MICROSCOPY RESEARCH AND TECHNIQUE 21:188-204 (1992)

Fine Structure of the Pinealopetal Innervation of the Mammalian Pineal Gland MORTEN M0LLER Institute of Medical Anatomy, Department B , University of Copenhagen, DK-2200 Copenhagen, Denmark

KEY WORDS

Ultrastructure, Central innervation, Sympathetic, Parasympathetic, Neuropeptides, Mammals, Human fetus

ABSTRACT

The mammalian pineal gland is innervated by peripheral sympathetic and parasympathetic nerve fibers as well as by nerve fibers originating in the central nervous system (central innervation). The perikarya of the sympathetic fibers are located in the superior cervical ganglia, while the fibers terminate in boutons containing small granular vesicles and a few large granular vesicles. Both noradrenaline and neuropeptide Y are contained in these neurons. The parasympathetic fibers originate from perikarya in the pterygopalatine ganglia. The neuropeptides, vasoactive intestinal peptide and peptide histidine isoleucine, are present in these fibers, the boutons of which contain small clear transmitter vesicles and larger granular vesicles. The fibers of the central innervation originate predominantly from perikarya located in hypothalamic and limbic forebrain structures as well as from perikarya in the optic system. These fibers terminate in boutons containing small clear and, in certain fibers, an abundant number of large granular vesicles. In rodents, the majority of the central fibers terminate in the deep pineal gland and the pineal stalk. From these areas impulses might be transmitted further caudally t o the superficial pineal gland via neuronal structures or processes from pinealocytes. Several hypothalamic neuropeptides and monoamines might be contained in the central fibers. The intrapineal nerve fibers are located both in the perivascular spaces and intraparenchymally. The majority of the intraparenchymally located fibers terminate freely between the pinealocytes. However, some nerve terminals make synaptic contacts with the pinealocytes and in some species with intrapineal neurons. In fetal mammals, sympathetic, parasympathetic, and central fibers are also present. In addition, an unpaired nerve, connecting the caudal part of the pineal gland with the extreme rostra1 part of the mesencephalon, is present. This nerve is a homologue to the pineal nerve (nervus pinealis) observed in lower vertebrates. o 1992 Wiley-Liss, Inc.

INTRODUCTION The pineal gland is a derivative of the prosencephalic brain vesicle of the neural tube. For several decades it was believed that the mammalian pineal gland during development loses all its neuronal connections with the rest of the neural tube and is innervated solely by sympathetic nerve fibers originating from perikarya located in the superior cervical ganglia (Kappers, 1965). However, neuronal tracing experiments and ultrastructural analysis of lesioned animals performed during the last decade have with certainty demonstrated that the above concept is an oversimplification and that the pinealopetal innervation also consists of other neuronal elements (for review see Korf and Moller, 1984, 1985). Firstly, the gland is innervated by pinealopetal fibers originating from perikarya in the forebrain and mesencephalon, penetrating into the pineal via the deep pineal (Mikkelsen and Moller, 1990; Mikkelsen et al., 1991; Moller and Korf, 1983a,b). This innervation has been called the “central innervation” of the pineal gland. Secondly, studies using lesion experiments, neuronal tracing, and immunohistochemistry have strongly indicated that the pineal is inner-

0 1992 WILEY-LISS, INC

vated by nerve fibers originating from extracerebral parasympathetic ganglia (Moller et al., 1987; Phansuwan-Pujito et al., 1991). However, recent anatomical studies of the pinealopeta1 innervation have also shown that considerable inter-species variations exist. Some of these variations can be understood in the light of the differences in gross morphology of the pineal complex. The differences are especially prominent between rodent and non-rodent species. Therefore, this review will start with a description of the anatomy of the pineal gland of rodents and non-rodents, with special emphasis on the deep pineal gland.

Received October 3, 1989; accepted in revised form June 25, 1990; revised August 11, 1991. Address reprint requests to Morten Mbller, M.D., Ph.D., Institute of Medical Anatomy, Department B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.

MAMMALIAN PINEALOPETAL INNERVATION

GROSS MORPHOLOGY OF THE MAMMALIAN PINEAL COMPLEX Rodents The pineal complex of most rodents consists of a superficial pineal gland, a deep pineal gland, and a stalk connecting these two parts (Vollrath, 1981). All three parts can vary in size; for example, the deep pineal in the rat (called lamina intercalaris; Kappers, 1960) is small, whereas in the gerbil it is larger (Moller, 1981). The pineal stalk connecting the two parts can be thick, thin, or even incomplete (see Vollrath, 1981). The deep pineal is closely associated with both the habenular and the posterior commissures (Figs. 1-8). In frontal sections, bilateral projections from the stria medullaris thalami project caudally (Figs. 5, 6) to merge in the habenular commissure (Figs. 2, 7, 13), to which the deep pineal gland is attached (Figs. 3, 7). In these stria medullaris projections, numerous myelinated nerve fibers and neurons are located (Figs. 5, 6, 16). The pinealocytes of the deep pineal gland are rostrally arranged in a ring-like structure around the suprapineal recess (Figs. 3, 8, 13). More caudally the suprapineal recess is obliterated by the pinealocytes of the deep pineal gland (Figs. 4, 7). Many myelinated nerve fibers with a rostro-caudal orientation are present also in the deep pineal (Figs. 10-15). Caudally, the deep pineal is in apposition to the posterior commissure (Fig. 7). The pineal stalk leaves the dorsal part of the deep pineal (Fig. 4) to enter the superficial pineal (Fig. 9) located between the caudal part of the hemispheres. The number of nerve fibers in the pineal stalk decreases towards the superficial pineal (Luo et al., 1984; Moller and Korf, 1983a), in which fewer myelinated fibers are present (Figs. 1720). The anatomical connection in the rodents between the deep pineal gland and the forebrain via the stria medullaris projections is slender. It is therefore not surprising that the number of central fibers entering the pineal gland of rodents via this route is less than in the non-rodents. Non-Rodents The gross morphology of the pineal complex of rodents described above differs from the morphology of most other mammalian species, e.g., bat (Bhatnagar et al., 1990), cat and monkey (Nielsen and Moller, 19751, fox (Karasek and Hansen, 1982), sheep (Anderson, 1965; Cozzi et al., 1989), horse (Cozzi, 19861, and cow (Phansuwan-Pujito et al., 1991). In the majority of mammals the pineal complex is not divided into a superficial and deep pineal gland, but it consists of a single larger cell mass situated on the dorsal part of the brain stem at the mesodiencephalic junction between the habenular and posterior commissures (Fig. 30). The shape of the pineal gland of these animals varies from slender and elongated to pyramidal with the base towards the brain stem. This base of the pineal varies in size and is penetrated by pinealopetal nerve fibers from the posterior and habenular commissures (Scharenberg and Liss, 1965). In certain species, e.g., human (Moller, 1978), sheep (Anderson, 1965), and cow (Phansuwan-Pujito et al., 1991), where the base of the pineal

