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Innervation of the pineal gland in the rat: An HRP study
EXPERIMENTAL
NEUROLOGY
95,207-2 15 ( 1987)
Innervation of the Pineal Gland in the Rat: An HRP Study JOHN W. PATRICK~ONAND THOMAS E. SMITH’ Department ofAnatomy, Loma Linda University School of Medicine, Loma Linda, California 92350 Received June 12, 1986; revision receivedAugust 18, 1986 Injections of horseradish peroxidase were made (a) stereotaxically and (b) directly (surgically exposed) into the pineal gland of Sprague-Dawley and Long-Evans rats weighing 30 to 500 g (20 to 130 days of age). Retrogradely labeled cells were seen in the superior cervical ganglia. Anterogradely labeled fibers were observed within the pineaf stalk, lamina intercalaris, and the medial habenular nuclei. Terminal fields were identified in lamina intercalaris and medial habenular nuclei. Labeled cells were not seen within the central nervous system. These results suggest that in the rat the pineal gland is not centrally innervated but in fact is innervated solely by the sympathetic postganghonic fibers. &I I987 Academic Press, Inc.
INTRODUCTION The innervation of the mammalian pineal gland has received renewed attention in recent years. In the past the gland was considered as being innervated solely by sympathetic fibers originating in the superior cervical ganglia (12). However, recent electrophysiological studies (5, 16, 2 1, 22), morphological studies (13, 18, 23, 24), and neurohistochemical studies (9, 18, 19) suggestedthat the gland may also be innervated by a direct central pathway-the epithalamoepiphyseal tract. These fibers are thought to be those found within the pineal stalk.The fibers within the stalk were at first thought of asbeing aberrant fibers exiting from the habenular and posterior commissures (12). Various attempts have been made to identify the perikaryon of these fibers in the guinea pig (13), Mongolian gerbil (19), and rat (9, 11). Abbreviations: DMB-diaminobenzidine, HRP-WGA-HRP-wheat germ agglutinin, SCG-superior cervical ganglion, TMB-tetramethylbenzidine. ’ We thank C. L. Wendtland, Linda Haines, and E. F. Reiber for the typing of the manuscript. This work was supported by the Samuel F. Crooks Chair of Anatomy Fund. 207 0014-4886/87 $3.00 Copyright Q 1987 by Academic Pms, Inc. AU rights of reproduction in any form reserved.
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Although there are species differences to be considered, these perikarya are widely distributed within the central nervous system (CNS). It has been suggested that these neurons provide an efferent pathway in the CNS for various modalities such as light, auditory, and olfactory stimuli (6, 16) that may influence pineal function. In studies using the rat model, Guerillot et al. (11) identified neurons in the habenulae, superior colliculi, amygdala, paraventricular and suprachiasmatic nuclei, preoptic area, and olfactory centers as projecting to the pineal. Dafny (9), using the same method, found labeled cells only in the medial habenular nuclei and stria medulllaris. The present study was conducted to determine the precise distribution of the neurons innervating the pineal gland and to evaluate possible strain variations. MATERIALS
AND
METHODS
A total of 85 rats (65 Sprague-Dawley and 20 Long-Evans) weighing 30 (weanlings) to 500 g representing an age range from 20 days to approximately 130 days, were used in the present study. The animals were exposed to a 12-h light: 12-h dark cycle. All animals were anesthetized with pentobarbital (30 m/kg). Stereotaxic Injections. Fifteen Sprague-Dawley rats (150 to 315 g) were prepared for placement of the micropipet within the pineal gland. This included making a small opening in the area of the lambda exposing the confluence of the sinuses. The micropipet was stereotaxically inserted in the midline 9.1 mm posterior to bregma, through the confluence of the sinuses to a depth of 2.5 mm from the overlying bone surface. A volume of 0.3 to 1 ~1 30% HRP in saline or a volume of 0.2 to 0.5 ~1 1% HRP-wheat germ agglutinin (WGA) in saline was hydraulically injected into the gland using a 1-pl Hamilton syringe. Surgical Exposure of the Pineal. In the remainder of animals, the confluence of the sinuses and the left limb of the transverse sinus were exposed and an incision made just left of the sinuses. The most distal end of the transverse sinus was cut after the placement of ligatures around the sinus. The sinus was then reflected to the right side thus exposing the pineal gland. The micropipet was visibly placed with the aid of a Zeiss dissecting microscope within the gland using a micromanipulator. Volumes of HRP and HRP-WGA as mentioned above were injected into the gland during a period of 15 to 30 min. After 12 to 72 h ( 10 to 24 h for the animals receiving the HRP-WGA), the animals were anesthetized and the head perfused via the aorta with 250 ml saline at 25°C then with 250 ml 1% paraformaldehyde and 1.25 glutaralde-
