THE ANATOMICAL RECORD 226:481-488 (1990)
UItrastructural Localization of Acety lcholinesterase in the Guinea Pig Pineal Gland ZHONGRONG LUO, ROBERT L. SCHULTZ, AND ERNEST F. WHITTER Department of Anatomy, Loma Linda University, School of Medicine, Loma Linda, California 92350
ABSTRACT The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.
The presence of acetylcholinesterase (AChE) is one indicator of the neural nature of a tissue (Cooper e t al., 1982). This cannot be the sole indicator, however; additional evidence is necessary, for exceptions to this generality are common. The study of Manocha (1970) demonstrated the complexity of the distribution of AChE (also called true or specific cholinesterase) and nonspecific cholinesterase (also called pseudo- or butyrlcholinesterase) in neural tissue (squirrel monkey brain). When considering the pineal organ, AChE has been shown in a number of nonmammalian animals, where pineal neurons are present and stain for AChE. Examples of these are anurans (Wake et al., 19741, goldfish (Wake, 1973), rainbow trout (Korf, 1974), eel (Matsuura and Herwig, 19811, and birds (Ueck and Kobayashi, 1972). In mammals, AChE is also present in the pineal gland in some species. The enzyme in the bovine pineal was detectable biochemically (23 kM/g/hr; LaBella and Shin, 1968). Rodriguez De Lores Arnaiz and Pellegrino De Iraldi (1972) estimated that the enzyme was equally distributed between pinealocytes and nerve fibers, since superior cervical ganglionectomy was followed by a 50% decrease of enzyme activity. Later the enzyme activity reverted to normal (Pellegrino De Iraldi and Rodriguez De Lores Arnaiz, 1976). Histochemically the enzyme was regularly demonstrated in intrapineal sympathetic nerve fibers (Eranko et al., 1970; Trueman and Herbert, 1970; Machado and Lemos, 1971). A mild to moderate reaction showed in the pinealocytes of the squirrel monkey (Manocha, 1970). However, the pinealocytes of rats (Arstila, 1967) and ferrets (Trueman and Herbert, 1970) are negative for AChE. Also, Vollrath (1981), citing unpublished work 0 1990 WILEY-LISS. INC
by Kohl, reported that guinea pig pinealocytes lacked AChE activity, even though the nerve fibers were positive. This was from a light microscopic study. We have investigated the AChE activity of the guinea pig pineal gland by electron microscopy, which allows a more precise localization of the enzyme to be made. We chose the guinea pig because of past extensive experience with this species. Also, the guinea pig pineal gland is relatively large and provides more tissue than some other animals such as the rat. MATERIAL AND METHODS Animals
Hartley strain guinea pigs of both sexes, weighing 500-700 g, were kept under normal laboratory conditions in a light-proof room. Lighting was controlled by a time clock set to give a 1ight:dark regimen (LD) of 12:12, with lights on from 06:OO to 18:OO and lights off from 18:OO to 06:00, at a room temperature of 22°C ? 2°C. The animals received Purina guinea pig chow and tap water ad libitum. Fixation
The four animals used in this experiment were anesthetized with urethane (1.5 g/kg) given intraperitoneally. The descending aorta was clamped just before the perfusion. In the perfusion procedure, gravity flow was used with a pressure head of 2.26 m. A tyrode solution, precooled to 4"C, was allowed to flow for 30 sec, which amounted to about 100 ml. This was immediately followed by a fixative solution consisting of 1% purified glutaraldehyde and 2% formaldehyde (freshly prepared from paraformaldehyde) buffered with 0.05M Received May 15, 1989; accepted August 21, 1989
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Fig. 1.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
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Fig. 2.A heavy AChE reaction was seen in the perinuclear cistern (arrowheads) and the cisternae of both smooth and rough ER (triangles). A mild to moderate electron-dense reaction is also present in the saccules of Golgi apparatus (arrows). x 28,000.
Fig. 3. A mild AChE reaction product is seen in the perinuclear cistern (arrowheads) and in the cisternae of the ER (triangles), but not in the “synaptic” ribbon vesicles (arrows). x 28,000.
sodium cacodylate a t pH 7.3, also precooled to 4°C. No attempt was made to control the colloid osmotic pressure by using a n additive such as dextran. This aldehyde solution was a t first allowed to flow freely (about 200 ml/min), and after 30 sec the flow rate was reduced to about 30 ml/min. The brains were removed and further fixed by immersion in 2% formaldehyde (from paraformaldehyde) buffered by 0.1 M sodium cacodylate a t pH 7.3 and cooled to 4°C. One hour was allowed for fixation, which included the perfusion, af-
ter which the pineal was dissected from the brain a t room temperature.
Fig. 1. This low-magnification electron micrograph shows a n AChE reaction-positive (ACHE-P) and neighboring AChE-negative pinealocytes. It also shows that the AChE-positive pinealocytes are rich in ER and near the blood vessels (V). A “synaptic” ribbon is present. (arrow). x 14,000.
