. This neurotransmitter stimulates the formation of a pineal hormone, melatonin, and therefore is of utmost importance in the regulation of this pineal function [Cardinali, 19831.

It has been demonstrated that the pineal gland of Felix catus domesticus is richly innervated by sympathetic postganglionic nerve fibers in compari- son to some other species examined [Karasek et al., 1983al. These nerve fibers contain a considerable number of small dark vesicles (45-60 nm), a few clear vesicles (45-60 nm), as well as a smaller number of large dark vesicles (80-120 nm) [Gonzilez et al., 1969, 1976; Gonzalez and Alvarez- Uria, 1984al. The small dark vesicles are storage sites for norepinephrine and serotonin Uaim-Etcheverry and Zieher, 1968, 1980; Pellegrino de Iraldi and Gueudet, 1969; Duffy and Markesbery, 1970; Huang et al., 1979; Hess, 1981; Matsuura et al., 19831. On the other hand, Jaim-Etcheverry and Zieher (1971) reported that “serotonin and noradrenaline are not only simultaneously present in these nerves but, as suggested by indirect evidence, may even coexist within their vesicles.”

Pineal synaptic ribbons (SR) are ultrastructural cell organelles of pineal- ocytes [Gonzilez et al., 19691 in all mammalian species studied so far [PCvet, 19791. Synaptic ribbons in the mammalian pinealocyte are apparently related to the pineal adrenergic innervation [Vollrath, 198 I]. This hypothesis has been examined under a wide variety of experimental conditions, including changes in light-dark cycle [Lues, 1971; Vollrath and Huss, 1973; Vollrath and Howe, 1976; Hewing, 1980; King and Dougherty, 1982b], blinding [Kurumado and Mori, 19801, pineal synipathectomy [Romijn, 1975, 1976; King and Dougherty, 1982a; Karesek et al., 1982, 1983b], and pineal organ culture [Karasek, 1976; Romijn and Gelsema, 19761. Additionally, an inverse correlation between SR number and the density of adrenergic nerve endings in the pineal gland of a diverse group of mammalian species has been demonstrated [Karasek et al., 1983al. These structures are relatively sparse in cat pinealocytes in comparison to some other mammalian species [Karasek et al., 1983al.

The purpose of our study was to perform a morphometric analysis of the nerve vesicles and synaptic ribbons in the cat pineal after electrical stimulation of the SCG.

Stimulation of the Pineal Sympathetic Nerves 17

MATERIALS AND METHODS

Sixteen adult male cats were housed in a room with controlled illumi- nation (LD 12:12, 8 AM to 8 PM) and temperature (22°C f 2°C) and had constant access to food and tap water for at least 2 weeks before used. Two groups consisting of eight animals each were formed. Cats were anesthetized with sodium pentobarbital (35 mg/kg IP). To expose the cervical sympathetic trunk for stimulation, a midline incision was made along the animal’s neck. The superior cervical ganglia and sympathetic nerve trunks were dissected free of the vagus nerves and carotid arteries, bilaterally exposed, and mounted on bipolar electrodes. The current used for stimulation of the cervical sym- pathetic trunks was that required to produce maximal exophthalmus and it was used to check the effectiveness of the stimulation.

Preganglionic trunks were bilaterally stimulated with square wave pulses generated by a constant current stimulator (25 Hz, 1 ms pulse duration) for 15 min. Bilateral stimulation was performed between 10 AM and 12 AM on eight animals. Whereas the other eight cats were treated exactly as outlined above except that the passage current through the electrodes was omitted (sham-stimulated animals). After the stimulation or sham-stimulation, the cats were killed by cardiac perfusion (between April 12-26, 1983). Four cats from each group were fixed by perfusion with 2.5% glutaraldehyde: 1% para- formaldehyde in 0.1 M Na cacodylate buffer, pH 7.5, at room temperature for 10 min and another 10 min at 4°C. All animals had assisted respiration using a respirator connected to a tracheal cannulae.