189

is broad, the number of pinealopetal central fibers is high.

INNERVATION OF THE MAMMALIAN PINEAL GLAND Sympathetic Innervation The pineal gland of mammals is heavily innervated by sympathetic nerve fibers originating from perikarya in the superior cervical ganglia (Kappers, 1960).These fibers contain noradrenalin as neurotransmitter and were early visualized by the classical Falck-Hallarp histofluorescence technique (Falck et a]., 1962) in a number of species (Moller and van Veen, 1981; Owman, 1965). Later the discovery of antibodies against the enzymes involved in the synthesis of catecholamines, e.g., tyrosine hydroxylase and dopamine phydroxylase (Hokfelt et al., 1978) made it possible to visualize these fibers by immunohistochemistry (Shiotani et al., 1989; Zhang et al., 1991). On the ultrastructural level the intrapineal sympathetic nerve fibers are endowed with terminal boutons and boutons en passage. These boutons contain transmitter vesicles with a diameter of 40-60 nm, of which some contain an eccentrically located dense core (Figs. 20,24). In addition, a few larger granular vesicles with a diameter of 100-200 nm are present in the terminals (Fig. 24). Several cytochemical studies have shown that the small transmitter vesicle with the eccentric dense core, and probably also the large dense core granules, contain noradrenalin (Hokfelt, 1968; Jaim-Etcheverry and Zieher, 1979; Pellegrino de Iraldi and Corazza, 1981; Wood, 1973). As in other sympathetic fiber systems, the large granular vesicles are thought to contain a neuropeptide, e.g., neuropeptide Y (NPY), which has been demonstrated in sympathetic intrapineal nerve fibers by immunohistochemistry (Chronwall et al., 1985; Schon et al., 1985; Schroder, 1986; Schroder and Vollrath, 1986; Zhang et al., 1991). The sympathetic nerve fibers are located both in the perivascular spaces and intraparenchymally between pinealocytes (Fig. 20) (Nielsen and Moller, 1978; Ronnekleiv and Moller, 1979). The majority of the fibers terminate with boutons located freely in the perivascular spaces or between the pinealocytes, without making synaptic junctional complexes with the pinealocytes. However, some synaptic junctions between the sympathetic terminals and pinealocytes have recently been demonstrated by electron microscopy (Bhatnagar, 1988; Huang and Lin, 1984). Although the number of such synaptic junctions is low, their functional role might be of importance for pineal physiology. Thus, the presence of electrotonic gap junctions between the pinealocytes (Fig. 33) (Huang and Taugner, 1984; Krstic, 1974; Moller, 1976; Taugner et al., 1981) makes it possible for an impulse to influence all the pinealocytes connected by gap junctions. With regard to the large number of perivascularly located nerve fibers, this location has to a morphologist always indicated an influence of these fibers on the vascular system of the gland, e.g., the blood flow. However, such an influence has still to be demonstrated. Some noradrenaline-containing nerve fibers innervating the pineal might not originate from the superior

190

M. M0LLER

Figs. 1-4. Frontal sections of paraffin-embedded material through the epithalamic area of the gerbil mounted in rostrocaudal direction. dp, deep pineal gland; hc, habenular commissure; pc, posterior commissure; PR, pineal recess; sco, subcommissural organ. Cresyl violet. x 90.

MAMMALIAN PINEALOPETAL INNERVATION

191

Figs. 5-8. Frontal sections of Epon-embedded material through the epithalamic area of the gerbil (Figs. 5, 6 , 8 ) and rat (Fig. 7). In Figures 5 and 6 the arrows point to neurons located in the projections from the stria medullaris. PR, pineal recess; DP, deep pineal gland;

HC, habenular commissure; PC, posterior commissure; PT,pretectal area; sco, subcommissural organ; SR, suprapineal recess. Cresyl violet. Figures 5 and 6 , x 300; Figure 7, x 380; Figure 8, x 180.

cervical ganglia. Thus, lesions of the habenular nuclei of the rat have resulted in degenerating intrapineal boutons exhibiting a morphology similar to that of the sympathetic boutons (Ronnekleiv and Moller, 1979). This has been interpreted as a possible innervation of

the pineal complex with fibers from the noradrenergic nuclei located in the brain stem. A part of the NPY-containing fibers in the pineal complex are also of extrasympathetic origin. Thus, in a series of ganglionectomized rats it was observed that

192

M. M0LLER

Fig. 9. Sagittal section through the superficial pineal gland of the Mongolian gerbil. Cresyl violet. x 230. Figs. 10, 11. Frontal sections through Epon-embedded material of the deep pineal gland of the gerbil. Note the rostrocaudally

oriented myelinated fibers (arrows) in the deep pineal. DP, deep pineal gland; PR, pineal recess. Cresyl violet. Figure 10, ~ 3 1 0 ; Figure 11, x 425.