Cytochemistty
For cytochemistry of AChE in the pineal glands, the copper-thiocholine method (Lewis and Shute, 1966, 1969) was modified and used in this study. In this method, acetylthiocholine iodide is the substrate. After dissection from the brain, the pineal glands were washed in a standard cacodylate washing solution (Lewis and Knight, 1986) overnight a t 4°C. This solution consisted of 24 ml of 0.2 M sodium cacodylate, 10 ml 0.2 M cacodylic acid, 1 ml 0.2 M calcium acetate, and isotonic sodium sulphate added to make 100 ml. To provide support, the glands were embedded in a 20% agar solution. Sections cut at 100 pm on a vibratome
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Fig. 4. AChE activity is seen in the cisternae of the ER of the pinealocyte processes (P) and also in the extracellular space (arrowheads)between the processes. A thin rim of reaction product is seen around the process terminal in the center of the field. The large irregular deposits (arrows) appear to be sites where AChE accumulates since such sites are not seen in any of the controls. x 9,000. Fig. 5. Note that reaction product is not found in the vesicles of the pinealocyte terminal (P)but in the intercellular space (arrowheads). x 34,000
were transferred to the substrate-free incubation medium for 30 min a t 4°C. These were then incubated at 4°C for 4 hr, with one change in the complete incubation medium. This medium contained (in ml) 0.4 M glycine, 2.0; 0.08 M copper sulfate, 2.0; isotonic sodium sulfate, 8.0; 0.5 M succinic acid, 1.0 (pH adjusted with 1 N NaOH); 10 mg/ml acetylthiocholine iodide (slowly added while stirring), 2.0; 10-5 M iso-OMPA or 10-5 M ethopropazine, 1.0. Distilled water was added to make up to 20 ml. For best results, the pH for the preincubation and incubation media was adjusted to 5.4. For controls the following media were used: 1) a medium containing 20 mg of butyrylthiocholine iodide (Sigma Chemical Co., St. Louis, MO) as a substrate and omission of inhibitors; 2) a medium without a substrate; 3) a medium containing 10-5 M eserine (physostigmine, Sigma), a n inhibitor of both AChE and pseudocholinesterase. Postincubation Processing
After incubation, sections were washed according to the procedure of Lewis and Shute (1966, 1969). The washed sections were postfixed in 1% osmium tetroxide buffered with 0.15 M sodium cacodylate for 2 h r at 4"C, dehydrated in a graded acetone series, and embedded in Poly/Bed (Polysciences, Inc., Warrington, PA). Semithin sections were cut at 2 pm on a Porter Blum MT-2 ultramicrotome and stained with polychrome staining solution (Alsop, 1974) and/or 1% toluidine blue solution. Ultrathin sections of 75 nm were cut on a MT 6000 microtome with a diamond knife, stained with lead citrate (Sato, 1968), and examined with a Zeiss EM 1OC electron microscope.
RESULTS
In semithin sections, a small number of pinealocytes near blood vessels and distributed in groups were AChE-positive, whereas neighboring pinealocytes were AChE-negative (Fig. 1).This was clearly shown at the ultrastructural level. The AChE-positive pinealocytes contained abundant ER. The AChE activity was mainly localized in the perinuclear cistern and the cisternae of the ER (Figs. 1-3). A light reaction product was also seen in the saccules of the Golgi apparatus (Fig. 2), but none in the "synaptic" ribbon vesicles (Fig. 3). In addition to the intrapinealocyte reaction sites, the intercellular space between pinealocyte processes was also AChE-positive (Figs. 4 and 5). In one unique case, the vesicles surrounding virus-like particles were labeled with AChE reaction. The axolemma of myelinated (Fig. 6) and nonmyelinated (Fig. 7) nerve fibers showed AChE activity. The basement membranes of Schwann cells likewise were AChE-positive. In sections incubated in the medium containing butyrylthiocholine iodide, but, without inhibitors to show pseudocholinesterase activity, pinealocytes showed no reaction. However, the Schwann cells exhibited this pseudocholinesterase activity in their perinuclear cis-
Fig. 6. The axolemma (triangles) and/or the Schwann cell membrane (arrowheads) of a myelinated nerve fiber shows AChE activity. x 75,000. Fig. 7. The axolemma (triangles) of unmyelinated nerve fibers shows AChE activity. The enzyme reaction product also can be seen in the basement membrane (arrows). x 14,000.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
Figs. 6 and 7.
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Figs. 8 and 9.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
ternae, ER cisternae, Golgi apparatus saccules, and the plasmalemma (Figs. 8 and 9). No AChE or pseudocholinesterase activities were shown in the sections incubated in either medium lacking substrate or that inhibited by eserine.
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among neighboring pinealocytes. A majority of bovine pinealocytes stained for both hydroxyindole O-methyltransferase (HIOMT) and neuron-specific enolase, but a few did not stain for either (Kuwano et al., 1983). However, relevant correlation between the heterogeneity shown by the AChE reaction in this study and the DISCUSSION heterogeneities revealed by other methods remains to The fact that some pinealocytes showed a n AChE- be shown. We conclude that the diversity of pinealocyte positive reaction in the study reported here lends sup- AChE activity demonstrated in this study augments port to the concept that pinealocytes are neuronal in the evidence accumulating concerning the morpholognature. Taken alone, AChE activity is not sufficient ical, cytochemical, and electrophysiological variability evidence for this concept, since AChE can be found in a of pinealocytes. Most likely this variability reflects the variety of cells and tissues. Thus the finding of S-an- functional states of these cells at the time of fixation. tigen in pinealocytes of the Djungarian hamster is par- Lack of AChE staining by a population of pinealocytes ticularly significant (Korf et al., 1986). The S-antigen cannot be interpreted a s showing a different or nonis a highly antigenic protein found only in retinal pho- neuronal origin for such cells. toreceptors, pineal photoreceptors of lower vertebrates, The localization of AChE activity in the perinuclear and pinealocytes. Another indication of the neuronal cisternae and ER cisternae in guinea pig pinealocytes nature of pinealocytes can be found in the study re- is a n indication that the enzyme is endogenous and is ported by McClure et al. (1986), where some pinealo- produced in the pinealocytes, since these organelles are cytes had neuron-specific enolase activity. responsible for protein synthesis (Palade, 1975).In that In some poikilothermic vertebrates, pineal photore- light reaction product for AChE was noticed in the sacceptor cells and neurons are found in the pineal organ. cules of the Golgi apparatus in this study, the Golgi It is interesting that AChE activity has been found in apparatus may be involved in the processing of the both cells (Vigh-Teichmann et al., 1982), which sug- enzyme. Besides intracytoplasmic localization, the gests a phylogenetic relationship for the AChE-positive AChE reaction product was also found in the intercelmammalian pinealocytes. lular space between pinealocyte processes. However, it