MICROSCOPY RESEARCH AND TECHNIQUE 21:124-135 (1992)
The Ultrastructure of Neuronal-Pinealocytic Interconnections in the Monkey Pineal TAKA0 ICHIMURA Department of Anatomy, Kagawa Medical School, Kagawa 761-07, Japan
KEY WORDS
Pinealocyte, Pineal-neuron, Synapses, Ribbon synapse, Innervation, Electron microscopy
ABSTRACT Recent ultrastructural studies of neuronal-pinealocytic interconnections in the monkey pineal are reviewed. The pinealocytes in the adult monkey show almost all of the cytological specializations known in subprimate mammals. Adjacent pinealocytes are functionally coupled through ribbon synapses on cell bodies and gap junctions on cell bodies and cell processes. The pinealocytes receive direct synaptic contacts of nerve fibers with cholinergic terminal morphology. Nerve cells restricted to the central portion of the pineal receive synaptic contacts with more than three different morphologically defined types of nerve terminals. In addition to nerve terminals containing small clear vesicles or vesicles of pleomorphic morphology, a pinealocyte’s terminal process containing the synaptic ribbon forms a true synaptic contact on the nerve cell body. The diversity of synapses on these nerve cells strongly suggests multiple origins of these neurons rather than a single peripheral parasympathetic origin. The possible involvement of pineal neurons in an intrinsic circuit that regulates the function of pinealocytes and integrates the neural input from the central as well as the peripheral nervous systems is discussed. INTRODUCTION
and Reiter, 19781,rabbit (Romijn, 1973a,b),and human (Bargmann, 1943), the monkey pineal has a group of Recent neuroanatomical studies of mammalian pi- interneurons or intrapineal nerve cells (Le Gros Clark, neal have revealed the diversity of neural connections 1940; Kenny, 1961). The parasympathetic nature of between the pineal and the central and peripheral ner- these cells was shown by a histochemical demonstravous systems. Structural findings on central connec- tion of acetylcholinesterase activity in the rabbit (Rotions of the mammalian pineal have been reviewed by mijn, 1975b) and in the monkey (David and Kumar, Korf and Mgller (198413). These authors emphasized 1978). Nerve fibers of central origin including peptithat a considerable number of nerve fibers in the mam- dergic fibers enter the parenchyma via the commismalian pineal belongs to a pinealopetal system of sures (Nielsen and Mgller, 1975; David et al., 1975, central origin (see Mgller, “Ultrastructure of the Mam- Ronnekleiv, 1988). Although target cells of none of malian Pineal Gland, Part II” (1992)).Peripheral sym- these fibers have been characterized, various types of pathetic fibers originating from the superior cervical synaptic morphologies and intercellular junctions are ganglion run deep into the parenchyma andlor perivas- observed on pinealocytes and intrapineal nerve cells cular space, and regulate major functional activity of (Ichimura et al., 1986) probably reflecting the diverse the mammalian pineal (Kappers, 1960; Klein, 1982). nature of the inputs. Recently, Ling et al. (1989) studParasympathetic nerve fibers originating in the me- ied topographical distribution of synaptic contacts on dulla oblongata also enter the pineal as demonstrated the Macaca fascicularis pinealocytes quantitatively in the monkey (Kenny, 1961) and rabbit (Romijn, and concluded that various types of synapses are con1973a,b). However, functional significance of parasym- centrated in the posterior third of the pineal. pathetic innervation of the mammalian pineal is still In this paper, recent ultrastructural findings on neuronal-pinealocytic interconnections will be briefly recontroversial. As t o ultrastructural aspects of interconnections be- viewed. Evidences showing direct synaptic contacts of tween the pineal and the central and peripheral ner- nerve terminals on the pinealocytes or pineal neurons vous systems, studies have been restricted to selected and of pinealocytic processes on the pineal neurons are species of mammals. Wide interspecies variation pre- surveyed. Multiple sources of inputs to the pineal neuvents us from generalizing our limited knowledge t o rons and the involvement of these neruons in the inother species of mammals. Thus, our knowledge of pineal innervation is still incomplete. In this respect, the monkey pineal is exceptional, because both the neural elements and synapses in the pineal have been analyzed at the ultrastructural level (Ichimura et al., 1986; Ling et al., 1989). As in the bat (Kenny, 1965; BhatnaReceived November 15,1989; accepted in revised form June 29,1990. gar, 1988; Bhatnagar et al., 1986, 1990), golden hamAddress reprint requests to Dr. Takao Ichimura, Kagawa Medical School, ster (Jin et al., 19881, ground squirrel (Matsushima Department of Anatomy, Kagawa 761-07, Japan.
0 1992 WILEY-LISS, INC.
Fig. 1. (Legend on overleaf.)
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Fig. 2. Microtubular sheaves in the Golgi field. A pair of microtubular sheaves (arrowheads) show a longitudinal cross section and a obliquely cut cross section in A. They are of the 9 x 3 + 0 type (B).
Incomplete microtubular assembly showing a transient form of the microtubular sheaf occurs in the perinuclear region (C). N, nucleus. A, ~17,000,B, x3O,OOO,C, ~ 3 5 , 0 0 0 .
trinsic circuitry of pinealocytes are suggested. Multiple innervation of the pineal by the sympathetic, parasympathetic, and central fibers is discussed. New evidences showing an additional group of nerve cells in the monkey pineal, presumably the same as those called Marburg’s ganglion, are presented.
Fig. 3. Vesicle crowned rodlets in the cell body (A), pineolocytic process (B),and terminal in the perivascular space (C). Large clusters of VCRs appear in the perikarya (A) or in the process (B),while a single or a pair of VCR a r e seen near the cell boundary (A) or in the terminal processes within the perivascular space (C). See the structural unit of the VCR-that is, rod (r),vesicles (v), and fine filamentous stalk (arrowhead in B f . N, nucleus; P, pinealocytic process; S, vesicle crowned spherule. A, x 38,000, B, x 64,000, C, X 27,000.
~~
~
Fig. 1. Cell body of a pinealocyte. Nucleus (N) with deeply invaginated nuclear envelope occupies almost one-third of the cell body in this section. Cisterns of rough endoplasmic reticulum (er) and Golgi apparatus tG) are accumulated on one side of the nucleus. Vesiclecrowned rodlets (vcr), a single cilium (0,and granular vesicles (gv) characterize the pinealocyte. Mitochondrion (m) with tubular cristae are widely distributed throughout the perikaryon. P, pinealocytic process. x 13,000. (See page 125.)
Fig. 4. A ribbon synapse mediating cell-to-cell communication between two pinealocytes. Two sets of VCRS are vertically applied to the part of the perikaryal cell mebrane faced to the cell body of adjacent pinealocyte on the left. Vesicles (v), filamentous stalks, and the membrane-contacting rods (r) show a real synaptic orientation. Possible fusion of vesicle membranes wit,h the cell membrane of the presynaptic side (arrowhead) suggests true synaptic release in close vicinity of this pair of synaptic ribbons. N, nucleus. x 41,000. (Reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.) Fig. 5. Gap junctions (G) between cell bodies (A) and between pinealocytic processes (B).The gap junction in A is accompanied by a n intermediate junction (I). In B, the clusters of vesicles and central dense bodies (vcr) shows that this terminal is indeed the pinealocytic process. These gap junctions have fine filamentous undercoating (arrowhead). A, x 55,000, B, x 69,000.
ULTRASTRUCTURE OF THE MONKEY PINEAL
Figs. 3-5.