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hyde in 0.1 A4 phosphate buffer (pH 7.4) at 25°C. This was followed by 250 ml 10% sucrose in 0.1 M phosphate buffer (PH 7.4) at 4°C. The brain and superior cervical ganglia (SCG) were removed and stored in the sucrose buffer solution for 12 to 24 h at 4°C. Frozen 30qm serial sections in the coronal or sag&al planes were made and incubated using tetramethyl benzidine (TMB) as the chromogen following the protocol of Mesulam (17). The sections were then mounted on gelatin-coated slides and coverslipped. The sections were examined under light- and dark-field illumination for the presence of HRP-positive neurons. RESULTS The injection of HRP, both conjugated and nonconjugated, into the pineal gland resulted in consistent labeling of postganglionic cells in the superior cervical ganglia, fibers within the pineal stalk, and a terminal field in lamina intercalaris (deep pineal) extending into the posterior area of the medial habenular nuclei. The anatomical relationship of the pineal gland to other adjacent CNS structures are shown in Fig. 1. Pineal Stalk, Fig. 2. Labeled fibers were consistently found in the stalk when HRP was injected into the rostral portion of the gland. These fibers coursed parallel within the stalk, giving the appearance of a small “tract.” Upon passing under the habenular commissure these fibers diverged, then entered the lamina intercalaris and the posterior region of the medial habenular nuclei. There were some fibers that seemed to terminate on the choroid plexus of the ventricular recess. Lamina Intercalaris, Figs. 3 and 4. Some labeled fibers of the stalk terminated within the lamina intercalaris and were visualized as a terminal field, i.e., an aggregation of terminal boutons and others terminated in the medial habenular nucleus. The terminal field was most pronounced when the conjugated form of the tracer was used. The terminal field included the entire region of lamina inter&&. Of the labeled fibers observed coursing the entire length of the pineal stalk, the majority seemed to terminate here. The number of fibers leaving the proximal end of the pineal was always greater than that entering the lamina intercalaris. Medial Habenular Nuclei, Fig. 5. Diverging fibers were seen leaving lamina intercalaris and entering the posterior region of the medial habenular nuclei. These fibers terminated within each nucleus forming terminal fields. Labeled fibers or terminal boutons were not observed in the surrounding brain tissue. Superior Cervical Ganglia, Fig. 6. Labeled perikarya were found consistently in the rostral third of the ganglia. There appeared to be no given pattern of distribution within the region. There were a few labeled cells scattered
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FIG. 1. A cresyl violet-stained paramedian section through the proximal end of the pineal gland showing its attachment to lamina intercalaris, habenular and posterior commissures via the pineal stalk. X65. FIG. 2. Dark-field photomicrograph of the pineal stalk showing HRP-labeled fibers. Spicular artifacts (A) were seen within the recess of the third ventricle. X325. FIG. 3. Dark-field photomicrograph of a paramedian section of the pineal stalk, habenular commissure, lamina intercalaris, and posterior commissure; showing HRP-labeled fibers within the pineal stalk and lamina intercalaris. X325.
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throughout the remainder of the ganglia with the frequency decreasingcaudally. In some animals in which the injections were made stereotaxically, one ganglion seemedto be more dominant than the other, i.e., having more labeled cells.Examination of the pineal gland showed complete filling of the gland with the tracer. In animals in which the gland was surgically exposed, which included the cutting of one limb of the transverse sinus, labeled cells were found only in the contralateral ganglion. Age Projile. Failure to find labeled perikarya within the CNS prompted the idea of a possibleage-related degeneration of centrally innervating fibers. Animals ranging from weanlings (20 to 25 days) to adults weighing approximately 500 g(130+ days) yielded the sameresults,i.e., labeled postganglionic cells,fibers in the stalk, and terminal fields in the lamina intercalaris. Subjectively the number of labeled fibers in the stalk remained relatively the same in all groups. Strain. Both Sprague-Dawley and Long-Evans (hooded) rats yielded similar results.In no instance were labeled cellsfound within the CNS. DISCUSSION The encapsulation and lack of rigid attachment of the pineal makesit very difficult to penetrate. This is acutely important if the injections are made stereotaxically. It was later observed that in such an approach the pineal oftentimes rotates along its anterior-posterior axis as the micropipet is advanced to its predetermined depth. The application of the tracer would not only partially enter the gland but would spill into the ventricular recessand/ or the transverse cerebral fissure. In a more direct approach, i.e., surgical exposure of the gland (seeMethods), the micropipet and gland could be manipulated so that one could visually record the entry and ultimate injection of the tracer material. However, despite such careful manipulation, there were times when the tracer would leak at the point of entry, spilling some
PIG. 4. Dark-field photomicrograph of a coronal section of lamina intercalaris. HRP-labeled fibers were seen diverging and traversing lamina intercalaris. Note the terminal field within the lamina (arrow). X600. FIG. 5. Dark-field photomicrograph of the posterior area of the medial habcnular nucleus. Anterogradely labeled fibers were seen in terminal fields as indicated (between the arrows). X750. FIG. 6. Dark-field photomicrograph of retrogradely labeled postganglionic neurons within the superior cervical ganglia. X255. Note: All pictures are from the Sprague-Dawley strain. Abbreviations: A-spicular artifacts, H-habenular commissure, LI-lamina intercalaris, Ppineal gland, PC-posterior commissure, S-pineal stalk, V3-third ventricle.