The negative unpublished report of Kohl for AChE in is not clear from this study whether AChE synthesized guinea pig pinealocytes as quoted by Vollrath (1981) in pinealocytes is released from the intracytoplasmic does not agree with our results. There are several pos- membrane system into the extracellular space. Biosible reasons for this discrepancy. Kohl used light mi- chemical studies (Massoulie, 1980) have shown that croscopy, which is less precise than electron micros- low-molecular-weight forms of AChE are readily solucopy. The techniques of fixation are another variable, bilized by water in the cytoplasm and are poorly for electron microscopy is more demanding, and vascu- stained cytochemically. The higher-molecular-weight lar perfusion is necessary for best results. Also, the forms are linked to the intracellular membranes and protocols used for demonstrating AChE vary in sensi- are demonstrable by existing cytochemical procedures. tivity. The collagen-tailed forms are believed to be extracelIn the present study neighboring pinealocytes were lularly localized, and they may be considered as a speshown to differ in their reaction for AChE activity. cial kind of secreted molecule. Since AChE activity was This agrees with the heterogeneity of pinealocytes re- not observed in any secretion vesicles, i t would appear ported by other investigators. Evidence for such mor- that the enzyme is secreted constituitively, i.e., exphological heterogeneity was seen in the staining prop- ported continuously without storage (Rivas and Moore, erties of pineal parenchyma1 cells (Tapp and Blumfield, 1989). 1970) and in the varying electron densities of light and The principal role of AChE is believed to be the terdark pinealocytes (Arstila, 1967; Benson and Satter- mination of nerve impulse transmission by hydrolysis field, 1975; Luo et al., 1984). Based on ultrastructural of the neurotransmitter acetylcholine. However, a chovariations (Pevet, 1977; Pevet and Kuyper, 1978) and a linergic parasympathetic innervation of the epiphysis possible link to photoreceptor cells (Pevet et al., 19771, cerebri, in contrast to the sympathetic supply, has been two mammalian pinealocyte populations (I and 11)were said not to be a regular feature in mammals (Kappers, identified. Electrophysiological data from guinea pig 1960, 1965, 1969). Species reported to have parasympineal glands showed a synchronization of several cell pathetic innervation are the macaque monkey (Kenny, types to different phases of the circadian rhythm 1961) and the rabbit (Romijn, 1973a,b, 1975a,b). In the (Semm and Vollrath, 1980). The localization of retinal guinea pig, definite parasympathetic nerve fibers have S-antigen (Korf et al., 1986) and neuron-specific eno- not been shown. One problem is that the sympathetic lase antigen (McClure e t al., 1986) in rats varied fibers of the mammalian pineal have parasympathetic and cholinergic characteristics. For example, AChE can be regularly demonstrated in intrapineal sympathetic nerve fibers (Eranko et al., 1970). Recently it has been reported that AChE has peptiFig. 8. Psuedocholinesterase reaction precipitate was seen in the perinuclear cistern (arrowheads) and the cisternae of the ER (trian- dase activity and can hydrolyze both substance P (Chubb et al., 1980) and leu- and metenkephalin gles) and on the plasmalemma (long arrow) of the Schwann cell. A light reaction product can be seen in the saccules of the Golgi appa- (Chubb et al., 1982). Substance P was revealed in the ratus (short arrows). x 28,000. pineal gland with radioimmunoassay studies (Powel et al., 1977), and substance P fibers were also found in the Fig. 9. Psuedocholinesterase activity is confined to the enveloping pineal organ, pineal stalk, and deep pineal glands membrane (triangles)of Schwann cell. x 75,000.
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(Ronnekleiv and Kelly, 1984).However, it is not known if the pineal AChE hydrolyzes the pineal substance P. ACKNOWLEDGMENTS
The authors thank Dr. Paul J. McMillan for his suggestions and discussion during this study. LITERATURE CITED Alsop, D.W. (1974) Rapid single-solution polychrome staining of semithin epoxy sections using polyethylene glycol 200 (PEG 200) a s a stain solvent. Stain Technol., 49t265-271. Arstila, A.U. (1967) Electron microscopic studies on the structure and histochemistry of the pineal gland of the rat. Neuroendocrinology, 2 [suppll:l-101. Benson. B., and V. Satterfield (1975) Ultrastructural characteristics of mouse pinealocytes following optic enucleation or continuous illumination. Anat. Rec., 181t312. Chubb, I.W., A.J. Hodgson, and G.H. White (1980) Acetylcholinesterase hydrolyses substance P. Neuroscience, 5t2065. Chubb, I.W., E. Ranieri, A.J. Hodgson, and G.H. White (1982) The hydrolysis of leu-and met-enkephalin by acetylcholine esterase. Neurosci. Lett, 8 lsuppll:539. Cooper, J.R., F.E. Bloom, and R.H. Roth (1982) The Biochemical Basis of Neuropharmacology. Oxford University Press, New York, pp. 83-85. Eranko, O., L. Rechardt, L. Eranko, and A. Cunningham (1970) Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibers in the pineal body of the rat. J . Histochem., 2t479-489. Kappers, J.A. (1960)Innervation of the epiphysis cerebri in the albino rat. Anat. Rec., 139t220-221. Kappers, J.A. (1965) Survey of the innervation of the epiphysis cerebri and the accessory pineal organs of vertebrates. Prog. Brain Res., 10t87-153. Kappers, _ _ J.A. (1969)The mammalian pineal organ. J. Neurovisc. Rel. 9 [suppll:140-184. Kenny, G.C.T. (1961) The “nervus conarii” of the monkey. (An experimental study). J . Neuropathol Exp. Neurol., 20t563-570. Korf, H.W. (1974) Acetylcholinesterase-positiveneurons in the pineal and parapineal organs of the rainbow trout, Salmo gairdneri (with special reference to the pineal tract). Cell Tissue Res., 155: 475-489. Korf, H.W., A. Oksche, P. Ekstrom, I. Gery, and J.S. Zigier (1986) Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum. Science, 231t735-737. Kuwano, R., T. Iwanaga, T. Nakajima, T. Masuda, and Y. Takahashi (1983) Immunocytochemical demonstration of hydroxyindole 0methyltransferase (HIOMT), neuron-specific enolase (NSE) and 5-100 protein in the bovine pineal gland. Brain Res., 274.171175. LaBella, F.S., and S. Shin (1968) Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body. J . Neurochem., 15t335-342. Lewis, P.R., and D.P. Knight (1986) Staining Methods for Sectioned Material. North-Holland Publishing Company, New York, pp. 289-294. Lewis, P.R., and C.C.D. Shute (1966) The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J . Cell Sci., It381. Lewis, P.R., and C.C.D. Shute (1969) An electron microscopic study of cholinesterase distribution in the rat adrenal medulla. J . Microscopy, 89t181. Luo, Z.R., R.L. Schultz, E.F. Whitter, and L. Vollrath (1984) Ultrastructural characterization of glial cells in the rat pineal gland with special reference to the pineal stalk. Anat. Rec., 210:663674. Machado, A.B.M., and V.P.J. Lemos (1971)Histochemical evidence for a cholinergic sympathetic innervation of the rat pineal body. J . Neurovisc. Rel. 32t104-111. Manocha, S.L. (1970) Histochemical distribution of acetylcholinesterase and simple esterases in the brain of squirrel monkey (Saimiri sciureus 1. Histochemie, 21.236-248.