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Fig. 6. Synaptic contacts of nerve terminals on pinealocytes. The cell body on the postsynaptic side is identified as that of the pinealocyte by the presence of VCR (vcr) (A). Boxed area in A is enlarged in B. Nerve terminals on the presynaptic side have small clear vesicles of 40-60 nm in diameters (sv) and a small number of large densecorded vesicles of 100-120 nm in diameters (dcv) (B,C). Homogeneity of vesicular sizes relative to those of pinealocytic processes and the absence of VCR clearly differentiate these nerve terminals from pin-
ealocytes’ terminals. Membrane specialization at the synaptic junction is not so prominent as seen in central synapses, but apposition of synaptic vesicles to axolemma (arrow) and subplasmalemmal accumulation of cytoplasmic filaments are clear on postsynaptic membranes (arrowhead). N, nucleus; m, mitochondria. A, x 12,000, B, X 50,000, C, X 33,000. (A and B reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.)
Fig. 7. The intrapineal neuron. The ultrastructure of this nerve cell is characterized by a dendritic process (D), oval-shaped nucleus (N)in the center of the cell body, a prominent nucleolus (n), a large mass of rough endoplasmic reticulum (er), and the cytoplasm rich in mitochondria (m). Some nerve terminals (asterisks) surround the cell body. x 11,000.
Fig. 8. A ribbon synapse on the intrapineal neuron. VCR in a slender pinealocytic process (vcr) closely applies to the terminal membrane. The postsynaptic membrane has filamentous undercoat (arrowhead). The cleft has fine filaments spanning the pre- and postsynaptic membranes. The cell body on the postsynaptic side is identified to be the pineal neuron as described in the text. x 50,000.(Reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.)
Pigs. 7 and 8.
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Fig. 9. Two types of synapses on the pineal neuron. Axo-somatic synapse of two nerve terminals (a) containing small clear vesicles are seen in A. Membrane specialization is not prominent except filamentous projection (arrow) on the presynaptic side. Axo-dendritic synapse shown in B has an asymmetric membrane specialization (arrowhead).
The presynaptic terminal (a) contains small clear vesicles and a few large dense-cored vesicles. The dendrite also has symmetric synaptic contacts. Round, elipsoid, and flat vesicles are seen in the axon terminal (C).D, dendrite; S, cell soma. A, ~50,000, B, ~39,000, C, x 53,000.
PINEALOCYTES The pinealocyte is the main parenchymal cell of the Mucucu fuscutu pineal. While reflecting its phylogenetic origins from the neurosensory photoreceptor cell, the pinealocyte of adult monkey has specific cell organelles which have been reported in subprimate mammals. These include unique intercellular junctions which can be used as cytological markers to differentiate the pinealocyte from other types of cells. These features are briefly surveyed below.
cleus also has a peculiar shape due to complicated invaginations of the nuclear envelope. The nucleus occupies an off-centered location in the cell body and has a unique nucleolus. Cisterns of rough endoplasmic reticulum and the Golgi apparatus are well developed, and their intracellular distribution appears uneven rather than homogeneous. Mitochondria with tubular cristae are distributed throughout the perikarya. In the periphery of the Golgi field, large granular vesicles, possible vesicular packages of indoles, occur frequently. Cross sections of single cilium are encountered (Fig. 1). Near the Golgi field, unique microtubular sheaves appear (Fig. 2). The microtubular sheaves are of the 9 x 3 + 0 type. They are believed to be involved in the formation of the sensory cilia. The most distinct organelle of the monkey pinealo-
Cytological Profile The monkey pinealocyte has a cell body of irregular shape and varying numbers of cell processes. The nu-
Fig. 10. Two nerve cell bodies in the proximal portion ofthe pineal near the ventricle. They are intermingled in a well-developed neuropi1 which contains rich myelinated fibers (M). There are at least two types of nerve cells: one of large spherical cell body with densely packed mitochondria, well-developed cisterns of the rough endoplasmie reticulum (er), and the Golgi apparatus (G)(B),and the other of
small elongated (bipolar-like) cell body with less developed network of the cisterns (A). Both types of neurons are different from the intrapineal neurons in the central portion of the pineal and referred to as Marburg’s ganglion cells. m, mitochondria; N, nucleus; n, nucleolus. A, x 7,600, B, x 8,100.
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INTRAPINEAL NEURONS “Pineal Neuron” A small group of isolated nerve cells has been observed in several species of mammals including human (Bargmann, 1943). In the macaque monkey, a large number of nerve cells (140 in one specimen) are found in the middle portion of the pineal (Ichimura et al., 1986). They are medium-sized (15-35 pm) multipolar neurons characterized by a central nucleus with a prominent nucleolus and few dendritic processes. Most dendritic processes are only few hundred microns long as traced on serial 4 pm sections. The perikarya is filled with extensive amounts of rough endoplasmic reticulum and the Golgi apparatus, making contacts with a few nerve terminals (Fig. 7). As in the rabbit, these Interpinealocytic Junctions neurons have been regarded as intrapineal parasymRibbon Synapses. Vesicle-crowned rodlets fre- pathetic neurons. However, they are not autonomic quently occur in close apposition to the plasma mem- parasympathetic ganglion cells in the true sense brane of the pinealocyte cell body (Fig. 4). Well-ori- (Smolen, 1988; see Discussion), but very unique nerve ented vesicles and a rodlet closely applied to cell cells characterized by complex synaptic formations on membranes are key structures in the true synaptic rib- their cell bodies and dendritic processes. bon in photoreceptor cells (Usukura and Yamada, 1987; Vollrath et al., 1989). Fine morphology and spePinealocytic-Neuronal Synapse cific orientation to cell membranes of VCR shown in As in other species of mammals, monkey pinealoFigure 4 are very similar to that of true ribbon synapse. Although the synaptic mechanism of this or- cytes have several processes. These processes form the ganelle in mammalian pinealocytes is not known, it so-called crab endings which carry and secrete synhas been suggested that this organelle is involved in thetic products including indole amines to the perivasintercellular communication between adjacent pineal- cular space. So far, no evidence has been obtained ocytes (Ueck and Wake, 1979). Together with gap junc- showing direct synaptic contact of these processes with tions, VCR might share the role of integration of pin- any type of cells in the pineal. The only exception is the ealocytes’ activity through establishing pinealocytic ribbon synapse formed on the nerve cell body in the monkey pineal. This synapse is characterized by a vescircuitry (Huang and Taugner, 1984; see below). Gap Junctions. The monkey pinealocytes also have icle-crowned rodlet in the terminal of pinealocytic progap junctions on their cell body as well as on the pro- cesses, subplasmalemmal densification of the postsyncesses. Gap junctions on cell bodies appear in close re- aptic nerve cell, and fine filaments spanning the lation to intermediate junctions and desmosomes, sug- synaptic cleft (Fig. 8). The presence of these synaptic gesting epithelium-like junctional complex between contacts suggests that even the primate pinealocyte is pinealocytes (Fig. 5). Fenestrated gap junctions are capable of transmitting a chemical signal to the inalso encountered on the cell body. All of these gap junc- trapineal neuron. It may also suggest that intrapineal tions are associated with subplasmalemmal accumula- neurons could relay synaptic input to the