Immediately after perfusion, the pineal glands were removed and one block of the central portion of each gland was dissected. The tissue blocks were postfixed for another 2 hr in the above fixative and then rinsed in the buffer for 12 hr at 4°C postfixed at OsO4 in 0.1 M cacodylate buffer for 2 hr, dehydrated in graded series of acetone, stained in 2% uranyl acetate in 70% acetone overnight, embedded in epoxy resin, and oriented in such a way that frontal sections were cut from the center of the blocks.

To determine whether the pleomorphic vesicle population occurring after stimulation of the pineal gland corresponds to one or more functional types, the triple fixation procedure of Tranzer and Richards [ 19761 was used to identify the dark vesicles (four cats per group were used). Sections of 60- 80 nm in thickness were obtained, stained with uranyl acetate and lead citrate, and examined with a Zeiss E.M. 10. Twenty micrographs of each animal were taken at a magnification of 12,000 diameters and photographi- cally enlarged to 36,000 diameters. The areas of the nerve vesicles were measured on the photographs by tracing onto milimeter-ruled graph paper. The calculated data were expressed as the total number of nerve vesicles per 1.25 pm2 of pineal tissue [Gonialez and Alvarez-Uria, 1984al.

Synaptic ribbons (SR) and ribbon fields (RF) were counted in the pineal tissue completely covering five randomly selected grid squares (200 mesh; total area of 45,125 pm2 per animal) and data were expressed as the number per 20,000 pm2 of pineal tissue [Karasek et al., 19821. A ribbon field was

18 Gonzalez and Alvarez-Uria

Stimulation of the Pineal Sympathetic Nerves 19

defined as one or more spatially related SR [Vollrath, 1973; Vollrath and Huss, 19731.

A statistical analysis was carried out on the resultant data to determine the mean, standard error, and significance according to Student’s t test.

RESULTS

Ultrastructural studies of the adrenergic endings originating in the SCG of the normal cat pineal gland showed characteristic populations of small and large vesicles, most of them displaying an osmiophilic dense core (Fig. 1). After electrical stimulation, the majority of the dense cores of the small vesicles were markedly depleted and their shape appeared rounded or ellipt- ical (Fig. 2). The number of large dark vesicles was modified and the shape of some of them was elongated (Fig. 2). No degeneration of axon processes or terminals was apparent after electrical stimulation of the pineal sympa- thetic nerves.

In pinealocyte of the controls, synaptic ribbons were observed lying on processes and perikarya. SR appeared either singly or in groups of two or three (Fig. 3). A maximum of 15 SR per RF were only occasionally observed.

SR were noted in processes and perikarya of the stimulated cats. SR were mostly lying in groups of five to ten elements, and only in some instances singly (Fig. 4). The matrixes of some of the mitochondria appeared swollen and clear.

The frequency of the respective types of nerve vesicles in the adrenergic endings of the gland were quantitatively determined in normal and stimu- lated pineal glands.

In sham-stimulated (control) cats, the different vesicle types showed an almost constant frequency. The number of small dark vesicles was higher than those of clear vesicles and large dark vesicles (Fig. 5).

After the stimulation, all the vesicles showed remarkable changes in their proportions. The numbers of clear and large dark vesicles increased (Fig. 5) . The small dark vesicles decreased under these experimental condi-

Fig. 1. Pineal sympathetic nerves of sham-stimulated (control) cats. The endings contain numerous vesicles of various size and dense cores (small, large, and clear). Tissue was pro- cessed according to the technique of Tranzer and Richards [1976]. X36,000.

Fig. 2 . Stimulated cats. Most of the small vesicles are empty and some of them contain a very small dense core. Several large dark vesicles are also present. Tissue processed as in Figure 1. x36,000.

Fig. 3. Synaptic ribbons of sham-stimulated (control) cats. One (SR) trilaminar rodlet is lying near the nucleus; the others, consisting of two SR, are apparently without relationship to the plasmalemma of the pinealocyte. ~ 4 0 , 0 0 0 .

Fig . 4. ribbons are attached to the cell plasmalemma (arrowhead). X 14,700.