Massoulie, J. (1980) The polymorphism of cholinesterases and its physiological significance. Trends Biochem. Sci. Pers. Ed., 5t160. Matsuura, T., and H.J. Herwig (1981) Histochemical and ultrastructural study of the nervous elements in the pineal organ of the eel, Anguilla unguilla. Cell Tissue Res., 261t544-555. McClure, C.D., P.J. McMillan, and A. Miranda C. (1986) Demonstration of differential immunohistochemical localization of the neuron-specific enolase antigen in rat pinealocytes. Am. J . Anat., I76t461-467. Palade, G.E. (1975) Intracellular aspects of the process of protein synthesis. Science, 189t347. Pellegrino De Iraldi, A., and G . Rodriguez De Lores Arnaiz (1976) Neural regulation of acetylcholinesterase in the superior cervical ganglia and the pineal gland of the rat. Brain Res., 113t435-440. Pevet, P. (1977) On the presence of different populations of pinealocytes in the mammalian pineal gland. J . Neural Transmiss., 40: 289-304. Pevet, P., J.A. Kappers, and A.M. Voute (1977) Morphological evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctalus noctula, Schreiber). Cell Tissue Res., 182t99-109. Pevet, P., and M. Kuyper (1978) The ultrastructure of pinealocytes in the golden mole iAmblysomus hottentotus ) with special reference to the granular vesicles. Cell Tissue Res., 191t39-56. Powel, O., S. Krabanek, and D. Cannon (1977) Substance P: Radioimmunoassay studies. In: Substance P.U. Von Euler and B. Pernow, eds. Raven Press, New York., pp. 35-40. Rivas, R.J., and H.H. Moore (1989) Spatial segregation of the regulated and constitutive secretory pathways. J. Cell Biol., 109t5160. Rodriguez De Lores Arnaiz, G., and A. Pellegrino De Iraldi (1972) Cholinesterase in cholinergic and adrenergic nerves: A study of the superior cervical ganglia and the pineal gland of the rat. Brain Res., 42t230-233. Romijn, H.J. (1973a) Structure and innervation of the pineal gland of the rabbit, Oryctolugus cuniculus (L.) I. A light microscopic investigation. Z. Zellforsch, 139t473-485. Romijn, H.J. (197313)Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.) 11. An electron microscopic investigation of the pinealocytes. Z. Zellforsch., 141t545-560. Romijn, H.J. (1975a) The ultrastructure of the rabbit pineal gland after sympathectomy, parasympathectomy, continuous illumination, and continuous darkness. J . Neural Transmiss., 36r183194. Romijn, H.J. (1975b) Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.) 111. An electron microscopic investigation of the innervation. Cell Tissue Res. 157t25-51. Ronnekleiv, O.K., and M.J. Kelly (1984) Distribution of substance P neurons in the epithalamus of the rat: An immunohistochemical investigation. J. Pineal Res., It355-370. Sato, T. (1968) A modified method for lead staining of thin sections. J . Electron Microsc. (Tokyo),17t158-159. Semm, P., and L. Vollrath (1980) Electrophysiological evidence for circadian rhythmicity in a mammalian pineal organ. J . Neural Transmiss., 47t181-191. Tapp, - . E., and M. Blumfield (1970) The parenchyma1 cells of the rat pineal gland. Acta. Morphol. Neerl.8cand.,~8:119-131. Trueman, T., and J. Herbert (1970) The distribution of monoamines and acetylcholinesterase in the pineal gland and habenula of the ferret. J . Anat., 106:406. Ueck, M., and H. Kobayashi (1972) Vergleichende untersuchungen uber acetylcholinesterase-haltigeneurone im pinealorgan der vogel. Z. Zellforsch., 129t140-160. Vigh-Teichmann, I., H.W. Korf, A. Oksche, and B. Vigh (1982) Opsinimmunoreactive outer segments and acetylcholinesterasepositive neurons in the pineal complex of Phox~nusphoxinus (Teleostei, Cyprinidae). Cell Tissue Res., 227t351-369. Vollrath, L. (1981) The Pineal Organ. Springer. New York, p. 106. Wake, K. (1973)Acetylcholinesterase-containingnerve cells and their distribution in the pineal organ of the goldfish, Carassius auratus. Z. Zellforsch., 145t287-298. Wake, K., M. Ueck, and A. Oksche (1974) Acetylcholinesterase-containing nerve cells in the pineal complex and subcommissural area of the frogs, Rana ridibundu and Rana esculenta. Cell Tissue Res., 154t423-424.