MAMMALIAN PINEALOPETAL INNERVATION

Figs. 12-14. Electron micrograph (Fig. 12) of the fibers in the deep pineal gland shown in Figure 13 (arrow)and, at higher magnification,in Figure 14. PR, pineal recess;hc, habenular commissure.Figures 13 and 14, cresyl violet. Figure 12, X 7,460; Figure 13, x 95; Figure 14, X 235. (Figure 13 reproduced from Mfiller, 1981, with permission of Elsevier Science Publishers.)

193

Fig. 15. Electron micrographs of the deep pineal gland of the gerbil. A classical axodendritic synapse (seen at higher magnification in the insert, right lower corner) is seen between the myelinated fibers close to the pinealocytes. Den, dendrite. x 10,025. (Figures 15 and 16 reproduced from Mgjller, 1981, with permission of Elsevier Science Publishers.)

Fig. 16. Electron micrograph of the stria medullaris projections of the mouse with a classical axodendritic synapse. Note the presence of clear small transmitter vesicles. Den, dendrite. x 13,100.

Fig. 17. Electron micrograph of a bundle of myelinated and unmyelinated nerve fibers in the superficial pineal gland of the gerbil. x 8,570. Fig. 18. Electron micrograph of the two myelinated fibers on the surface of the superficial pineal gland. x 6,425.

Fig. 19. A bundle of myelinated and unmyelinated nerve fibers located just outside the superficial pineal gland in the subarachnoidal space. x 7,100.

M.M0LLER

Fig. 20. Intraparenchymal sympathetic nerve fiber with small dense core transmitter vesicles (Sym) in the superficial pineal gland of the gerbil. x 15,000.

Figs. 21, 22. Intraparenchymal nerve terminals (Clear) with small clear transmitter vesicles and some large granular vesicles in the superficial pineal gland of the gerbil. Figure 21, x 20,000; Figure 22, ~ 8 , 8 5 0 .

Fig. 23. Nerve terminals with clear transmitter vesicles making synaptic contacts with pinealocytes in the superficial pineal gland of the Mongolian gerbil. Pin, pinealocytes. x 17,700. (Reproduced from Merller, 1985, with permission of Chapman & Hall, Ltd.) Fig. 24. Sympathetic nerve terminal located intraparenchymally in the superficial pineal gland of the rat. ~ 4 7 , 1 2 5 .(Reproduced from Korf and Mfiller, 1984, with permission of the publisher.)

Fig. 25. Synaptic contact between a nerve fiber with clear vesicles and a pinealocyte (Pin) in the perivascular space of the Mongolian gerbil. x 34,740. (Reproduced from Moller, 1985, with permission of Chapman & Hall, Ltd.)

198

M. MBLLER

Fig. 26. Degenerating nerve fibers (f) in the superficial pineal gland of the Mongolian gerbil 1 week after lesioning of the habenular nuclei. A large cellular structure with many lysosomes is seen (arrows). x 12,800. (Reproduced from Meller and Korf, 1983a, with permission of Springer-Verlag.)

Figs. 27, 28. Degenerating nerve terminals (arrows) with clear transmitter vesicles in the superficial pineal gland of the Mongolian gerbil 3 days after lesion of the medial habenular nuclei. Figure 27, X 41,200; Figure 28, X 35,125. (Figure 28 reproduced from Korf and M@ller,1984, with permission of the publisher.)

MAMMALIAN PINEALOPETAL INNERVATION

199

Fig. 29. Midsagittal section through the caudal part of the pineal gland (PG) of the human fetus (CRL = 248 mm). The human fetal nerve (arrows) is located in the midline just caudal to the gland, connecting the gland and the most rostral area of the mesencephalon. Epon, cresyl violet. x 510.

Fig. 30. Midsagittal section through the pineal gland (PG) of the fetal sheep (fetal day 60). The fetal pineal nerve (arrows) is observed just caudal to the gland. Ha, habenula; sco, subcommissural organ. Epon, cresyl violet. x 380. (Reproduced from Meller et al., 1975, with permission of Springer-Verlag.)

several NPY-containing nerve fibers remain in the gland after the ganglionectomy (Zhang et al., 1991). Some of these NPYergic fibers originate from perikarya located in the forebrain. In the forebrain, NPYcontaining neurons are present in the intergeniculate leaflet of the lateral geniculate body (Card and Moore, 1989). Anterograde neuronal tracings from the intergeniculate leaflet of the rat (Mikkelsen and Moller, 1990) and gerbil (Mikkelsen et al., 1991) have shown a direct projection from this subnucleus to the rostral part of the pineal complex. Recent investigations in our laboratory by using combined immunohistochemistry and neuronal tracings have indicated that some pinealopetal fibers from the intergeniculate leaflet actually contain NPY (unpublished). The NPYergic neurons in the intergeniculate leaflet have been shown to influence circadian rhythmicity (Johnson et al., 1988).

