Journal of Pineal Research 4367-376 (1987)

Effects of Superior Cervical Preganglionectomy on Nerve Vesicles and Synaptic Ribbons in the Cat Pineal Gland

Gabino Gonzalez and Manuel Alvarez-Uria Cajal Institute, Consejo Superior de Investigaciones Cientificas (G.G.), and Department of Microscopic Morphology, University of Oviedo (M.A-U.),

Oviedo, Spain

Previous studies have shown that bilateral preganglionectomy of the superior cervical ganglia (SCGp) modifies the nerve vesicles and synaptic ribbons in the cat pineal gland. In the present study the nerve vesicles (NV) and synaptic ribbons (SR) were analyzed morphometrically after chronic preganglionectomy of the pineal sympathetic nerve fibers. It was shown that bilateral sympathectomy of the preganglionic fibers innervating the superior cervical ganglia (SCG) markedly reduces the number of dense cores of small dark vesicles (SDV) and, on the other band, modifies the number and shape of the large dark vesicles (LDV). An increase in the number of the synaptic ribbons after chronic preganglionectomy of the SCG supports the hypothesis that the numerical reduction of dense cores of nerve vesicles in the cat pineal gland influences the level of synaptic ribbon formation.

Key words: noradrenergic innervation, morphometrics

INTRODUCTION

Our earlier studies on the adrenergic innervation of the cat pineal gland have shown that the nerve vesicles (NV) and synaptic ribbons (SR) are altered by preganglionectomy [Gonzilez et al., 1976; Gonzhlez and Alvarez-Uria, 19841 or electrical stimulation of the superior cervical ganglia (SCG) [Gonzi- lez and Alvarez-Uria, 1986al. The sympathetic innervation of the mammalian pineal organ was initially studied in rabbit by Ramon y Cajal [ 18951 and later confirmed in rat by Kappers [ 19601 and Bowers and Zigmond [ 19821. The existence of this innervation has been confirmed in all mammals studied up to the present [Korf and Moller, 19841. Photic information is transmitted via

Received June 20, 1986; accepted December 8, 1986.

Address reprint requests to G. Gonzilez, Department of Microscopic Morphology, University of Oviedo, 33006 Oviedo, Spain.

@I 1987 Alan R. Liss, Inc.

368 Gonzalez and Alvarez-Uria

the sympathetic route to the pineal gland [Moore, 19781, and the release of noradrenaline from the nerve vesicles of the adrenergic boutons stimulates the formation of synaptic ribbons [Gonzdez and Alvarez-Uria, 1986bI and the synthesis of melatonin, involving a beta-receptor in the cell membrane and the cyclic-AMP system [Zatz et al. 19781.

Superior cervical ganglionectomy in several mammalian species has furnished convincing evidence of the sympathetic origin of most, if not all, of the intrapineal nerve fibers [Kappers, 1960; King and Dougherty, l982aI. Furthermore, fluorescence microscopy [Moller and Van Veen, 1981; Mat- suura and Sano, 19831, cytochemical techniques [Romijn, 19751, immunocy- tochemical studies [Matsurra et al., 19831, and autoradiography [Taxi and Droz, 19661 have demonstrated the noradrenergic nature of these fibers.

It has been demonstrated that the pineal gIand of FeZix cutus domesticus is richly innervated by sympathetic postganglionic nerve fibers in compari- son to other species examined [Karasek et al., 1983al. These fibers contain a considerable number of small dark vesicles (SDV) (45-60 nm), a few clear vesicles (CV) (45-60 nm), as well as a smaller number of large dark vesicles (LDV) (800-120 nm) [Gonzhlez et al., 1969, 1976; Gonzhlez and Alvarez- Uria, 19841. The SDV are storage sites for norepinephrine and serotonin Uaim-Etcheverry and Zieher, 1968, 1980; Matsuura et al., 19831. Jaim-Etch- everry and Zieher [ 19711 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 SR are ultrastructural cell organelles of pinealocytes [GonzPlez et al., 19691 in all mammalian species studied so far. SR in the mammalian pinealocyte are apparently related to pineal adrenergic innervation [King and Dougherty, 19801. This hypothesis has been examined under a wide variety of experimental conditions, including changes in the light-dark cycle [Voll- rath and Howe, 1976; Hewing, 1980; King and Dougherty, 1982b], blinding [Kurumado and Mori, 19801, pineal sympathectomy [Romijn, 1975; King and Dougherty, 1982a; Karasek et al., 1982, 1983b], pineal organ culture [Romijn and Gelsema, 19761, and electrical stimulation of the pineal sympathetic nerves [Gonzalez and Alvarez-Uria, 1986al. Additionally, an inverse correla- tion between SR number and the density of adrenergic nerve endings in the pineal gland of a diverse group of mammalian species has been reported by Karasek et al. [ 1983al. SR are relatively sparse in cat pinealocytes in compar- ison to other mammalian species [Karasek et al., 1983al.