UItrastructural Localization of Acety lcholinesterase in the Guinea Pig Pineal Gland ZHONGRONG LUO, ROBERT L. SCHULTZ, AND ERNEST F. WHITTER Department of Anatomy, Loma Linda University, School of Medicine, Loma Linda, California 92350
ABSTRACT The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.
The presence of acetylcholinesterase (AChE) is one indicator of the neural nature of a tissue (Cooper e t al., 1982). This cannot be the sole indicator, however; additional evidence is necessary, for exceptions to this generality are common. The study of Manocha (1970) demonstrated the complexity of the distribution of AChE (also called true or specific cholinesterase) and nonspecific cholinesterase (also called pseudo- or butyrlcholinesterase) in neural tissue (squirrel monkey brain). When considering the pineal organ, AChE has been shown in a number of nonmammalian animals, where pineal neurons are present and stain for AChE. Examples of these are anurans (Wake et al., 19741, goldfish (Wake, 1973), rainbow trout (Korf, 1974), eel (Matsuura and Herwig, 19811, and birds (Ueck and Kobayashi, 1972). In mammals, AChE is also present in the pineal gland in some species. The enzyme in the bovine pineal was detectable biochemically (23 kM/g/hr; LaBella and Shin, 1968). Rodriguez De Lores Arnaiz and Pellegrino De Iraldi (1972) estimated that the enzyme was equally distributed between pinealocytes and nerve fibers, since superior cervical ganglionectomy was followed by a 50% decrease of enzyme activity. Later the enzyme activity reverted to normal (Pellegrino De Iraldi and Rodriguez De Lores Arnaiz, 1976). Histochemically the enzyme was regularly demonstrated in intrapineal sympathetic nerve fibers (Eranko et al., 1970; Trueman and Herbert, 1970; Machado and Lemos, 1971). A mild to moderate reaction showed in the pinealocytes of the squirrel monkey (Manocha, 1970). However, the pinealocytes of rats (Arstila, 1967) and ferrets (Trueman and Herbert, 1970) are negative for AChE. Also, Vollrath (1981), citing unpublished work 0 1990 WILEY-LISS. INC
by Kohl, reported that guinea pig pinealocytes lacked AChE activity, even though the nerve fibers were positive. This was from a light microscopic study. We have investigated the AChE activity of the guinea pig pineal gland by electron microscopy, which allows a more precise localization of the enzyme to be made. We chose the guinea pig because of past extensive experience with this species. Also, the guinea pig pineal gland is relatively large and provides more tissue than some other animals such as the rat. MATERIAL AND METHODS Animals
Hartley strain guinea pigs of both sexes, weighing 500-700 g, were kept under normal laboratory conditions in a light-proof room. Lighting was controlled by a time clock set to give a 1ight:dark regimen (LD) of 12:12, with lights on from 06:OO to 18:OO and lights off from 18:OO to 06:00, at a room temperature of 22°C ? 2°C. The animals received Purina guinea pig chow and tap water ad libitum. Fixation
The four animals used in this experiment were anesthetized with urethane (1.5 g/kg) given intraperitoneally. The descending aorta was clamped just before the perfusion. In the perfusion procedure, gravity flow was used with a pressure head of 2.26 m. A tyrode solution, precooled to 4"C, was allowed to flow for 30 sec, which amounted to about 100 ml. This was immediately followed by a fixative solution consisting of 1% purified glutaraldehyde and 2% formaldehyde (freshly prepared from paraformaldehyde) buffered with 0.05M Received May 15, 1989; accepted August 21, 1989
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Fig. 1.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
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Fig. 2.A heavy AChE reaction was seen in the perinuclear cistern (arrowheads) and the cisternae of both smooth and rough ER (triangles). A mild to moderate electron-dense reaction is also present in the saccules of Golgi apparatus (arrows). x 28,000.
Fig. 3. A mild AChE reaction product is seen in the perinuclear cistern (arrowheads) and in the cisternae of the ER (triangles), but not in the “synaptic” ribbon vesicles (arrows). x 28,000.
sodium cacodylate a t pH 7.3, also precooled to 4°C. No attempt was made to control the colloid osmotic pressure by using a n additive such as dextran. This aldehyde solution was a t first allowed to flow freely (about 200 ml/min), and after 30 sec the flow rate was reduced to about 30 ml/min. The brains were removed and further fixed by immersion in 2% formaldehyde (from paraformaldehyde) buffered by 0.1 M sodium cacodylate a t pH 7.3 and cooled to 4°C. One hour was allowed for fixation, which included the perfusion, af-
ter which the pineal was dissected from the brain a t room temperature.
Fig. 1. This low-magnification electron micrograph shows a n AChE reaction-positive (ACHE-P) and neighboring AChE-negative pinealocytes. It also shows that the AChE-positive pinealocytes are rich in ER and near the blood vessels (V). A “synaptic” ribbon is present. (arrow). x 14,000.