brain like the tion of fine filaments, which is characteristic of gap second-order neurons of the pineal ganglion in lower junctions in the central nervous system. Tight junc- vertebrates. tions are not found on parenchymal cells. Nerve Terminals Synapsing on Pineal Neurons Neuronal-Pinealocytic Synapses. True synaptic contacts by nerve terminals on pinealocytes have been In addition to the ribbon synapse, the pineal neuron observed only in the vampire bat (Bhatnagar, 19881, receives a number of synaptic contacts on its cell body ground squirrel (Matsushima and Reiter, 1978), rat and on its dendritic processes. They are characterized (Huang and Lin, 1984) and monkey (Ichimura et al., by a package of small clear vesicles, a few large vesicles 1986; Ling et al., 1989; see also Wood, 1973). This par- with dense cores, pre- and postsynaptic membrane speticular type of synaptic contact has not been reported in cializations, filamentous cleft substance, and subsurother species of mammals. In the rat, sympathetic face cisterns. Both symmetric and asymmetric type adrenergic nerve fibers form en passant synapse on membrane specializations are encountered. Terminals pinealocytes, while in the monkey the synapse is the including pleomorphic synaptic vesicles are also found bouton synapse like that with a typical cholinergic on dendrites (Fig. 9). Acetylcholinesterase-positive morphology (Fig. 6). Recently, synapse-like contacts of nerve terminals and neurons demonstrated in the monnerve bouton with pinealocytes were also observed in key (David and Kumar, 1978) and in the rabbit (Rohuman pineal (Hasegawa et al., 1990). The functional mijn, 1975b) have been regarded as pre- and postgansignificance of these synapses in neural control of pin- glionic parasympathetic nerve fibers and intrapineal ealocytic activity is unknown. However, as will be dis- parasympathetic neurons, respectively. However, the cussed, these synapses are possibly involved not only in diversity of synaptic morphologies seen in the monkey the parasympathetic input but also in the central in- pineal a t the ultrastructural level suggests multiple nervation of the pineal. origins of these neurons rather than a single, parasym-
cyte is, as in other mammals, the vesicle-crowned rodlet (VCR). The structural unit of this rod-vesicle complex is a number of vesicles bound to a rod with fine filamentous stalks. The VCR form a large cluster in the perinuclear region or in the cell processes. Only one or two units occur in close apposition to cell membranes or in terminal processes (Fig. 3). Pevet (1983) argued that the precursor of this organelle might be the microtubular sheave. Thus, this organelle is thought to be formed in the perikarya and transported to the periphery of the cell body and to terminal processes. Whirl bodies or lamellar bodies, which usually appear in subprimate pineals, are not encountered in the monkey pineal.
ULTRASTRUCTURE OF THE MONKEY PINEAL
pathetic origin. Recently, neuron-like cells which show immunoreactivity to tyrosine hydroxylase, a catecholamine synthesizing enzyme, were demonstrated in the pineal of golden hamster (Jin et al., 1988). This suggests the presence of DOPA or dopaminergic neuron in the mammalian pineal, at least in the golden hamster.
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to the pinealocyte. This would function in synchronizing metabolism and secretion of indoles by the pinealocyes. It is still unknown whether electrical coupling through gap junctions and chemical coupling through ribbon synapses represent independent regulatory mechanisms of pinealocyte function. Nerve cell bodies in the pineal parenchyma have Marburg's Ganglion been described in the ferret (David and Herbert, 1973; Near the proximal end of the pineal subjacent to the David et al., 1973),bat (Kenny, 1965; Bhatnagar, 1988; subependymal glial cell layers, another group of nerve Bhatnagar et al., 1986, 19901, golden hamster (Jin et cells is observed. These are intermingled in a well-de- al., 19881, ground squirrel (Matsushima and Reiter, veloped neuropil and a dense mass of myelinated fi- 19781, rabbit (Romijn, 1973a,b), monkey (Le Gros bers. They have spherical or elipsoid cell bodies with a Clark, 1940; Kenny, 1961; Ichimura et al., 1986; Ling few processes, a centrally located and round-shaped nu- et al., 1989), and human (Bargmann, 1943). In the fercleus with a prominent nucleolus, a well-developed net- ret there are intrapineal neurons homologous to those work of Golgi cisterns, and rough endoplasmic retic- in the pineal of lower vertebrates. In the rabbit they ulum (Fig. 10). Only a few nerve terminals form have been regarded as intrapineal parasympathetic synaptic contact on these cell bodies and dendrites. neurons receiving pre- and postganglionic fibers. The These cytological profiles resemble those of nerve cells cholinergic nature of these cell bodies and nerve fibers in the parasympathetic ganglion (see Discussion). involved has been demonstrated by acetylcholinestTheir topographical distribution is quite different from erase technique. Also in the monkey, the nerve cell intrapineal neurons. Thus, another functional role, bodies have been shown to be acetylcholinesterase posperhaps including parasympathetic innervation of the itive (David and Kumar, 19'78).Although the origin of monkey pineal, might be attributed to these neurons. the parasympathetic fibers has been traced experimentally to the superior salivatory nucleus (Romijn, DISCUSSION 19'75a1, after bilateral removal of facial nerves only In this brief review, morphological evidence for the minor changes were detected in the pinealocyte ultrasynaptic organization interconnecting pinealocytes structure (Romijn, 197513).It has been argued that the and intrapineal neurons in the monkey pineal has been acetylcholinesterase reactivity in the rat and rabbit surveyed. Connections between the peripheral and cen- might be due to non-specific staining or the presence of tral nervous systems with the pineal was also re- carnitine acetyltransferase (Schrier and Klein, 1974). Therefore, parasympathetic innervation of the mamviewed. Cell-to-cell contacts of pinealocytes through ribbon malian pineal is not widely accepted. Recent immunohistochemical studies by Jin et al. synapse have been known to occur in almost all mammalian pineals so far examined (for review see Voll- (1988) demonstrated the presence of catecholaminerath, 1981; PBvet, 1983). The chemical nature of the containing neuron-like cells in the pineal of the golden vesicles associated with rodlets is different from that of hamster. Various peptidergic nerve fibers originating true synaptic vesicles, because in the former neither from the pterygopalatine ganglion and trigeminal ganthiamine pyrophosphatase, Na+, nor Ca' have been glion were also shown in the pineal of the gerbil (Shioreported. The vesicles are neither cholinergic nor tani et al., 1986). Although little is known about the adrenergic. Although the mechanism of synaptic trans- anatomical and functional relationships of these neumission may differ from that of the other synaptic junc- ron-like cells and fibers to the sympathetic neurons in tions, the vesicle-crowned rodlets (VCR) membrane the superior cervical ganglion, their involvement in complex in the monkey pineal may establish a chemi- special pineal activity such as regression of the gonads cal link between adjacent pinealocytes (Ueck and during hibernation was suggested. Another group of nerve cells observed in a well-deWake, 19'79). Immunocytochemical identification of transmitter substance is crucial to understand the veloped neuropil in the proximal monkey pineal is comfunction of this synaptic link, The functional signifi- pared to Marburg's ganglion described in the human cance of VCRs in the processes is not well understood; pineal (Kenny, 1965; Mollgaard and MZller, 1973). The however, they might be involved in the release of