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tracer onto the adjacent structures. Whenever such spillage occurred, the uptake of the tracer was more widely distributed within the CNS. These structures include those described by Dafny (9) and most of those listed by Gukrillot et al. (1 I), except the supraoptic nucleus and preoptic area. It is noteworthy that even though both investigators used basically the same method, including the samechromogen, diaminobenzidine (DMB); their results,except for the medial habenular nucleus, do not correspond. Gu&illot et al. (11) described thesecellsas “isolated and rare,” suggestinginconsistenciesin the location of labeled perikarya within the CNS. To suggestthat these cells are the origin of centrally innervating fibers would be impractical. The chromogen DMB has been shown to be lesssensitive than TMB ( 17). In the present study, however, not only was TMB used, but in addition to the regular HRP, the conjugated form-HRP-WGA was also utilized. The conjugated form of HRP has been demonstrated as having a much higher affinity to neurons than the regular form (10). The combination of both the conjugated HRP and TMB maximized the transport and ultimate visualization of labeled neurons. The termination of the labeled fibers within the lamina intercalaris suggeststhat they are sympathetic fibers similar to those described by Bjorklund et al. (1) and Wiklund (26). The labeling of these fibers also suggeststhat they do make synaptic contact (synapseenpassant) within the pineal, although it is possiblethat some of them may have taken up HRP at points of injury as they passthrough the gland. It is indeed conceivable that the degenerating fibers and terminals observed by Rdnnekleiv and Mdller (23) within the pineal following medial habenular lesion are sympathetic nerves which not only innervate the pineal but also the medial habenular nuclei. Not only are thesefibers and terminals found in known regions of sympathetic innervation within the gland, but the terminals undergo similar degenerative changesasseenin superior cervical ganglionectomy (20). During the initial segment of the present study, failure to locate labeled perikarya within the CNS prompted the idea of a possibleage-related degeneration of the central fibers sincethe animals used thus far weighed approximately 300 g (70 to 75 days of age). The study was therefore expanded to include weanlings (20 to 25 days, weighing 30 to 50 g) and older animals weighing approximately 500 g ( 130 days). The number of labeled fibers remained relatively constant throughout the agegroups. This suggeststhat the fibers of the pineal stalk that communicate synaptically with the gland do not undergo a maturation degeneration as is the possible casein man (25). Various electrophysiological studies have been made in an effort to verify the existenceof a central pathway to the rat pineal gland. These include extracellular recordings within the pineal in responseto electrical stimulation of distal CNS structures (the medial habenular nuclei, hypothalamus, and hippocampus) (2 1,22) and the recording of evoked potentials in responseto
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acoustic and photic stimulation (6, 7). The fact that the discharge patterns of pineal elements can be altered by stimulation of the medial habenular nucleus does not rule out the possibility that these elements are responding to antidromic stimulation of sympathetic fibers. These fibers communicate with the pineal elements via “synapse en passant,” then through the pineal stalk to enter the medial habenular nucleus. These elements would therefore respond as if they were conducting orthodromically as observed by Reuss et al. (2 1). The latency of evoked potentials recorded on the pineal surface has been used as a criterion in postulating the existence of a direct central innervation of the pineal. In order to utilize such latencies, it is very important to be able to recognize the waveforms generated by the various known generators within that given pathway (15). Based on the latency considered, the recording parameters should be such as to visualize the early field potentials. These waveforms are not visible in the recordings reported by Dafny (5-8). In addition, the waveform that is used to determine the latency has to be generated within the pineal. Although the configuration of the waveform basically remains the same, it has not been demonstrated that the pineal is the generator. It