cies, a large intrapineal ganglion, observable in the light microscope, is present, e.g., in the ferret (David et al., 1973), ground squirrel (Matsushima and Reiter, 19781, bats (Bhatnagar et al., 1986, 19901, macaque (Hartmann, 1957; Hosaka et al., 1957; Ichimura et al., 1986; Kolmer, 1929; Le Gros Clark, 1940; Levin, 19381, and Rhesus monkey (David et al., 1975), and in humans (Bargmann, 1943;Josephy, 1920) and the human fetus (Moller, 1976, 1986; Mollgdrd and Moller, 1973). The presence of such a ganglion is strongly indicative of a parasympathetic innervation. Further, careful experiments performed 30 years ago showed that lesions of the greater petrosal nerve in the monkey were followed by degeneration of intrapineal fibers visualized by silver impregnation (Kenny, 1961). At the ultrastructural level, the intrapineal nerve cells have been studied in the rabbit (Romijn, 19731, macaque (Ichimura et al., 1986), and ferret (David et al., 1973). In all these animal species, electron microscopy revealed the presence of intrapineal neurons on which axodendritic and axosomatic synapses were observed. The presynaptic boutons contained 40-60 nm transmitter vesicles of the electron-lucent type (with-

Innervation of the Pineal From Non-Sympathetic Ganglia Strong morphological indications for a parasympathetic innervation of the mammalian pineal have existed for many years. Thus, in certain non-rodent spe-

200

M. M0LLER

Fig. 31. Electron micrograph from the pineal fetal nerve of the rabbit (fetal day 24). A classical axodendritic synapse with clear transmitter vesicles and a few large granular vesicles is observed. Den, dendrite. x 61,000. (Reproduced from Mdler et al., 1975, with permission from Springer-Verlag.)

Fig. 32. Electron micrograph of the human fetal pineal nerve showing a classical axodendritic synapse with clear small vesicles in the presynaptic element. Den, dendrite. x 25,000. Fig. 33. Electron micrograph of a gap junction (arrows) between two pinealocytes of the human fetal pineal gland. x 30,000.

MAMMALIAN PINEALOPETAL INNERVATION

out eccentric dense cores) (Figs. 21, 22). However, it must be emphasized that the perikaryal origin of these presynaptic boutons has yet to be established. Only in the ferret has the perikaryal origin of the presynaptic input to some of the intrapineal neurons been documented. In this species, the fibers originated in the central nervous system (see later for discussion). Immunohistochemical studies performed during the last 5 years have provided further evidence for a parasympathetic innervation of the pineal. Thus, recently nerve fibers immunoreactive to choline acetyltransferase, the rate limiting enzyme in acetylcholine synthesis, have been demonstrated in the bovine pineal gland (Phansuwan-Pujito et al., 1991). Further, nerve fibers immunoreactive to vasoactive intestinal peptide (VIP) have in several studies been observed in the mammalian pineal (Cozzi et al., 1989; Mikkelsen et al., 1987;Mgller et al., 1985; Shiotani et al., 1986; Uddman et al., 1980). VIP is generally considered to be a parasympathetic transmitter (Rosthe, 1984). A large number of VIP-containing perikarya are located in the parasympathetic pterygopalatine and otic ganglia Wemura et al., 1988). Further, a combined immunohistochemical study and tracer study has indicated (Shiotani et al., 1986) that intrapineal VIPergic nerve fibers originate from perikarya in that ganglion. However, both the superior cervical ganglia (Sasek and Zigmond, 1989) and the paraventricular hypothalamic nucleus (Mikkelsen and Mgller, 1988) contain VIPergic neurons. The possibility exists that some VIPergic fibers in the pineal might originate from these places. As discussed above, the fine structure of the parasympathetic nerve terminals has never been demonstrated with certainty because lesions of this system are very difficult to perform. However, after removal of the sympathetic input to the pineal gland, degeneration is seen solely in the boutons containing the 40-60 nm granular transmitter vesicles (Mgller and Korf, 1983a; Romijn, 1975). Therefore, both parasympathetic and central nerve fibers must terminate with boutons endowed with 40-60 nm electron-lucent transmitter vesicles.

Central Innervation of the Mammalian Pineal Gland The central innervation of the pineal gland has been verified in a number of species by several neuroanatomical methods. This innervation was first demonstrated in the ferret (David and Herbert, 19731, in which lesions of the habenula were followed by a degeneration of intrapineal boutons terminating on the intrapineal ganglion. Later, lesions of the habenula of the gerbil verified the presence of a central innervation of this species (Mgller and Korf, 1983a). In the gerbil the degenerating boutons (Figs. 27, 28) contained the 40-60 nm electron-lucent transmitter vesicles and few larger granular vesicles where in the same species bilateral removal of the superior cervical ganglia (Mgller and Korf, 1983a) resulted in degeneration of the boutons containing 40-60 nm transmitter vesicles with an eccentrically located dense core. In the same lesion study of the gerbil, a prominent feature was the degeneration of myelinated fibers (Fig. 26) which is never observed after ganglionectomy. The number of large

201

dense core vesicles in the boutons of the central innervation vary from a few to many. The latter boutons containing a high number of the large dense core vesicles might represent a peptidergic projection, e.g., from the paraventricular nucleus to the pineal gland (Buijs and Pevet, 1980; Niirnberger and Korf, 1981). Neuronal retrograde tracing studies of the guinea pig (Korf and Wagner, 1980) and gerbil pineal (Moller and Korf, 1983a) with horseradish peroxidase have revealed that the central fibers originate from perikarya in the posterior commissure, lateral geniculate body, habenula, pretectum, and paraventricular nucleus. Recent anterograde tracing studies in the rat and gerbil have verified the presence of a central innervation to the pineal gland from the paraventricular nucleus (Larsen et al., 19911,from the intergeniculate leaflet of the lateral geniculate body (Mikkelsen and Mgller, 1990; Mikkelsen et al., 1991), and from the lateral hypothalamic area (Fink-Jensen and Moller, 1990). Also the serotoninergic raphe nuclei might innervate the pineal gland. Thus, in the dog (Matsuura and Sano, 1983), serotoninergic nerve fibers have been observed to penetrate into the pineal through the stalk, after removal of the sympathetic input to the gland. The anterograde tracing of the central innervation of rodents as well as electron microscopical studies (Luo et al., 1984; Mikkelsen and Mgller, 1990) have indicated that the majority of the fibers terminate in the deep pineal and the pineal stalk. An innervation of only the rostral part of the pineal complex of rodents does not exclude an influence on the whole pineal. The pinealocytes of the rodent superficial and deep pineal (Cozzi and Mgller, 1988; Korf et al., 1986) are endowed with long processes. Pinealocytes from the superficial pineal might reach the deep pineal to be influenced by the central innervation in that region. The second possibility is the following: in rodents, several neurons are located in the deep pineal area, especially in the stria medullaris projections and in the border area between the habenular and the deep pineal (Mgller and Korf, 1983a). These neurons are endowed with several axodendritic synapses of which the presynaptic boutons contain clear vesicles (Mgller, 1981). These neurons in the deep pineal area might function as a rostral pineal ganglion which projects further caudally toward the superficial pineal. Also, in several non-rodent species, huge amounts of fibers penetrate directly into the pineal gland from both the posterior and habenular commissures (Mgller, 1978; Nielsen and Moller, 1975; Pastori, 1928; Phansuwan-Pujito et al., 1991). However, tracing studies have not been performed in any of these species.