syn- present observation that only limited numbers of synthetic products into perivascular space (Vollrath, aptic contacts are seen on these neurons sugests their morphological similarity to parasympathetic ganglion 1981). Electrical coupling of pinealocytes through gap junc- cells (Smolen, 1988; see also below). Although it is not tions and their possible involvement in functional yet clear whether the cell bodies of these neurons lie compartmentalization have been described in the rat inside the pineal parenchyma, they may participate in (Taugner et al., 1981) and the guinea pig (Jung and the parasympathetic innervation of the pineal as sugVollrath, 1982; Huang and Taugner, 1984). In these gested by Kenny (1965). Their involvement in the cenrodents pinealocytes form isolated cell clusters, while tral innervation is unknown. Various types of synaptic morphologies present on in the monkey lobular compartmentalization is not distinct as in the rat. Even without apparent lobular pineal neurons now raise the question as to the origin structure, however, electrical coupling could facilitate of the inputs. The ribbon synapse found on the nerve cell body is a propagation of neural input to adjacent pinealocytes following synaptic transmission from nerve terminals unique structure showing a direct synaptic coupling +
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between the pinealocyte and the neuron. This is the form of a synaptic link between the photoreceptive pinealocyte and the second-order neuron in lower vertebrates. In this respect, functional significance of the 9 x 3 + 0 type microtubular sheaves and the cilia found in the monkey pineal needs reevaluation. The presence of these synaptic contacts and the cilia might suggest that pinealocytes are actually sensory cells and that intrapineal neurons in the monkey, at least in part, are the second-order neurons. In higher mammals, however, there is no electrophysiological evidence which shows that pinealocytes respond directly to photic stimulation. Morphological evidence showing the presence of pinealofugal elements in the habenular and posterior commissures is not convincing. Although a possibility that the monkey pineal has sensory pinealocytes may not be eliminated, it seems more acceptable to suspect that the pinealocytic-neuronal ribbon synapse has acquired a different function in the monkey pineal. Regulation of the indoles’ metabolism through a pinealocytic-neuronal feedback loop might be a possible function acquired during phylogenetic evolution of the mammalian pineal. The varieties of synaptic terminals on pineal neurons strongly suggest that nerve fibers as well as nerve cell bodies, a t least in part, arise elsewhere than the autonomic nervous system. For peripheral parasympathetic neurons, all of the pre- and postganglionic axons are of the same biochemical and morphological type (i-e., cholinergic) with small clear vesicles and a few large dense corded vesicles (Smolen, 1988). Each postganghonic neuron receives synaptic connection from only one preganglionic axon (Lichtman, 1977). These neurons contain enkephalins, substance P (Erichsen et al., 19821, and somatostatin (Kondo, 1977). Immunocytochemical examination of coexistence of these peptides with acetylcholine in pineal neurons might provide evidence for their parasympathetic nature, if they are, in fact, parasympathetic in nature. Cumulating evidence suggests that central nerve fibers enter the mammalian pineal via the habenular and/or posterior commissures (Kappers, 1960; Nielsen and Mdler, 1975; Korf and Mprller, 1984a; Reuss and Mdler, 1986; Mprller and Korf, 1987; Ronnekleiv, 1988). Fibers containing arginine vasotosin, a- MSH, LHRH, or somatostatin have been identified in the rat, and fibers containing substance P, vasopressin, oxytocin, or neurophysins have been found in the monkey pineal. These peptidergic central fibers resemble neurosecretory fibers in the neurophypophysis. As suggested in rodents (Korf and Mprller, 1984a), these central pinealopetal fibers in the monkey may also be neurosecretory. However, the possibility that they are involved in direct central regulation and/or modulation of the pineal function by the central nervous system cannot be eliminated. Intrapineal neurons and/or pinealocytes themselves might be target cells of these central fibers. Anatomical evidence of their origin in the brain and their connections with target cells in the pineal, combined with immunocytochemical identification of transmitters, would greatly improve our understanding of innervation of the mammalian pineal. In conclusion it should be emphasized that extensive
study of the monkey pineal by means of anatomical, ultrastructural, immunocytochemical, biochemical, as well as electrophysiological techniques would provide us the needed information on the brain-pineal intercommunication in the mammal.
REFERENCES Bargmann, W. (1943) Die epiphysis cerebri. In: Handbuch der mikroskopische Anatomie des Menschen, Vol. 6, No. 4. W.V. Mollendorff, ed. Springer Verlag, Berlin, p. 309. 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., Frahn, H.D., and Stephan, H. (1990) The megachiropteran pineal organ: A comparative morphological and volurnetric investigation with special emphasis on the remarkably large pineal of Dobsoniu pruedatrix. J. Anat., 168:143-166. David, G.F.X., and Herbert, J. (1973) Experimental evidence for a 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., 11579-97. David, G.F.X., and Kumar, T.C.A. (1978) Histochemical localization of cholinesterase in the neural tissue of the pineal in the rhesw monkey. Experientia, 34:1067-1068. David, G.F.X., Umberkoman, B., Kumar, K., and Anad Kumar, T.C. (1975) Neuroendocrine significance of the pineal. In: Brain-Endocrine Interaction 11: The Ventricular System in Neuroendocrine Mechanism. K.M. Knigge, D.E. Scott, H. Kobayashi, and S. Ishii, eds. Karger, Basel. Erichsen, J.T., Karten, H.J., Eldred, W.D., and Brecha, N.C. (1982) Localization of substance P-like and enkephalin-like immunoreactivity within preganglionic terminals of the avian ciliary ganglion: Light and electron microscopy. J. Neurosci., 2:994-1003. Hasegawa, A., Ohtsubo, K., Izumiyama, N., and Shimada, H. (1990) Ultrastructural study of the human pineal gland in aged patients including a centenarian. Acta Pathol. Jpn., 4030-40. 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., 244:569576. Jin, K.L., Shiotani, Y., Kawai, Y., Kiyama, H. (1988) Immunohistochemical demonstration of tyrosine hydroxylase (THI-positive but dopamine B-hydroxylase (DBH)-negative neuron-like cells in the pineal gland of golden hamsters. Neurosci. Lett., 93:28-31. Jung, D., and Vollrath, L. (1982) Structural dissimilarities in different regions of the pineal gland of Pirbright White guinea-pigs. J. Neural Transm., 54:117-128. Kappers, J.A. (1960) The development, topographical relations, and innervation of the epiphysis cerebri in the albino rat. 2. Zellforsch., 52:163-215. Kenny, G.C.T. (1961) The “nervus conarii” of the monkey (an experimental study). J. Neuropath. Exp. Neurol., 20563-570. Kenny, G.C.T. (1965) The innervation of the mammalian pineal body (a comparative study). Roc. Aust. Assoc. Neurol., 3:133-140. Klein, D.C. (1982) Melatonin rhythm generating system. Developmental aspects. Karger, Basel. Kondo, H. (1977) Innervation of SIF cells in the superior cervical and nodose ganglia: An ultrastructural study with serial sections. Biol. Cell, 30:253-264. Korf, H.W., and Mdler, M. (1984a) The innervation of the mammalian pineal gland with special reference to central pinealopetal projections. In: Pineal Research Reviews, Vol. 2. R.J. Reiter, ed. Alan Liss, New York, pp. 41-86. Korf, H.W., and Mprller, M. (1984b) The central innervation of the mammalian pineal organ. In: The Pineal Gland. Current status of pineal research. B. Mess, C. Ruzsas, T. Tima, and P. Pevet, eds. Elsevier, Amsterdam, New York, pp. 47-69.