The purpose of our study was to perform a morphometric analysis of the NV and SR in the cat pineal gland after chronic bilateral preganglionec- tomy of the superior cervical ganglia (SCGp); results have been previously reported in an abstract [Gonzhlez and Alvarez-Uria, 1986b1, published with erroneous graphics. To determine whether the pleomorphic NV population occurring after SCGp of the pineal gland corresponded to one or more functional types, the triple-fixation procedure of Tranzer and Richards [ 19761 was used to identify the dark vesicles.

MATERIALS AND METHODS

Forty-eight adult male cats were housed in a room with controlled illumination (LD 12:12, 8 A.M. to 8 P.M. and temperature (22°C f 2°C) and

Preganglionectomy of Pineal Sympathetic Nerves 369

had constant access to food and tapwater for at least 2 w prior to use. Twelve groups consisting of four animals each were formed.

Cats were anesthetized with sodium pentobarbital (35 mg/kg i.p.). An anterior midline incision was made along the animal’s neck in order to expose the cervical sympathetic trunk for preganglionectomy. The SCG and sympathetic nerve trunks were dissected free of the vagus nerves and carotid arteries, bilaterally exposed on both sides, and a 2-mm piece of each cervical sympathetic trunk was removed about 5 mm proximal to the ganglion. Once recovered preganglionectomized cats were returned to the same 12: 12 LD photoperiod. Bilateral ptosis was apparent following the surgery. SCGp was performed on 24 animals, whereas the other 24 cats were treated exactly as outlined above except that each ganglion and its sympathetic nerve trunk was visualized but not removed (sham-operated animal).

Groups of four cats were sacrificed between 10 A.M. and 12 A.M., at 15 days, 1 mo, 3 mo, 6 mo, 9 mo, and 1 yr postoperation (between May 15 and 30, 1983, 1985). The cats were perfused [according to Tranzer and Richards, 19761 with 3% glutaraldehyde and 0.4% paraformaldehyde buffered to pH 7.2 with 0.1 M potassium chromate-dichromate at room temperature for 10 min and another 10 min at 4°C. All animals had respiration assisted by means of a respirator connected to a tracheal cannula.

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 h in the same fixative and subsequently incu- bated overnight with continuous stirring at 4°C in 0.2 M potassium chro- mate-dichromate solution adjusted to pH 6 to which 7% sucrose was added, followed by postfixation at room temperature with OsO4 in 0.1 M potassium chromate-dichromate buffered to pH 7.2. Slices were dehydrated in a graded series of acetone, stained in 2% uranyl acetate in 70% acetone overnight, oriented in such a way that frontal sections were cut from the center of the blocks. Sections 60-80-nm thick were obtained, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 10. Twenty micrographs of each gland were taken at a magnification of 12,000 diameters and photo- graphically enlarged to 36,000 diameters.

The areas of the NV were measured on the photographs by tracing onto millimeter-ruled graph paper. The calculated data were expressed as the total number of NV per 1.25 pm2 of pineal tissue [Gonzilez and Alvarez-Uria, 19841. Elongated LDV exhibiting an elongated dense core were examined with a goniometer in a Zeiss EM 10.

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 [Karasek et al., 19821. An RF was defined as one or more spatially related SR [Vollrath, 19731.

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

RESULTS

Adrenergic nerve fibers in the cat pineal organ terminate either in the perivascular spaces or between adjacent pinealocytes. Ultrastructural studies

370 Gonzalez and Alvarez-Uria

on 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 SCGp, the majority of the dense cores of the small vesicles were markedly depleted and their shape appeared rounded or elliptical (Fig. 2). The number of LDV was modified and the shape of some of them was elongated (Fig. 2) . These vesicles were examined with a goniometer to assure that their shape was elongated. N o degeneration profiles of axon processes or terminals of the pineal sympathetic nerves were apparent after SCGp.

In the pinealocytes of the controls, SR were observed lying in processes and perikarya. SR appeared either singly or in groups of two to five (Fig. 3). A maximum of 15 SR per RF was only occasionally observed. SR were noted in processes and perikarya of the operated cats, mostly lying in groups of 1- 25 elements (Fig. 4); some of them formed large aggregates (Fig. 4 , inset).

The frequency of the respective types of NV in the adrenergic endings of the gland were quantitatively determined in normal and operated pineal glands. In each sham-operated animal (control cats) the different types of vesicles showed an almost constant frequency. The number of SDV was

Fig. 1. Pineal sympathetic fibers. Sham-operated cat. The endings contain numerous vesicles of various sizes and dense cores (small, large, and clear). Tissue processed according to the technique of Tranzer and Richards [1976]. x 36,000.

Fig. 2. Preganglionectomized cat (6 mo). 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,OOO.

Preganglionectomy of Pineal Sympathetic Nerves 371

Fig. 3. perikarya. X 30,000.

Fig. 4. number of the synaptic ribbons are aggregated. X 30,000.