Cytochemistty
For cytochemistry of AChE in the pineal glands, the copper-thiocholine method (Lewis and Shute, 1966, 1969) was modified and used in this study. In this method, acetylthiocholine iodide is the substrate. After dissection from the brain, the pineal glands were washed in a standard cacodylate washing solution (Lewis and Knight, 1986) overnight a t 4°C. This solution consisted of 24 ml of 0.2 M sodium cacodylate, 10 ml 0.2 M cacodylic acid, 1 ml 0.2 M calcium acetate, and isotonic sodium sulphate added to make 100 ml. To provide support, the glands were embedded in a 20% agar solution. Sections cut at 100 pm on a vibratome
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Fig. 4. AChE activity is seen in the cisternae of the ER of the pinealocyte processes (P) and also in the extracellular space (arrowheads)between the processes. A thin rim of reaction product is seen around the process terminal in the center of the field. The large irregular deposits (arrows) appear to be sites where AChE accumulates since such sites are not seen in any of the controls. x 9,000. Fig. 5. Note that reaction product is not found in the vesicles of the pinealocyte terminal (P)but in the intercellular space (arrowheads). x 34,000
were transferred to the substrate-free incubation medium for 30 min a t 4°C. These were then incubated at 4°C for 4 hr, with one change in the complete incubation medium. This medium contained (in ml) 0.4 M glycine, 2.0; 0.08 M copper sulfate, 2.0; isotonic sodium sulfate, 8.0; 0.5 M succinic acid, 1.0 (pH adjusted with 1 N NaOH); 10 mg/ml acetylthiocholine iodide (slowly added while stirring), 2.0; 10-5 M iso-OMPA or 10-5 M ethopropazine, 1.0. Distilled water was added to make up to 20 ml. For best results, the pH for the preincubation and incubation media was adjusted to 5.4. For controls the following media were used: 1) a medium containing 20 mg of butyrylthiocholine iodide (Sigma Chemical Co., St. Louis, MO) as a substrate and omission of inhibitors; 2) a medium without a substrate; 3) a medium containing 10-5 M eserine (physostigmine, Sigma), a n inhibitor of both AChE and pseudocholinesterase. Postincubation Processing
After incubation, sections were washed according to the procedure of Lewis and Shute (1966, 1969). The washed sections were postfixed in 1% osmium tetroxide buffered with 0.15 M sodium cacodylate for 2 h r at 4"C, dehydrated in a graded acetone series, and embedded in Poly/Bed (Polysciences, Inc., Warrington, PA). Semithin sections were cut at 2 pm on a Porter Blum MT-2 ultramicrotome and stained with polychrome staining solution (Alsop, 1974) and/or 1% toluidine blue solution. Ultrathin sections of 75 nm were cut on a MT 6000 microtome with a diamond knife, stained with lead citrate (Sato, 1968), and examined with a Zeiss EM 1OC electron microscope.
RESULTS
In semithin sections, a small number of pinealocytes near blood vessels and distributed in groups were AChE-positive, whereas neighboring pinealocytes were AChE-negative (Fig. 1).This was clearly shown at the ultrastructural level. The AChE-positive pinealocytes contained abundant ER. The AChE activity was mainly localized in the perinuclear cistern and the cisternae of the ER (Figs. 1-3). A light reaction product was also seen in the saccules of the Golgi apparatus (Fig. 2), but none in the "synaptic" ribbon vesicles (Fig. 3). In addition to the intrapinealocyte reaction sites, the intercellular space between pinealocyte processes was also AChE-positive (Figs. 4 and 5). In one unique case, the vesicles surrounding virus-like particles were labeled with AChE reaction. The axolemma of myelinated (Fig. 6) and nonmyelinated (Fig. 7) nerve fibers showed AChE activity. The basement membranes of Schwann cells likewise were AChE-positive. In sections incubated in the medium containing butyrylthiocholine iodide, but, without inhibitors to show pseudocholinesterase activity, pinealocytes showed no reaction. However, the Schwann cells exhibited this pseudocholinesterase activity in their perinuclear cis-
Fig. 6. The axolemma (triangles) and/or the Schwann cell membrane (arrowheads) of a myelinated nerve fiber shows AChE activity. x 75,000. Fig. 7. The axolemma (triangles) of unmyelinated nerve fibers shows AChE activity. The enzyme reaction product also can be seen in the basement membrane (arrows). x 14,000.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
Figs. 6 and 7.
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Figs. 8 and 9.
LOCALIZATION OF ACETYLCHOLINESTERASE IN PINEAL
ternae, ER cisternae, Golgi apparatus saccules, and the plasmalemma (Figs. 8 and 9). No AChE or pseudocholinesterase activities were shown in the sections incubated in either medium lacking substrate or that inhibited by eserine.
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among neighboring pinealocytes. A majority of bovine pinealocytes stained for both hydroxyindole O-methyltransferase (HIOMT) and neuron-specific enolase, but a few did not stain for either (Kuwano et al., 1983). However, relevant correlation between the heterogeneity shown by the AChE reaction in this study and the DISCUSSION heterogeneities revealed by other methods remains to The fact that some pinealocytes showed a n AChE- be shown. We conclude that the diversity of pinealocyte positive reaction in the study reported here lends sup- AChE activity demonstrated in this study augments port to the concept that pinealocytes are neuronal in the evidence accumulating concerning the morpholognature. Taken alone, AChE activity is not sufficient ical, cytochemical, and electrophysiological variability evidence for this concept, since AChE can be found in a of pinealocytes. Most likely this variability reflects the variety of cells and tissues. Thus the finding of S-an- functional states of these cells at the time of fixation. tigen in pinealocytes of the Djungarian hamster is par- Lack of AChE staining by a population of pinealocytes ticularly significant (Korf et al., 1986). The S-antigen cannot be interpreted a s showing a different or nonis a highly antigenic protein found only in retinal pho- neuronal origin for such cells. toreceptors, pineal photoreceptors of lower vertebrates, The localization of AChE activity in the perinuclear and pinealocytes. Another indication of the neuronal cisternae and ER cisternae in guinea pig pinealocytes nature of pinealocytes can be found in the study re- is a n indication that the enzyme is endogenous and is ported by McClure et al. (1986), where some pinealo- produced in the pinealocytes, since these organelles are cytes had neuron-specific enolase activity. responsible for protein synthesis (Palade, 1975).In that In some poikilothermic vertebrates, pineal photore- light reaction product for AChE was noticed in the sacceptor cells and neurons are found in the pineal organ. cules of the Golgi apparatus in this study, the Golgi It is interesting that AChE activity has been found in apparatus may be involved in the processing of the both cells (Vigh-Teichmann et al., 1982), which sug- enzyme. Besides intracytoplasmic localization, the gests a phylogenetic relationship for the AChE-positive AChE reaction product was also found in the intercelmammalian pinealocytes. lular space between pinealocyte processes. However, it