ULTRASTRUCTURE OF THE MONKEY PINEAL Le Gros Clark, W.E. (1940)The nervous and vascular relations of the pineal gland. J. Anat., 74:471-492. Lichtman, J . (1977) The reorganization of synaptic connections in the rat submandibular ganglion during postnatal development. J. Physiol. (Lond.), 273:155-177. Ling, E.A., Tan, T.Y., Yick, T.Y., and Wong, W.C. (1989) Ultrastructure of the pineal gland of the monkey, Macaca fascicularis, with special reference to the presence of synaptic junctions on pinealocytes. Anat. Embliol. 180:151-158. 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. M@ller,M. (1992) Fine structure of the pinealopetal innervation of the mammalian pineal gland. Microsc. Res. Tech., in press. Mplller, M., and Korf, H.W. (1987) Neural connections between the brain and the pineal gland of the golden hamster (Mesocricetus euratus). Tracer studies by use of horseradish peroxidase in vivo and in vitro. Cell Tissue Res., 247:145-153. Mollgard, K., and Mplller, M. (1973) On the innervation of the human fetal pineal gland. Brain Res., 52:428-432. Nielsen, J.T., and Mplller, M. (1975) Nervous connections between the brain and the pineal in the cat (Felis catus) and the monkey (Cercopithecus aethiops). Cell Tissue h s . , 161:293-301. Pevet, P. (1983) Anatomy of the pineal gland of mammals. In: The Pineal Gland. R. Relkin, ed. Elsevier, New York, Amsterdam, Oxford, pp. 1-75. Reuss, S., and Meller, M. (1986) Direct projections to the rat pineal gland via the stria medullaris thalami. An anterograde tracing study by use of horseradish peroxidase. Cell Tissue Res., 244:691694. Romijn, H.J. (1973a) Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.). I. A light microscopic investigation. Z. Zellforsch., 139:473-485. Romijn, H.J. (1973b) Parasympathetic innervation of the rabbit pineal gland. Brain Res.. 55:431-436. Romijn7H.J. (1975a) Str&ture and innervation of the pineal gland of
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the rabbit, Oryctolagus cuniculus (L.) 111. An electron microscopic investigation of the innervation. Cell Tissue Res., 157:25-51. Romijn, H.J. (1975b) The ultrastructure of the rabbit pineal gland after sympathectomy, parasympathectomy, continuous illumination, and continuous darkness. J. Neural Transm., 36:183. Ronnekleiv, O.K. (1988) Distribution in the macaque pineal of nerve fibers containing immunoreactive substance P, vasopressin, oxytocin, and neurophysins. J. Pineal Res., 5:259-271. Schrier, B.K., and Klein, D.C. (1974) Absence of choline acetyltransferase in rat and rabbit pineal gland. Brain Res., 79:347-351. Shiotani, Y., Yarnano, M., Shiosaka, S., Emson, P.C., Hillyard, C.J., Girgis, S.,and MacIntyre, I. (19863 Distribution and origins of substance P (SP)-, calcitonin gene-related peptide (CGRPI-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the pineal gland of gerbils. Neurosci. Lett., 70:187192. Smolen, A. (1988) Morphology of synapses in the autonomic nervous system. J. Electron Microsc. Tech., 10:187-204. Taugner, R., Schiller, A., and Rix, E. (1981) Gap junctions between pinealocytes. A freeze fracture study of the pineal gland in rats. Cell Tissue Res., 218:303-314. Ueck, M., and Wake, K. (1979) The pinealocyte-a paraneuron. Prog. Brain Res., 52:141-147. Usukura, J., and Yamada, E. (1987) Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution. Cell Tissue Res., 247:483488. Vollrath, L. (1981) The pineal organ. In: Handbuch der mikroskopischen Anatomie des Menschen, Vol. VI, No. 7. A. Oksche, and L. Vollrath, eds. Springer, Heidelberg, pp. 186-215. Vollrath, L., Meyer, A,, and Buschmann, F. (1989) Ribbon synapses of the mammalian retina contain two types of synaptic bodies-ribbons and spheres. J. Neurocytol., 18:115-120. Wood, J.G. (1973) The effect of niamid and reserpine on the nerve endings - of the pineal gland. Z. Zellforsch., 145:151-166.
The Ultrastructure of Neuronal-Pinealocytic Interconnections in the Monkey Pineal TAKA0 ICHIMURA Department of Anatomy, Kagawa Medical School, Kagawa 761-07, Japan
KEY WORDS
Pinealocyte, Pineal-neuron, Synapses, Ribbon synapse, Innervation, Electron microscopy
ABSTRACT Recent ultrastructural studies of neuronal-pinealocytic interconnections in the monkey pineal are reviewed. The pinealocytes in the adult monkey show almost all of the cytological specializations known in subprimate mammals. Adjacent pinealocytes are functionally coupled through ribbon synapses on cell bodies and gap junctions on cell bodies and cell processes. The pinealocytes receive direct synaptic contacts of nerve fibers with cholinergic terminal morphology. Nerve cells restricted to the central portion of the pineal receive synaptic contacts with more than three different morphologically defined types of nerve terminals. In addition to nerve terminals containing small clear vesicles or vesicles of pleomorphic morphology, a pinealocyte’s terminal process containing the synaptic ribbon forms a true synaptic contact on the nerve cell body. The diversity of synapses on these nerve cells strongly suggests multiple origins of these neurons rather than a single peripheral parasympathetic origin. The possible involvement of pineal neurons in an intrinsic circuit that regulates the function of pinealocytes and integrates the neural input from the central as well as the peripheral nervous systems is discussed. INTRODUCTION
and Reiter, 19781,rabbit (Romijn, 1973a,b),and human (Bargmann, 1943), the monkey pineal has a group of Recent neuroanatomical studies of mammalian pi- interneurons or intrapineal nerve cells (Le Gros Clark, neal have revealed the diversity of neural connections 1940; Kenny, 1961). The parasympathetic nature of between the pineal and the central and peripheral ner- these cells was shown by a histochemical demonstravous systems. Structural findings on central connec- tion of acetylcholinesterase activity in the rabbit (Rotions of the mammalian pineal have been reviewed by mijn, 1975b) and in the monkey (David and Kumar, Korf and Mgller (198413). These authors emphasized 1978). Nerve fibers of central origin including peptithat a considerable number of nerve fibers in the mam- dergic fibers enter the parenchyma via the commismalian pineal belongs to a pinealopetal system of sures (Nielsen and Mgller, 1975; David et al., 1975, central origin (see Mgller, “Ultrastructure of the Mam- Ronnekleiv, 1988). Although target cells of none of malian Pineal Gland, Part II” (1992)).Peripheral sym- these fibers have been characterized, various types of pathetic fibers originating from the superior cervical synaptic morphologies and intercellular junctions are ganglion run deep into the parenchyma andlor perivas- observed on pinealocytes and intrapineal nerve cells cular space, and regulate major functional activity of (Ichimura et al., 1986) probably reflecting the diverse the mammalian pineal (Kappers, 1960; Klein, 1982). nature of the inputs. Recently, Ling et al. (1989) studParasympathetic nerve fibers originating in the me- ied topographical distribution of synaptic contacts on dulla oblongata also enter the pineal as demonstrated the Macaca fascicularis pinealocytes quantitatively in the monkey (Kenny, 1961) and rabbit (Romijn, and concluded that various types of synapses are con1973a,b). However, functional significance of parasym- centrated in the posterior third of the pineal. pathetic innervation of the mammalian pineal is still In this paper, recent ultrastructural findings on neuronal-pinealocytic interconnections will be briefly recontroversial. As t o ultrastructural aspects of interconnections be- viewed. Evidences showing direct synaptic contacts of tween the pineal and the central and peripheral ner- nerve terminals on the pinealocytes or pineal neurons vous systems, studies have been restricted to selected and of pinealocytic processes on the pineal neurons are species of mammals. Wide interspecies variation pre- surveyed. Multiple sources of inputs to the pineal neuvents us from generalizing our limited knowledge t o rons and the involvement of these neruons in the inother species of mammals. Thus, our knowledge of pineal innervation is still incomplete. In this respect, the monkey pineal is exceptional, because both the neural elements and synapses in the pineal have been analyzed at the ultrastructural level (Ichimura et al., 1986; Ling et al., 1989). As in the bat (Kenny, 1965; BhatnaReceived November 15,1989; accepted in revised form June 29,1990. gar, 1988; Bhatnagar et al., 1986, 1990), golden hamAddress reprint requests to Dr. Takao Ichimura, Kagawa Medical School, ster (Jin et al., 19881, ground squirrel (Matsushima Department of Anatomy, Kagawa 761-07, Japan.