Innervation of the Pinealocytes The majority of nerve terminals present in the parenchyma of the pineal gland terminate freely between the pinealocytes. Therefore, the neurotransmitters/ modulators must diffuse through the extracellular space before reaching the receptors on the pinealocytic cell membrane. Such neurotransmission requires a time delay. The fast responses in the pineal caused by light might need a faster route of transmission. HOWever, as described above during recent years, several

202

M. M0LLER

electron microscopical studies have shown synapse-like contacts between the nerve terminals and the pinealocytes. Such synaptic contacts have been observed both between the sympathetic terminals and pinealocytes in the rat (Huang and Lin, 1984) and the nonsympathetic terminals in ground squirrel (Matsushima and Reiter, 1978), gerbil (Fig. 25) (Moller, 1985), and macaque (Ichimura et al., 1986). This direct innervation of the pinealocyte via a synaptic complex is probably a fast transmitting system. Physiological responses in the pineal caused by light can be observed seconds after the light has activated the retinal photoreceptors. The above direct innervation of the pinealocyte might be the neural route for such an influence.

INNERVATION O F THE MAMMALIAN FETAL PINEAL GLAND During our earlier investigations of the ultrastructure of the mammalian pineal gland we discovered a hitherto undescribed fetal cranial nerve in the human fetus (Moller, 1978, 1979; Mollghd and Moller, 1973). This nerve was later confirmed to be present in sheep and rabbit fetuses (Moller, 1979; Moller et al., 1975). The human fetal pineal gland exhibits a pyramidal shape with a broad base towards the brain stem. It develops with an anterior and posterior lobe which merge in later fetuses. The human fetal pineal nerve is located in the midsagittal plane just caudal to the pineal gland (Fig. 29). The nerve connects the pineal with the extreme rostra1 part of the tectal area. It emerges from the dorsal part of the posterior commissural area (Fig. 29). After penetrating the external limiting membrane, the nerve is located in the subarachnoidal space and follows the caudal side of the pineal gland in the direction of the pineal apex. The nerve decreases in thickness towards the tip of the pineal, and fibers can be followed from the nerve into the caudal part of the pineal gland. In the light microscope, as well as in the electron microscope (Fig. 321, neurons were observed in the nerve. In the older fetuses, the neurons were organized in a ganglion-like structure (Mgller, 1978). Contrarily, in younger fetuses, the neurons were observed along the nerve. The fetal nerve was observed in fetuses with a CRL between 56 mm and 248 mm. Autopsy performed on newborn infant brains did not reveal the nerve. The nerve was observed in the sheep fetus at 60 days of gestation (Fig. 30) and in rabbit fetuses a t fetal days 23 and 24 (Fig. 31) (Moiler et al., 1975). In addition to the ganglion located in the pineal nerve, other collections of nerve cells are present in relation to the human fetal pineal gland. Thus, at the apex of the gland, above the great cerebral vein of Galen, a ganglion is located (Mollgiird and Moller, 1973) which was first described by Marburg (1909). In the subarachnoidal space, just a t the apex of the pineal, a collection of nerve cells previously described by Pastori (1928) is located (Mprllgbrd and Moiler, 1973). Finally, a collection of nerve cells at the anterior surface of the pineal was observed (Moller, 1979). In the pineal itself, between the cords of pinealocytes, an intrapineal ganglion is located in some fetuses (Moller, 1986). In other

fetuses intraparenchymal single nerve cells are present (Moller, 1986). At the ultrastructural level, the human fetal pineal nerve contains unmyelinated nerve fibers (Fig. 32) with a few fibers beginning to myelinate in the oldest fetuses (Moller, 1978). The perikarya in the nerve are neuroblasts endowed with many polyribosomes but without a well-developed endoplasmic reticulum. Both axodendritic and axosomatic synapses are present on the neuroblasts. The presynaptic terminals are endowed with 40-60 nm clear vesicles (Fig. 32). The ultrastructural investigation of the human fetal pineal gland supports the concept of a diversified innervation of the mammalian pinealocyte coming from the sympathetic system, the parasympathetic system, and the central nervous system.