The negative unpublished report of Kohl for AChE in is not clear from this study whether AChE synthesized guinea pig pinealocytes as quoted by Vollrath (1981) in pinealocytes is released from the intracytoplasmic does not agree with our results. There are several pos- membrane system into the extracellular space. Biosible reasons for this discrepancy. Kohl used light mi- chemical studies (Massoulie, 1980) have shown that croscopy, which is less precise than electron micros- low-molecular-weight forms of AChE are readily solucopy. The techniques of fixation are another variable, bilized by water in the cytoplasm and are poorly for electron microscopy is more demanding, and vascu- stained cytochemically. The higher-molecular-weight lar perfusion is necessary for best results. Also, the forms are linked to the intracellular membranes and protocols used for demonstrating AChE vary in sensi- are demonstrable by existing cytochemical procedures. tivity. The collagen-tailed forms are believed to be extracelIn the present study neighboring pinealocytes were lularly localized, and they may be considered as a speshown to differ in their reaction for AChE activity. cial kind of secreted molecule. Since AChE activity was This agrees with the heterogeneity of pinealocytes re- not observed in any secretion vesicles, i t would appear ported by other investigators. Evidence for such mor- that the enzyme is secreted constituitively, i.e., exphological heterogeneity was seen in the staining prop- ported continuously without storage (Rivas and Moore, erties of pineal parenchyma1 cells (Tapp and Blumfield, 1989). 1970) and in the varying electron densities of light and The principal role of AChE is believed to be the terdark pinealocytes (Arstila, 1967; Benson and Satter- mination of nerve impulse transmission by hydrolysis field, 1975; Luo et al., 1984). Based on ultrastructural of the neurotransmitter acetylcholine. However, a chovariations (Pevet, 1977; Pevet and Kuyper, 1978) and a linergic parasympathetic innervation of the epiphysis possible link to photoreceptor cells (Pevet et al., 19771, cerebri, in contrast to the sympathetic supply, has been two mammalian pinealocyte populations (I and 11)were said not to be a regular feature in mammals (Kappers, identified. Electrophysiological data from guinea pig 1960, 1965, 1969). Species reported to have parasympineal glands showed a synchronization of several cell pathetic innervation are the macaque monkey (Kenny, types to different phases of the circadian rhythm 1961) and the rabbit (Romijn, 1973a,b, 1975a,b). In the (Semm and Vollrath, 1980). The localization of retinal guinea pig, definite parasympathetic nerve fibers have S-antigen (Korf et al., 1986) and neuron-specific eno- not been shown. One problem is that the sympathetic lase antigen (McClure e t al., 1986) in rats varied fibers of the mammalian pineal have parasympathetic and cholinergic characteristics. For example, AChE can be regularly demonstrated in intrapineal sympathetic nerve fibers (Eranko et al., 1970). Recently it has been reported that AChE has peptiFig. 8. Psuedocholinesterase reaction precipitate was seen in the perinuclear cistern (arrowheads) and the cisternae of the ER (trian- dase activity and can hydrolyze both substance P (Chubb et al., 1980) and leu- and metenkephalin gles) and on the plasmalemma (long arrow) of the Schwann cell. A light reaction product can be seen in the saccules of the Golgi appa- (Chubb et al., 1982). Substance P was revealed in the ratus (short arrows). x 28,000. pineal gland with radioimmunoassay studies (Powel et al., 1977), and substance P fibers were also found in the Fig. 9. Psuedocholinesterase activity is confined to the enveloping pineal organ, pineal stalk, and deep pineal glands membrane (triangles)of Schwann cell. x 75,000.
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(Ronnekleiv and Kelly, 1984).However, it is not known if the pineal AChE hydrolyzes the pineal substance P. ACKNOWLEDGMENTS
The authors thank Dr. Paul J. McMillan for his suggestions and discussion during this study. LITERATURE CITED Alsop, D.W. (1974) Rapid single-solution polychrome staining of semithin epoxy sections using polyethylene glycol 200 (PEG 200) a s a stain solvent. Stain Technol., 49t265-271. Arstila, A.U. (1967) Electron microscopic studies on the structure and histochemistry of the pineal gland of the rat. Neuroendocrinology, 2 [suppll:l-101. Benson. B., and V. Satterfield (1975) Ultrastructural characteristics of mouse pinealocytes following optic enucleation or continuous illumination. Anat. Rec., 181t312. Chubb, I.W., A.J. Hodgson, and G.H. White (1980) Acetylcholinesterase hydrolyses substance P. Neuroscience, 5t2065. Chubb, I.W., E. Ranieri, A.J. Hodgson, and G.H. White (1982) The hydrolysis of leu-and met-enkephalin by acetylcholine esterase. Neurosci. Lett, 8 lsuppll:539. Cooper, J.R., F.E. Bloom, and R.H. Roth (1982) The Biochemical Basis of Neuropharmacology. Oxford University Press, New York, pp. 83-85. Eranko, O., L. Rechardt, L. Eranko, and A. Cunningham (1970) Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibers in the pineal body of the rat. J . Histochem., 2t479-489. Kappers, J.A. (1960)Innervation of the epiphysis cerebri in the albino rat. Anat. Rec., 139t220-221. Kappers, J.A. (1965) Survey of the innervation of the epiphysis cerebri and the accessory pineal organs of vertebrates. Prog. Brain Res., 10t87-153. Kappers, _ _ J.A. (1969)The mammalian pineal organ. J. Neurovisc. Rel. 9 [suppll:140-184. Kenny, G.C.T. (1961) The “nervus conarii” of the monkey. (An experimental study). J . Neuropathol Exp. Neurol., 20t563-570. Korf, H.W. (1974) Acetylcholinesterase-positiveneurons in the pineal and parapineal organs of the rainbow trout, Salmo gairdneri (with special reference to the pineal tract). Cell Tissue Res., 155: 475-489. Korf, H.W., A. Oksche, P. Ekstrom, I. Gery, and J.S. Zigier (1986) Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum. Science, 231t735-737. Kuwano, R., T. Iwanaga, T. Nakajima, T. Masuda, and Y. Takahashi (1983) Immunocytochemical demonstration of hydroxyindole 0methyltransferase (HIOMT), neuron-specific enolase (NSE) and 5-100 protein in the bovine pineal gland. Brain Res., 274.171175. LaBella, F.S., and S. Shin (1968) Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body. J . Neurochem., 15t335-342. Lewis, P.R., and D.P. Knight (1986) Staining Methods for Sectioned Material. North-Holland Publishing Company, New York, pp. 289-294. Lewis, P.R., and C.C.D. Shute (1966) The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J . Cell Sci., It381. Lewis, P.R., and C.C.D. Shute (1969) An electron microscopic study of cholinesterase distribution in the rat adrenal medulla. J . Microscopy, 89t181. Luo, Z.R., R.L. Schultz, E.F. Whitter, and L. Vollrath (1984) Ultrastructural characterization of glial cells in the rat pineal gland with special reference to the pineal stalk. Anat. Rec., 210:663674. Machado, A.B.M., and V.P.J. Lemos (1971)Histochemical evidence for a cholinergic sympathetic innervation of the rat pineal body. J . Neurovisc. Rel. 32t104-111. Manocha, S.L. (1970) Histochemical distribution of acetylcholinesterase and simple esterases in the brain of squirrel monkey (Saimiri sciureus 1. Histochemie, 21.236-248.