0 1992 WILEY-LISS, INC.
Fig. 1. (Legend on overleaf.)
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Fig. 2. Microtubular sheaves in the Golgi field. A pair of microtubular sheaves (arrowheads) show a longitudinal cross section and a obliquely cut cross section in A. They are of the 9 x 3 + 0 type (B).
Incomplete microtubular assembly showing a transient form of the microtubular sheaf occurs in the perinuclear region (C). N, nucleus. A, ~17,000,B, x3O,OOO,C, ~ 3 5 , 0 0 0 .
trinsic circuitry of pinealocytes are suggested. Multiple innervation of the pineal by the sympathetic, parasympathetic, and central fibers is discussed. New evidences showing an additional group of nerve cells in the monkey pineal, presumably the same as those called Marburg’s ganglion, are presented.
Fig. 3. Vesicle crowned rodlets in the cell body (A), pineolocytic process (B),and terminal in the perivascular space (C). Large clusters of VCRs appear in the perikarya (A) or in the process (B),while a single or a pair of VCR a r e seen near the cell boundary (A) or in the terminal processes within the perivascular space (C). See the structural unit of the VCR-that is, rod (r),vesicles (v), and fine filamentous stalk (arrowhead in B f . N, nucleus; P, pinealocytic process; S, vesicle crowned spherule. A, x 38,000, B, x 64,000, C, X 27,000.
~~
~
Fig. 1. Cell body of a pinealocyte. Nucleus (N) with deeply invaginated nuclear envelope occupies almost one-third of the cell body in this section. Cisterns of rough endoplasmic reticulum (er) and Golgi apparatus tG) are accumulated on one side of the nucleus. Vesiclecrowned rodlets (vcr), a single cilium (0,and granular vesicles (gv) characterize the pinealocyte. Mitochondrion (m) with tubular cristae are widely distributed throughout the perikaryon. P, pinealocytic process. x 13,000. (See page 125.)
Fig. 4. A ribbon synapse mediating cell-to-cell communication between two pinealocytes. Two sets of VCRS are vertically applied to the part of the perikaryal cell mebrane faced to the cell body of adjacent pinealocyte on the left. Vesicles (v), filamentous stalks, and the membrane-contacting rods (r) show a real synaptic orientation. Possible fusion of vesicle membranes wit,h the cell membrane of the presynaptic side (arrowhead) suggests true synaptic release in close vicinity of this pair of synaptic ribbons. N, nucleus. x 41,000. (Reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.) Fig. 5. Gap junctions (G) between cell bodies (A) and between pinealocytic processes (B).The gap junction in A is accompanied by a n intermediate junction (I). In B, the clusters of vesicles and central dense bodies (vcr) shows that this terminal is indeed the pinealocytic process. These gap junctions have fine filamentous undercoating (arrowhead). A, x 55,000, B, x 69,000.
ULTRASTRUCTURE OF THE MONKEY PINEAL
Figs. 3-5.
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Fig. 6. Synaptic contacts of nerve terminals on pinealocytes. The cell body on the postsynaptic side is identified as that of the pinealocyte by the presence of VCR (vcr) (A). Boxed area in A is enlarged in B. Nerve terminals on the presynaptic side have small clear vesicles of 40-60 nm in diameters (sv) and a small number of large densecorded vesicles of 100-120 nm in diameters (dcv) (B,C). Homogeneity of vesicular sizes relative to those of pinealocytic processes and the absence of VCR clearly differentiate these nerve terminals from pin-
ealocytes’ terminals. Membrane specialization at the synaptic junction is not so prominent as seen in central synapses, but apposition of synaptic vesicles to axolemma (arrow) and subplasmalemmal accumulation of cytoplasmic filaments are clear on postsynaptic membranes (arrowhead). N, nucleus; m, mitochondria. A, x 12,000, B, X 50,000, C, X 33,000. (A and B reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.)
Fig. 7. The intrapineal neuron. The ultrastructure of this nerve cell is characterized by a dendritic process (D), oval-shaped nucleus (N)in the center of the cell body, a prominent nucleolus (n), a large mass of rough endoplasmic reticulum (er), and the cytoplasm rich in mitochondria (m). Some nerve terminals (asterisks) surround the cell body. x 11,000.
Fig. 8. A ribbon synapse on the intrapineal neuron. VCR in a slender pinealocytic process (vcr) closely applies to the terminal membrane. The postsynaptic membrane has filamentous undercoat (arrowhead). The cleft has fine filaments spanning the pre- and postsynaptic membranes. The cell body on the postsynaptic side is identified to be the pineal neuron as described in the text. x 50,000.(Reproduced from Ichimura et al., 1986, with permission of Springer-Verlag.)
Pigs. 7 and 8.
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Fig. 9. Two types of synapses on the pineal neuron. Axo-somatic synapse of two nerve terminals (a) containing small clear vesicles are seen in A. Membrane specialization is not prominent except filamentous projection (arrow) on the presynaptic side. Axo-dendritic synapse shown in B has an asymmetric membrane specialization (arrowhead).
The presynaptic terminal (a) contains small clear vesicles and a few large dense-cored vesicles. The dendrite also has symmetric synaptic contacts. Round, elipsoid, and flat vesicles are seen in the axon terminal (C).D, dendrite; S, cell soma. A, ~50,000, B, ~39,000, C, x 53,000.
PINEALOCYTES The pinealocyte is the main parenchymal cell of the Mucucu fuscutu pineal. While reflecting its phylogenetic origins from the neurosensory photoreceptor cell, the pinealocyte of adult monkey has specific cell organelles which have been reported in subprimate mammals. These include unique intercellular junctions which can be used as cytological markers to differentiate the pinealocyte from other types of cells. These features are briefly surveyed below.
cleus also has a peculiar shape due to complicated invaginations of the nuclear envelope. The nucleus occupies an off-centered location in the cell body and has a unique nucleolus. Cisterns of rough endoplasmic reticulum and the Golgi apparatus are well developed, and their intracellular distribution appears uneven rather than homogeneous. Mitochondria with tubular cristae are distributed throughout the perikarya. In the periphery of the Golgi field, large granular vesicles, possible vesicular packages of indoles, occur frequently. Cross sections of single cilium are encountered (Fig. 1). Near the Golgi field, unique microtubular sheaves appear (Fig. 2). The microtubular sheaves are of the 9 x 3 + 0 type. They are believed to be involved in the formation of the sensory cilia. The most distinct organelle of the monkey pinealo-
Cytological Profile The monkey pinealocyte has a cell body of irregular shape and varying numbers of cell processes. The nu-
Fig. 10. Two nerve cell bodies in the proximal portion ofthe pineal near the ventricle. They are intermingled in a well-developed neuropi1 which contains rich myelinated fibers (M). There are at least two types of nerve cells: one of large spherical cell body with densely packed mitochondria, well-developed cisterns of the rough endoplasmie reticulum (er), and the Golgi apparatus (G)(B),and the other of
small elongated (bipolar-like) cell body with less developed network of the cisterns (A). Both types of neurons are different from the intrapineal neurons in the central portion of the pineal and referred to as Marburg’s ganglion cells. m, mitochondria; N, nucleus; n, nucleolus. A, x 7,600, B, x 8,100.