Stimulated cats. There are numerous ribbon fields (arrow), and some of the synaptic

20 Gonzalez and Alvarez-Uria

CONTROL

70-

60-

50-

40 - 30-

20-

10-

-

STIMULATED

OJ

Fig. 5 .

LDV SDV 3 cv

100

50

0

r . .:. : : j

;:: . . . _... ...:.;

.*.. :;: >

.I;,.. . '. ..:*::,.:;

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LDV SDV CV

- Numbers of vesicles of the adrenergic nerve terminals in the pineal gland of sham- stimulated (control) and stimulated cats. The vertical lines from the top of the bars indicate SEM. LDV, large dark vesicles; SDV, small dark vesicles; CV, clear vesicles.

RIBBON FIELDS

3 C

SYNAPTIC RIBBONS

rl C

T

ST

Fig. 6. Numbers of ribbon fields (RF) and synaptic ribbons (SR) in the pineal gland of sham- operated (control) and stimulated cats. The vertical lines from the top of the bars indicate SEM. C, sham stimulated (control); ST, stimulated.

Stimulation of the Pineal Sympathetic Nerves 2 1

tions. The number of SR and RF significantly (P<O.OOl) increased after stimulation (Fig. 6).

DISCUSSION

According to our results, it seems clear that electrical stimulation simul- taneously induces both qualitative and quantitative changes in nerve vesicles and in the number of synaptic ribbons of the cat pineal gland. The qualitative modifications produced in the shape and size of the SDV are similar to those described in adrenergic fibers of the rat pineal gland after electrical stimula- tion Wdim-Etcheverry and Zieher, 1980, 1983; Pellegrino de Iraldi and Cor- azza, 19811. The results of the present study show that the electrical stimulation of the preganglionic fibers from the SCG dramatically decreases the number of dense cores in the small dark vesicles of adrenergic terminals of the cat pineal gland and also reduces the size of the few remaining cores. These modifications of SDV suggest that the neurotransmitter, norepineph- rine, present in the cores of SDV, might be released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo.

The frequency of LDV was statistically different between stimulated and control nerves. This fact indicates that they were not depleted and that their number increases during stimulation, some of them being elongated. N o large elongated vesicles were found in the pineal sympathetic nerves of sham-stimulated cats (controls).

Finally, the neural stimulation of the pineal gland can be abolished without denervating the gland by electrical stimulation or by cutting the cervical sympathetic trunk [GonzBlez et al., 19761, which contains the pre- ganglionic input to the SCG. In spite of this fact, we hypothesize that after a bilateral electrical stimulation of the SCG as well as following bilateral pre- ganglionectomy [GonzBlez et al., 19761 the pineal sympathetic nerves do not receive the type of neural information from the central nervous system that is necessary to regulate the level of nerve vesicles.

Quantitative analysis indicates that the numbers of both RF and SR significantly (P < 0.00 1) increase after electrical stimulation. Although a rela- tionship between the adrenergic innervation of the pineal gland and SR formation has been hypothesized [Vollrath, 1981; Karasek et al., 1983a1, the nature of this relationship is still being questioned.

Although the functional significance of SR in the mammalian pinealo- cyte is obscure, the numerical changes in these structures under different natural and experimental conditions have resulted in various hypotheses concerning their role in the pineal function [see Karasek et al., 1983al. Common to these studies, King and Dougherty [1982a,b] suggest that the formations of SR may be involved in the regulation of beta-adrenergic recep- tors along the plasmalemma of the pinealocyte.

Our study suggests an inverse numerical relationship between dense cores of nerve vesicles and SR or RF in the cat pineal gland after electrical stimulation. This idea has been suggested for the cat pineal gland after superior cervical preganglionectomy [GonzAlez and Alvarez-Uria, 1984bl.

22 Gonzalez and Alvarez-LTria

ACKNOWLEDGMENTS

We are grateful to Armando MenCndez, Jorge Arias, Puri Arribas, and Beatriz G6mez for technical assistance. This work was supported by C.AkI.C,.Y.’I’. grant 4461.

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