Massoulie, J. (1980) The polymorphism of cholinesterases and its physiological significance. Trends Biochem. Sci. Pers. Ed., 5t160. Matsuura, T., and H.J. Herwig (1981) Histochemical and ultrastructural study of the nervous elements in the pineal organ of the eel, Anguilla unguilla. Cell Tissue Res., 261t544-555. McClure, C.D., P.J. McMillan, and A. Miranda C. (1986) Demonstration of differential immunohistochemical localization of the neuron-specific enolase antigen in rat pinealocytes. Am. J . Anat., I76t461-467. Palade, G.E. (1975) Intracellular aspects of the process of protein synthesis. Science, 189t347. Pellegrino De Iraldi, A., and G . Rodriguez De Lores Arnaiz (1976) Neural regulation of acetylcholinesterase in the superior cervical ganglia and the pineal gland of the rat. Brain Res., 113t435-440. Pevet, P. (1977) On the presence of different populations of pinealocytes in the mammalian pineal gland. J . Neural Transmiss., 40: 289-304. Pevet, P., J.A. Kappers, and A.M. Voute (1977) Morphological evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctalus noctula, Schreiber). Cell Tissue Res., 182t99-109. Pevet, P., and M. Kuyper (1978) The ultrastructure of pinealocytes in the golden mole iAmblysomus hottentotus ) with special reference to the granular vesicles. Cell Tissue Res., 191t39-56. Powel, O., S. Krabanek, and D. Cannon (1977) Substance P: Radioimmunoassay studies. In: Substance P.U. Von Euler and B. Pernow, eds. Raven Press, New York., pp. 35-40. Rivas, R.J., and H.H. Moore (1989) Spatial segregation of the regulated and constitutive secretory pathways. J. Cell Biol., 109t5160. Rodriguez De Lores Arnaiz, G., and A. Pellegrino De Iraldi (1972) Cholinesterase in cholinergic and adrenergic nerves: A study of the superior cervical ganglia and the pineal gland of the rat. Brain Res., 42t230-233. Romijn, H.J. (1973a) Structure and innervation of the pineal gland of the rabbit, Oryctolugus cuniculus (L.) I. A light microscopic investigation. Z. Zellforsch, 139t473-485. Romijn, H.J. (197313)Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.) 11. An electron microscopic investigation of the pinealocytes. Z. Zellforsch., 141t545-560. Romijn, H.J. (1975a) The ultrastructure of the rabbit pineal gland after sympathectomy, parasympathectomy, continuous illumination, and continuous darkness. J . Neural Transmiss., 36r183194. Romijn, H.J. (1975b) Structure and innervation of the pineal gland of the rabbit, Oryctolagus cuniculus (L.) 111. An electron microscopic investigation of the innervation. Cell Tissue Res. 157t25-51. Ronnekleiv, O.K., and M.J. Kelly (1984) Distribution of substance P neurons in the epithalamus of the rat: An immunohistochemical investigation. J. Pineal Res., It355-370. Sato, T. (1968) A modified method for lead staining of thin sections. J . Electron Microsc. (Tokyo),17t158-159. Semm, P., and L. Vollrath (1980) Electrophysiological evidence for circadian rhythmicity in a mammalian pineal organ. J . Neural Transmiss., 47t181-191. Tapp, - . E., and M. Blumfield (1970) The parenchyma1 cells of the rat pineal gland. Acta. Morphol. Neerl.8cand.,~8:119-131. Trueman, T., and J. Herbert (1970) The distribution of monoamines and acetylcholinesterase in the pineal gland and habenula of the ferret. J . Anat., 106:406. Ueck, M., and H. Kobayashi (1972) Vergleichende untersuchungen uber acetylcholinesterase-haltigeneurone im pinealorgan der vogel. Z. Zellforsch., 129t140-160. Vigh-Teichmann, I., H.W. Korf, A. Oksche, and B. Vigh (1982) Opsinimmunoreactive outer segments and acetylcholinesterasepositive neurons in the pineal complex of Phox~nusphoxinus (Teleostei, Cyprinidae). Cell Tissue Res., 227t351-369. Vollrath, L. (1981) The Pineal Organ. Springer. New York, p. 106. Wake, K. (1973)Acetylcholinesterase-containingnerve cells and their distribution in the pineal organ of the goldfish, Carassius auratus. Z. Zellforsch., 145t287-298. Wake, K., M. Ueck, and A. Oksche (1974) Acetylcholinesterase-containing nerve cells in the pineal complex and subcommissural area of the frogs, Rana ridibundu and Rana esculenta. Cell Tissue Res., 154t423-424.