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INTRAPINEAL NEURONS “Pineal Neuron” A small group of isolated nerve cells has been observed in several species of mammals including human (Bargmann, 1943). In the macaque monkey, a large number of nerve cells (140 in one specimen) are found in the middle portion of the pineal (Ichimura et al., 1986). They are medium-sized (15-35 pm) multipolar neurons characterized by a central nucleus with a prominent nucleolus and few dendritic processes. Most dendritic processes are only few hundred microns long as traced on serial 4 pm sections. The perikarya is filled with extensive amounts of rough endoplasmic reticulum and the Golgi apparatus, making contacts with a few nerve terminals (Fig. 7). As in the rabbit, these Interpinealocytic Junctions neurons have been regarded as intrapineal parasymRibbon Synapses. Vesicle-crowned rodlets fre- pathetic neurons. However, they are not autonomic quently occur in close apposition to the plasma mem- parasympathetic ganglion cells in the true sense brane of the pinealocyte cell body (Fig. 4). Well-ori- (Smolen, 1988; see Discussion), but very unique nerve ented vesicles and a rodlet closely applied to cell cells characterized by complex synaptic formations on membranes are key structures in the true synaptic rib- their cell bodies and dendritic processes. bon in photoreceptor cells (Usukura and Yamada, 1987; Vollrath et al., 1989). Fine morphology and spePinealocytic-Neuronal Synapse cific orientation to cell membranes of VCR shown in As in other species of mammals, monkey pinealoFigure 4 are very similar to that of true ribbon synapse. Although the synaptic mechanism of this or- cytes have several processes. These processes form the ganelle in mammalian pinealocytes is not known, it so-called crab endings which carry and secrete synhas been suggested that this organelle is involved in thetic products including indole amines to the perivasintercellular communication between adjacent pineal- cular space. So far, no evidence has been obtained ocytes (Ueck and Wake, 1979). Together with gap junc- showing direct synaptic contact of these processes with tions, VCR might share the role of integration of pin- any type of cells in the pineal. The only exception is the ealocytes’ activity through establishing pinealocytic ribbon synapse formed on the nerve cell body in the monkey pineal. This synapse is characterized by a vescircuitry (Huang and Taugner, 1984; see below). Gap Junctions. The monkey pinealocytes also have icle-crowned rodlet in the terminal of pinealocytic progap junctions on their cell body as well as on the pro- cesses, subplasmalemmal densification of the postsyncesses. Gap junctions on cell bodies appear in close re- aptic nerve cell, and fine filaments spanning the lation to intermediate junctions and desmosomes, sug- synaptic cleft (Fig. 8). The presence of these synaptic gesting epithelium-like junctional complex between contacts suggests that even the primate pinealocyte is pinealocytes (Fig. 5). Fenestrated gap junctions are capable of transmitting a chemical signal to the inalso encountered on the cell body. All of these gap junc- trapineal neuron. It may also suggest that intrapineal tions are associated with subplasmalemmal accumula- neurons could relay synaptic input to the brain like the tion of fine filaments, which is characteristic of gap second-order neurons of the pineal ganglion in lower junctions in the central nervous system. Tight junc- vertebrates. tions are not found on parenchymal cells. Nerve Terminals Synapsing on Pineal Neurons Neuronal-Pinealocytic Synapses. True synaptic contacts by nerve terminals on pinealocytes have been In addition to the ribbon synapse, the pineal neuron observed only in the vampire bat (Bhatnagar, 19881, receives a number of synaptic contacts on its cell body ground squirrel (Matsushima and Reiter, 1978), rat and on its dendritic processes. They are characterized (Huang and Lin, 1984) and monkey (Ichimura et al., by a package of small clear vesicles, a few large vesicles 1986; Ling et al., 1989; see also Wood, 1973). This par- with dense cores, pre- and postsynaptic membrane speticular type of synaptic contact has not been reported in cializations, filamentous cleft substance, and subsurother species of mammals. In the rat, sympathetic face cisterns. Both symmetric and asymmetric type adrenergic nerve fibers form en passant synapse on membrane specializations are encountered. Terminals pinealocytes, while in the monkey the synapse is the including pleomorphic synaptic vesicles are also found bouton synapse like that with a typical cholinergic on dendrites (Fig. 9). Acetylcholinesterase-positive morphology (Fig. 6). Recently, synapse-like contacts of nerve terminals and neurons demonstrated in the monnerve bouton with pinealocytes were also observed in key (David and Kumar, 1978) and in the rabbit (Rohuman pineal (Hasegawa et al., 1990). The functional mijn, 1975b) have been regarded as pre- and postgansignificance of these synapses in neural control of pin- glionic parasympathetic nerve fibers and intrapineal ealocytic activity is unknown. However, as will be dis- parasympathetic neurons, respectively. However, the cussed, these synapses are possibly involved not only in diversity of synaptic morphologies seen in the monkey the parasympathetic input but also in the central in- pineal a t the ultrastructural level suggests multiple nervation of the pineal. origins of these neurons rather than a single, parasym-
cyte is, as in other mammals, the vesicle-crowned rodlet (VCR). The structural unit of this rod-vesicle complex is a number of vesicles bound to a rod with fine filamentous stalks. The VCR form a large cluster in the perinuclear region or in the cell processes. Only one or two units occur in close apposition to cell membranes or in terminal processes (Fig. 3). Pevet (1983) argued that the precursor of this organelle might be the microtubular sheave. Thus, this organelle is thought to be formed in the perikarya and transported to the periphery of the cell body and to terminal processes. Whirl bodies or lamellar bodies, which usually appear in subprimate pineals, are not encountered in the monkey pineal.
ULTRASTRUCTURE OF THE MONKEY PINEAL
pathetic origin. Recently, neuron-like cells which show immunoreactivity to tyrosine hydroxylase, a catecholamine synthesizing enzyme, were demonstrated in the pineal of golden hamster (Jin et al., 1988). This suggests the presence of DOPA or dopaminergic neuron in the mammalian pineal, at least in the golden hamster.