ACKNOWLEDGMENTS The author is indebted to Mrs. U. Rentzman for technical assistance, to Mrs. G. Hahn and Mrs. B. Houlind for photographic assistance, and to Mrs. J. Korner for typing the manuscript. Work by the author was supported by grants from the Danish Biotechnology Programme (1987-1990) to the “Biotechnology Centre for Neuropeptide Research,” and by grants from the Danish Research Foundation (gr. nos. 12-8815 and 1202361, Jacob Madsen and Hustru Olga Madsens Fond, NOVO’s Fond, P. Carl Petersens Fond, Fonden ti1 Laegevidenskabens Fremme, and Landsforeningen ti1 bekaempelse af 0jensygdomme og Blindhed. REFERENCES Anderson, E. (1965) The anatomy of bovine and ovine pineals. Light and electron microscopic studies. J . Ultrastruct. Res., suppl. 8:l-80. Bargmann, W. (1943) Die Epiphysis Cerebri. In: Handbuch der mikroskopischen Anatomie des Menschen. W. von Mdlendorf, ed. Springer-Verlag, Berlin, Vol. VUI, pp. 309-502. Bhatnagar, K.P. (1988) Ultrastructure of the pineal body of the common vampire bat, Desmodus rotundus. Am. J. Anat., 181:163-178. Bhatnagar, K.P., Frahm, H.D., and Stephan, H. (1986) The pineal organ of bats: A comparative morphological and volumetric investigation. J. Anat., 147:143-161. Bhatnagar, K.P., Frahm, H.D., and Stephan, H. (1990) The megachiropteran pineal organ: A comparative morphological and volumetric investigation with special emphasis on the remarkably large pineal of Dobsoniu pruedutrix. J . Anat., 168:143-166. Buijs, R.M., and Pevet, P. (1980) Vasopressin- and oxytocincontaining fibers in the pineal gland and subcommissural organ of the rat. Cell Tissue Res., 20511-17. Card, J.P., and Moore, R.Y. (1989) Organization of lateral geniculatehypothalamic connections in the rat. J . Comp. Neurol., 284:135147. Chronwall, B.M., DiMaggio, D.A., Massari, V.J., Pickels, V.M., Ruggiero, D.A., and ODonohue, T.L. (1985) The anatomy of neuropeptide-Y-containing neurons in the rat brain. Neuroscience, 15:11591181. Cozzi, B. (1986) Cell types in the pineal gland of the horse: An ultrastructural and immunocytochemical study. Anat. Rec., 216:165174. Cozzi, B., Mikkelsen, J.D., Merati, D., Capsoni, S., and Moller, M. (1989) Vasoactive intestinal peptide (VIP)-like immunoreactive nerve fibers in the pineal gland of the sheep. J . Pineal Res., 8: 41-47. Cozzi, B., and M@ller,M. (1988) Indications for the presence of two populations of serotonin containing pinealocytes in the pineal complex of the golden hamster (Mesocricetus uurutus). An immunohistochemical study. Cell Tissue Res., 252:115-122. David, G.F.X., and Herbert, J . (1973) Experimental evidence for a

MAMMALIAN PINEALOPETAL INNERVATION synaptic connection between habenula and pineal ganglion in the ferret. Brain Res., 64:327-343. David, G.F.X., Herbert, J., and Wright, G.D.S. (1973) The ultrastructure of the pineal ganglion in the ferret. J. Anat., 115:79-97. David, G.F.X., Umberkoman, B., Kumar, K., and Anand Kumar, T.C.A. (1975) Neuroendocrine significance of pineal. In: Brain Endocrine Interaction 11. K.M. Knigge and D.E. Scott, eds. Karger, Basel, pp. 365-375. Falck, B., Hillarp, N.A., Thieme, G., and Thorp, A. (1962) Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10:348-354. Fink-Jensen, A., and Mfiller, M. (1990) Direct projections from the anterior and tuberal regions of the lateral hypothalamus to the rostra1 part of the pineal complex of the rat. An anterograde neuron-tracing study by using Phaseolus vulgaris leucoagglutinin. Brain Res., 522:337,:341. Hartmann, F. (1957) Uber die Innervation der Epiphysis cerebri einiger Saugetiere. Z. Zellforsch., 46:416-429. Hokfelt, T. (1968) In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch., 91:l-74. Hokfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, A,, Goldstein, M., Luft, R., Efendic, s.,Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora, K., Possani, L., Tapia, R., Teran, L., and Palacios, R. (1978) Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In: The Hypothalamus. S. Reichlin, R.J. Baldessarini, and J.B. Martin, eds. Raven Press, New York, pp. 69-135. Hosaka, T., Nakai, A., and Kushima, S. (1957) Innervation of the pineal body and the supracommissural and subcommissural organs of the Japanese monkey. Bull. Tokyo Med. Dent. Univ., 4:365-378. Huang, H.T., and Lin, H.S. (1984) Synaptic junctions between the adrenergic axon varicosity and the pinealocyte in the rat. J. Pineal Res., 1:281-291. Huang, S.K., and Taugner, R. (1984) Gap junctions between guineapig pinealocytes. Cell Tissue Res., 235:137-141. Ichimura, T., Arikuni, T., and Hashimoto, P.H. (1986) Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations. Cell Tissue Res., 244569576. Jaim-Etcheverry, G., and Zieher, L.M. (1979) Ultrastructural cytochemistry and pharmacology of 5-hydroxytryptamine in adrenergic nerve endings. 111. Selective increase of norepinephrine in the rat pineal gland consecutive to depletion of neuronal 5-hydroxytryptamine. J . Pharmacol. Exp. Ther., 178:42-48. Johnson, R.F., Smale, L., Moore, R.Y., and Morin, L.P. (1988) Lateral geniculate lesions block circadian phase-shift responses to a benzodiazepine. Proc. Natl. Acad. Sci. (USA), 85:5301-5304. Josephy, H. (1920) Die feinere Histologie der Epiphyse. Z. Ges. Neurol. Psychiatr., 62:91-119. Kappers, J.A. (1960) The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch., 52:163-215. Kappers, J.A. (1965) Survey of the innervation of the epiphysis cerebri and the accessory pineal organ of vertebrates. Prog. Brain Res., 10237-151. Karasek, M., and Hansen, J . (1982) Ultrastructure of the pineal gland of the fox. Am. J . Anat., 163:257-267. Kenny, G.C.T. (1961) The “nervus conarii” of the monkey. An experimental study. J . Neuropathol. Exp. Neurol., 20563-570. Kolmer, W. (1929) Ganglienzellen als konstanter Bestandteil der Zirbe1 von Affen. Z. Ges. Neurol. Psychiatr., 121:423-428. Korf, H.-W., and Mgller, M. (1984) The innervation of the mammalian pineal gland with special reference to central pinealopetal projections. Pineal Res. Rev., 2:41-86. Korf, H.-W., and Mgller, M. (1985) The central innervation of the mammalian pineal organ. In: The Pineal Gland. B. Mess, Cs. Rdzsas, L. Tima, and P. Pevet, eds. Akademia Kiado, Budapest, pp. 47-69. Korf, H.W., Oksche, A,, Ekstrom, P., Gery, I., Zigler, J.S., and Klein, D.C. (1986) Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum. Science, 231:735-737. Korf, H.W., and Wagner, U. (1980) Evidence for a nervous connection between the brain and the pineal organ in the guinea pig. Cell Tissue Res., 209505-510. Krstic, R.V. (1974) Ultrastructure of the rat pineal gland after preparation by freeze-etching technique. Cell Tissue Res., 148:371-379. Larsen, P.J., Mpiller, M., and Mikkelsen, J.D. (1991) Efferent projections from the periventricular and medial parvocellular subnuclei