Synaptic ribbons of sham-operated cat. Five trilaminar rodlets (SR) are lying near the

Preganglionectomized cat (6 mo). Numerous synaptic ribbons X25,OOO. Inset: A large

higher than those of CV and LDV (Fig. 5). After each SCGp experiment, all the NV showed remarkable changes in their proportions. Under these exper- imental conditions the number of CV and LDV increased (Fig. 5), and the SDV decreased. The number of SR and RF significantly (P<O.OOl) increased after each pregangliomctomy relative to sham-operated controls (P < 0.00 1) (Figs. 6, 7).

DISCUSSION

The cat pineal gland is actually a peripheral organ which is richly innervated by sympathetic postganglionic nerve fibers coming from the SCG [Bowers and Zigmond, 19821. On the other hand, SR in the cat pinealocyte are apparently related to the pineal adrenergic innervation [Karasek et al., 1983al.

According to our results, it seems clear that bilateral preganglionectomy simultaneously induces both qualitative and quantitative changes in NV and in the number of SR 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 pineal gland after electrical stimulation Uaim-Etch- everry and Zieher, 1980; Pellegrino de Iraldi and Corazza, 1981; Gonzhlez

372 Gonzalez and Alvarez-Uria

Fig. 5 . Number of vesicles of the adrenergic nerve terminals in the pineal gland of sham- operated (C) and preganglionectomized (Exp) cats. The vertical lines above the bars indicate SEM. LDV, large dark vesicles; SDV, small dark vesicles; CV, clear vesicles.

and Alvarez-Uria, 1986aI. SCGp dramatically decreases the number of dense cores in the SDV of adrenergic terminals and also reduces the size of the few remaining cores. These modifications of SDV suggest that the neurotransmit- ter, norepinephrine, present in the cores of SDV, might be released from their storage sites in the pineal sympathetic nerves after SCGp.

The frequency of LDV was significantly different between operated and control nerves. LDV were not depleted and their number increased during the experiments, some of them being elongated. No large elongated vesicles were found in the pineal sympathetic nerves of sham-operated cats (controls).

Quantitative analysis indicates that the number of both RF and SR significantly (P < 0.001) increase after SCGp; although a relationship between the adrenergic innervation of the pineal gland and SR formation has been hypothesized [Karasek et al., 1983a1, the nature of this relationship is still being questioned.

Our study suggests that the SR formation is not intrinsic to pineal parenchymal cells, but depends on information carried to the pineal via its sympathetic nerves. It is still possible that the mechanism responsible for the SR formation could be intrinsic to the pineal but requires intact sympathetic nerves for its expression. Alternatively, the control mechanism might be in the SCG, or the nerve impulses responsible for the SR formation could have originated in the central nervous system (CNS), reaching the SCG via their preganglionic fibers. To examine both possibilities, the preganglionic nerves to the SCG were cut and the cats were killed after SCGp. Quantitative

Preganglionectomy of Pineal Sympathetic Nerves 373

i 15

T

T

T I

T

T

aya 1 month 3 months 6 months 9 months I year

Fig. 6. Number of synaptic ribbons (SR) in the pineal gland of sham-operated and pregangli- onectomized cats. The vertical lines above the bars indicate SEM. Black columns, sham- operated; white columns, preganglionectomized.

analyses show that the number of both SR and RF significantly increased after SCGp; these results indicate that the stimulus responsible for SR forma- tion originated somewhere in the CNS, and was transmitted to the pineal via preganglionic fibers to the SCG.

The neural stimulation of the pineal gland can be abolished, without denervating the gland, by electrical stimulation [Gonzalez and Alvarez-Uria, 1986aI or by cutting the cervical sympathetic trunk, which contains the preganglionic input to the SCG. Subsequently, we hypothesize that after bilateral electrical stimulation of the SCG [Gonzalez and Alvarez-Uria, 1986al as well as following SCGp, the pineal and its sympathetic nerves do not receive the type of neural information from the CNS that is necessary to regulate the number of dense cores of NV and the SR or RF.

In conclusion, our study suggests an inverse numerical relationship between dense cores of NV and the SR or RF in the cat pineal gland after chronic SCGp. Although the functional significance of SR in the mammalian pinealocyte is obscure, the numerical changes in these structures under different natural and experimental conditions have resulted in various hy- potheses concerning their role in the pineal function [see Karasek et al., 1983al. Common to these studies, King and Dougherty [ 1982a,b] suggest that the SR may be involved in the regulation of beta-adrenergic receptors

374 Gonzalez and Alvarez-Uria

30 1 T ‘I 3

T

15 d a p I month S months 6 month. 9 months I year

Fig. 7. Number of ribbon fields (RF) in the pineal gland of sham-operated and preganglionec- tomized cats. The vertical lines above the bars indicate SEM. Black columns, sham-operated; white columns, preganglionectomized.

along the plasmalemma of the pinealocyte. Further biochemical and immu- nocytochemical studies will be necessary, however, to elucidate the mecha- nism which brings about these quantitative and morphological changes in NV and SR after chronic SCGp in the pineal gland of the rat.

ACKNOWLEDGMENTS

We are grateful to Puri Arribas and Robert Moyer for technical assistance and to Prof. J. Taxi for his valuable comments.

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