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to the pinealocyte. This would function in synchronizing metabolism and secretion of indoles by the pinealocyes. It is still unknown whether electrical coupling through gap junctions and chemical coupling through ribbon synapses represent independent regulatory mechanisms of pinealocyte function. Nerve cell bodies in the pineal parenchyma have Marburg's Ganglion been described in the ferret (David and Herbert, 1973; Near the proximal end of the pineal subjacent to the David et al., 1973),bat (Kenny, 1965; Bhatnagar, 1988; subependymal glial cell layers, another group of nerve Bhatnagar et al., 1986, 19901, golden hamster (Jin et cells is observed. These are intermingled in a well-de- al., 19881, ground squirrel (Matsushima and Reiter, veloped neuropil and a dense mass of myelinated fi- 19781, rabbit (Romijn, 1973a,b), monkey (Le Gros bers. They have spherical or elipsoid cell bodies with a Clark, 1940; Kenny, 1961; Ichimura et al., 1986; Ling few processes, a centrally located and round-shaped nu- et al., 1989), and human (Bargmann, 1943). In the fercleus with a prominent nucleolus, a well-developed net- ret there are intrapineal neurons homologous to those work of Golgi cisterns, and rough endoplasmic retic- in the pineal of lower vertebrates. In the rabbit they ulum (Fig. 10). Only a few nerve terminals form have been regarded as intrapineal parasympathetic synaptic contact on these cell bodies and dendrites. neurons receiving pre- and postganglionic fibers. The These cytological profiles resemble those of nerve cells cholinergic nature of these cell bodies and nerve fibers in the parasympathetic ganglion (see Discussion). involved has been demonstrated by acetylcholinestTheir topographical distribution is quite different from erase technique. Also in the monkey, the nerve cell intrapineal neurons. Thus, another functional role, bodies have been shown to be acetylcholinesterase posperhaps including parasympathetic innervation of the itive (David and Kumar, 19'78).Although the origin of monkey pineal, might be attributed to these neurons. the parasympathetic fibers has been traced experimentally to the superior salivatory nucleus (Romijn, DISCUSSION 19'75a1, after bilateral removal of facial nerves only In this brief review, morphological evidence for the minor changes were detected in the pinealocyte ultrasynaptic organization interconnecting pinealocytes structure (Romijn, 197513).It has been argued that the and intrapineal neurons in the monkey pineal has been acetylcholinesterase reactivity in the rat and rabbit surveyed. Connections between the peripheral and cen- might be due to non-specific staining or the presence of tral nervous systems with the pineal was also re- carnitine acetyltransferase (Schrier and Klein, 1974). Therefore, parasympathetic innervation of the mamviewed. Cell-to-cell contacts of pinealocytes through ribbon malian pineal is not widely accepted. Recent immunohistochemical studies by Jin et al. synapse have been known to occur in almost all mammalian pineals so far examined (for review see Voll- (1988) demonstrated the presence of catecholaminerath, 1981; PBvet, 1983). The chemical nature of the containing neuron-like cells in the pineal of the golden vesicles associated with rodlets is different from that of hamster. Various peptidergic nerve fibers originating true synaptic vesicles, because in the former neither from the pterygopalatine ganglion and trigeminal ganthiamine pyrophosphatase, Na+, nor Ca' have been glion were also shown in the pineal of the gerbil (Shioreported. The vesicles are neither cholinergic nor tani et al., 1986). Although little is known about the adrenergic. Although the mechanism of synaptic trans- anatomical and functional relationships of these neumission may differ from that of the other synaptic junc- ron-like cells and fibers to the sympathetic neurons in tions, the vesicle-crowned rodlets (VCR) membrane the superior cervical ganglion, their involvement in complex in the monkey pineal may establish a chemi- special pineal activity such as regression of the gonads cal link between adjacent pinealocytes (Ueck and during hibernation was suggested. Another group of nerve cells observed in a well-deWake, 19'79). Immunocytochemical identification of transmitter substance is crucial to understand the veloped neuropil in the proximal monkey pineal is comfunction of this synaptic link, The functional signifi- pared to Marburg's ganglion described in the human cance of VCRs in the processes is not well understood; pineal (Kenny, 1965; Mollgaard and MZller, 1973). The however, they might be involved in the release of syn- present observation that only limited numbers of synthetic products into perivascular space (Vollrath, aptic contacts are seen on these neurons sugests their morphological similarity to parasympathetic ganglion 1981). Electrical coupling of pinealocytes through gap junc- cells (Smolen, 1988; see also below). Although it is not tions and their possible involvement in functional yet clear whether the cell bodies of these neurons lie compartmentalization have been described in the rat inside the pineal parenchyma, they may participate in (Taugner et al., 1981) and the guinea pig (Jung and the parasympathetic innervation of the pineal as sugVollrath, 1982; Huang and Taugner, 1984). In these gested by Kenny (1965). Their involvement in the cenrodents pinealocytes form isolated cell clusters, while tral innervation is unknown. Various types of synaptic morphologies present on in the monkey lobular compartmentalization is not distinct as in the rat. Even without apparent lobular pineal neurons now raise the question as to the origin structure, however, electrical coupling could facilitate of the inputs. The ribbon synapse found on the nerve cell body is a propagation of neural input to adjacent pinealocytes following synaptic transmission from nerve terminals unique structure showing a direct synaptic coupling +
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T.ICHIMURA
between the pinealocyte and the neuron. This is the form of a synaptic link between the photoreceptive pinealocyte and the second-order neuron in lower vertebrates. In this respect, functional significance of the 9 x 3 + 0 type microtubular sheaves and the cilia found in the monkey pineal needs reevaluation. The presence of these synaptic contacts and the cilia might suggest that pinealocytes are actually sensory cells and that intrapineal neurons in the monkey, at least in part, are the second-order neurons. In higher mammals, however, there is no electrophysiological evidence which shows that pinealocytes respond directly to photic stimulation. Morphological evidence showing the presence of pinealofugal elements in the habenular and posterior commissures is not convincing. Although a possibility that the monkey pineal has sensory pinealocytes may not be eliminated, it seems more acceptable to suspect that the pinealocytic-neuronal ribbon synapse has acquired a different function in the monkey pineal. Regulation of the indoles’ metabolism through a pinealocytic-neuronal feedback loop might be a possible function acquired during phylogenetic evolution of the mammalian pineal. The varieties of synaptic terminals on pineal neurons strongly suggest that nerve fibers as well as nerve cell bodies, a t least in part, arise elsewhere than the autonomic nervous system. For peripheral parasympathetic neurons, all of the pre- and postganglionic axons are of the same biochemical and morphological type (i-e., cholinergic) with small clear vesicles and a few large dense corded vesicles (Smolen, 1988). Each postganghonic neuron receives synaptic connection from only one preganglionic axon (Lichtman, 1977). These neurons contain enkephalins, substance P (Erichsen et al., 19821, and somatostatin (Kondo, 1977). Immunocytochemical examination of coexistence of these peptides with acetylcholine in pineal neurons might provide evidence for their parasympathetic nature, if they are, in fact, parasympathetic in nature. Cumulating evidence suggests that central nerve fibers enter the mammalian pineal via the habenular and/or posterior commissures (Kappers, 1960; Nielsen and Mdler, 1975; Korf and Mprller, 1984a; Reuss and Mdler, 1986; Mprller and Korf, 1987; Ronnekleiv, 1988). Fibers containing arginine vasotosin, a- MSH, LHRH, or somatostatin have been identified in the rat, and fibers containing substance P, vasopressin, oxytocin, or neurophysins have been found in the monkey pineal. These peptidergic central fibers resemble neurosecretory fibers in the neurophypophysis. As suggested in rodents (Korf and Mprller, 1984a), these central pinealopetal fibers in the monkey may also be neurosecretory. However, the possibility that they are involved in direct central regulation and/or modulation of the pineal function by the central nervous system cannot be eliminated. Intrapineal neurons and/or pinealocytes themselves might be target cells of these central fibers. Anatomical evidence of their origin in the brain and their connections with target cells in the pineal, combined with immunocytochemical identification of transmitters, would greatly improve our understanding of innervation of the mammalian pineal. In conclusion it should be emphasized that extensive
study of the monkey pineal by means of anatomical, ultrastructural, immunocytochemical, biochemical, as well as electrophysiological techniques would provide us the needed information on the brain-pineal intercommunication in the mammal.
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