203

of the hypothalamic paraventricular nucleus to the circumventricular organs of the rat. A Phaseolus vulgaris leucoagglutinin (PHAL) tracing study. J . Comp. Neurol., 306:462-479. Le Gros Clark, W.E. (1940) The nervous and vascular relations of the pineal gland. J . Anat. (Lond.), 74:470-492. Levin, P.M. (1938) A nervous structure in the pineal body of the monkey. J . Comp. Neurol., 68:405-409. LUO,Z.R., Schultz, R.L., Whitter, E.F., and Vollrath, L. (1984) The ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk. J . Pineal Res., 1:313-337. Marburg, 0. (1909) Zur Kenntnis der normalen und pathologischen Histologie der Zirbeldruse. Die Adipositas cerebralis. Arb. Neurol. Inst. Univ. Wien., 17:217-279. Matsushima, S., and Reiter, R.J. (1978) Electron microscopic observations on neuron-like cells in the ground squirrel pineal gland. J. Neural Transm., 42:223-237. Matsuura, T., and Sano, Y. (1983) Distribution of monoamine-containing nerve fibers in the pineal organ of untreated and sympathectomized dogs. Fluorescence and immunohistochemical studies. Cell Tissue Res., 234:519-531. Mikkelsen, J.D., Cozzi, B., and Mgller, M. (1991) Efferent projections from the lateral geniculate nucleus to the pineal complex of the Mongolian gerbil (Meriones unguiculatus). Cell Tissue Res., 264: 95-102. Mikkelsen, J.D., Korf, H.W., and Mgller, M. (1987) Vasoactive intestinal peptide (VIP) in the pineal gland of the rat. In: Fundamentals and Clinics in Pineal Research. Serono Symposia Publications, Vol. 44. G.P. Trentini, C. de Gaetani, and P. Pevet, eds. Raven Press, New York, pp. 87-90. Mikkelsen, J.D., and Mpiller, M. (1988) Vasoactive intestinal peptide in the hypothalamohypophysial system of the Mongolian gerbil. J . Comp. Neurol., 273:87-98. Mikkelsen, J.D., and Mpiller, M. (1990) A direct neuronal projection from the intergeniculate leaflet of the lateral geniculate nucleus to the deep pineal gland of the rat, demonstrated with Phuseolus uulgaris leucoagglutinin (PHA-L). Brain Res., 520:342-347. Mgller, M. (1976) The ultrastructure of the human fetal pineal gland. 2. Innervation and cell junctions. Cell Tissue Res. 169:7-21. Mgller, M. (1978) Presence of a pineal nerve (n. pinealis) in the human fetus; a light and electron microscopical study of the innervation of the pineal gland. Brain Res., 154:l-12. Mgller, M. (1979) Presence of a pineal nerve (nervus pinealis) in fetal mammals. Prog. Brain Res., 52:103-106. Mpiller, M. (1981) The ultrastructure of the deep pineal gland of the Mongolian gerbil and mouse: Granular vesicle localisation and innervation. In: Pineal Function. C.D. Matthews and R.F. Seamark, eds. Elsevier, Amsterdam, pp. 257-266. Mdler, M. (1985) Non-sympathetic synaptic innervation of the pinealocyte of the Mongolian gerbil (Meriones unguiculatus): An electron microscopic study. J . Neurocytol., 14541-550. M$ller, M. (1986) The human fetal pineal gland. Morphological indications of a photoreceptive capacity. In: The Pineal Gland During Development. D. Gupta and R.J. Reiter, eds. Croom-Helm Ltd., London, Sydney, pp. 80-88. Mpiller, M., Cozzi, B., Schroder, H., and Mikkelsen, J.D. (1987) The peptidergic innervation of the mammalian pineal gland. In: Fundamentals and Clinics in Pineal Research. Serono Symposia Publications, Vol. 44. G.P. Trentini, c. de Gaetani, and P. Pevet, eds. Raven Press, New York, pp. 71-77. Moller, M., and Korf, H.W. (1983a) Central innervation of the pineal organ of the Mongolian gerbil. A histochemical and lesion study. Cell Tissue Res., 230:259-272. Mpiller, M., and Korf, H.W. (1983b) The origin of central pinealopetal nerve fibers in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. Cell Tissue Res., 230: 273-287. Mgller, M., Mikkelsen, J.D., Fahrenkrug, J., and Korf, H.W. (1985) The presence of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerve fibres and VIP-receptors in the pineal gland of the Mongolian gerbil (Meriones unguiculatus). An immunohistochemical and receptor-autoradiographic study. Cell Tissue Res., 241:333340. Mfiller, M., Mfillgird, K., and Kimble, J.K. (1975) Presence ofa pineal nerve in sheep and rabbit fetuses. Cell Tissue Res., 158:451-459. Mgller, M., and van Veen, Th. (1981) Fluorescence histochemistry of the pineal gland. In: The Pineal Gland, Vol. 1. Anatomy and Biochemistry. R.J. Reiter, ed. CRC Press, Boca Raton, Florida, pp. 69-93.

204

M. M0LLER

Mprllgbrd, K., and Mprller, M. (1973) On the innervation of the human fetal pineal gland. Brain Res., 52:428-432. Nielsen, J.T., and Mbller, M. (1975) Nervous connections between the brain and the pineal gland in the cat (Felis catus) and the monkey (Cercopithecus aethiops). Cell Tissue Res., 161:293-301. Nielsen, J.T., and Mbller, M. (1978) Innervation of the pineal gland in the Mongolian gerbil (Meriones unguiculutus). A fluorescence microscopical study. Cell Tissue Res., 187:235-250. Nurnberger, F., and Korf, H.W. (1981) Oxytocin- and vasopressinimmunoreactive nerve fibers in the pineal gland of the hedgehog, Erznaceus europaeus L. Cell Tissue Res., 220:87-97. Owman, Ch. (1965) Localization of neuronal and parenchymal monoamines under normal and experimental conditions in the mammalian pineal gland. Prog. Brain Res., 10:423-453. Pastori, G. (1928) Uber Nervenfasern und Nervenzellen in der “Epiphysis Cerebri.” Z. Ges. Neurol. Psychiatr., 117:202-211. Pellegrino de Iraldi, A,, and Corazza, J.P. (1981) A calcium-binding site modifiable by electrical stimulation in the monoaminergic vesicles of rat pineal nerves. Cell Tissue Res., 216:625-634. Phansuwan-Pujito, P., Mikkelsen, J.D., Govitrapong, P., and Mbller, M. (1991) A cholinergic innervation of the bovine pineal gland visualized by immunohistochemical detection of choline acetyltransferase-immunoreactive nerve fibers. Brain Res., 54549-59. Romijn, H.J. (1973) Parasympathetic innervation of the rabbit pineal gland. Brain Res., 55:431-436. Romijn, H.J. (1975) Structure and innervation of the pineal gland of the rabbit, Oryctolugus cuniculus (L.) 111. An electron microscopical investigation of the innervation. Cell Tissue Res., 157:25-51. Rbnnekleiv, O., and Mprller, M. (1979) Brain-pineal nervous connections in the rat: An ultrastructural study following habenular lesion. Exp. Brain Res., 37:551-562. Rostene, W.H. (1984) Neurobiological and neuroendocrine functions of the vasoactive intestinal peptide (VIP). Prog. Neurobiol., 22:103129. Sasek, C.A., and Zigmond, R.E. (1989) Localization of vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities in the rat superior cervical ganglion and its nerve trunks. J . Comp. Neurol., 280522-523. Scharenberg, K., and Liss, L. (1965) The histologic structure of the human pineal body. Prog. Brain Res., 10:193-217.

Schon, F., Allen, J.M., Yeats, J.C., Allen, Y.S., Ballesta, J., Polak, J.M., Kelly, J.S., and Bloom, S.R. (1985) Neuropeptide Y innervation of the rodent pineal gland and cerebral vessels. Neurosci. Lett., 57:65-71. Schroder, H. (1986) Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation. Histochemistry, 85:321-325. Schroder, H., and Vollrath, L. (1986) Neuropeptide Y (NPY)-like immunoreactivity in the guinea pig pineal organ. Neurosci. Lett., 63: 285-289. Shiotani, Y., Jin, K.L., Kawai, Y., and Kiyama, H. (1989) Immunohistochemical studies on the innervation of the mammalian pineal gland. In: Advances in Pineal Research, Vol. I. R.J. Reiter and S.F. Pang, eds. John Libbey & Co., Ltd., London, Paris, pp. 49-54. Shiotani, Y., Yamano, M., Shisaka, S., Emson, P.C., Hillyard, C.G., Girgis, S., and MacIntyre, I. (1986) Distribution and origins of substance P (SPI-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the pineal gland of gerbils. Neurosci. Lett., 70:187192. Taugner, R., Schiller, A., and Rix, E. (1981) Gap junctions between pinealocytes. A freeze fracture study of the pineal gland in rat. Cell Tissue Res., 218:303-314. Uddman, R., Alumets, J., Hbkanson, R., Loren, I., and Sundler, F. (1980) Vasoactive intestinal peptide (VIP) occurs in nerves of the pineal gland. Experientia, 36:1119-1120. Uemura, Y., Sugimoto, T., Kikuchi, H., and Mizuno, J. (1988)Possible origins of cerebrovascular nerve fibers showing vasoactive intestinal polypeptide-like immunoreactivity: An immunohistochemical study in the dog. Brain Res., 448:98-105. Vollrath, L. (1981) The pineal organ. In: Handbuch der mikroskopischen Anatomie des Menschen, Bd VIi7. A. Oksche and L. Vollrath, eds. Springer-Verlag, Berlin, pp. 1-665. Wood, J.G. (1973) The effect of niamid and reserpine on the nerve endings of the pineal gland. Z. Zellforsch., 145:151-166. Zhang, E.-T., Mikkelsen, J.D., and Mbller, M. (1991)Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglia. Cell Tissue Res., 265:63-71.