is also possible that the evoked potential assigned to the pineal could be generated by either the superior or inferior colliculus in response to photic or auditory stimulus, respectively. In addition, the evoked potential attributed to the superior cervical ganglia is in conflict with the studies of Brooks et al. (4). They demonstrated that photic or electrical stimulation of the visual pathway inhibits sympathetic discharge to the pineal gland. The evidence further suggests that the inhibition occurs centrally and not in the SCG (3). The present study does suggest however, that not all fibers of the pineal stalk have synaptic communication with the pineal and those that have are “efferents” of the pineal. In a recent ultrastructural study, Lou et al. (14) showed that the number of fibers contained within the stalk decreased from its proximal end to its distal pineal connection. It is possible that these fibers may loop on themselves in the stalk and proximal pineal, returning to the epithalamic regions as suggested by Rappers ( 12). If bouton terminaux exist for these fibers within the pineal, the presence of HRP at the terminals would be picked up and transported to the perikarya of each fiber. This is borne out with respect to the sympathetic fibers. In all injections made within the pineal parenchyma, labeled cells were observed in the SCG. Postganglionic neurons in the SCG that innervate the pineal are dispersed throughout the ganglia, the majority in the rostral one-third. A topographic pattern of organization of these neurons as it relates to the pineal gland could not be discerned. However the localization of these neurons in the SCGcontralateral to the ligated transverse sinus, suggests that the projection of the SCG to the pineal gland is ipsilateral and travels in close proximity to the
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transverse sinus where it enters the gland as the nervi conarii, concurring with similar observations made by Bowers et al. (2). Although our study has identified fibers leaving the pineal by way of the stalk to innervate the lamina intercalaris and the medial habenular nuclei, it is yet to be determined if these fibers represent all the sympathetic fibers that are present in the stalk. In conclusion, although there is ample evidence that the pineal stalk in the rat contains nerve fibers, this study suggeststhat not all these fibers synapse in the pineal gland. The fibers that do communicate with the gland also terminate in the lamina intercalaris (deep pineal) and medial habenular nuclei. It further suggeststhat these fibers are postganglionic sympathetics whose somasare situated in the SCG. REFERENCES I. BJ~RKLUND, A., C. OWMAN, AND K. A. WEST. 1972. Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain. Z. Zellforsch. Microsk. Anat. [Histochem.] 127~570-579. 2. BOWERS, C. W., L. M. DAHM, AND R. E. ZIGMOND. 1984. The number and distribution of sympathetic neurons that innervate the rat pineal gland. Neuroscience 13:87-96. 3. BOWERS, C. W., AND R. E. ZIGMOND. 1982. The influence ofthe frequency and pattern of sympathetic nerve activity on serotonin N-acetyltransferase in the rat pineal. J. Physiol. (London) 330: 279-296. 4. BROOKS, C. McC., T. ISHIKAWA, AND K. KOIZUMI. 1975. Autonomic system control of the pineal gland and the role of this complex in the integration of body function. Bruin Res. 87: 181-190. 5. DAFNY, N., R. M&LUNG, AND S. J. STRADA. 1975. Neurophysiological properties of the pineal body. 1. Field potentials. L@ Sci. 16:61l-620. 6. DAFNY, N. 1977. Electrophysiological evidence of photic, acoustic, and central input to the pineal body and hypothalamus. Exp. Neurol. 55: 449-457. 7. DAFNY, N. 1980. Two photic pathways contribute to pineal evoked responses. Life Sci. 26:
737-742. 8. DAFNY, N. 1980. Photic input to rat pineal gland conveyed by both sympathetic and central alferents. J. Neural Transm. 48: 203-208. 9. DAFNY, N. 1983. Evidence that the rat has neuronal connections via the pineal stalk. Exp. Neurol. 79: 858-86 1. 10. GONATAS, N. K., C. HARPER, T. MIZUTANI, AND J. 0. GONATAS. 1979. Superior sensitivity of conjugates of horseradish with wheat germ agglutinin for studies of retrograde axonal transport. J. Histochem. Cytochem. 27: 728-734. 1I. GU~?RILLOT,C., A. PFISTER,J. MILLER, AND C. DA LAGE. 1982. Recherche de l’origine des fibres nerveuses extraorthosympathiques innervant l’epiphyse du rat (etude du transport retrograde de la peroxydase). Reprod. Nutr. Dev. 22: 37 1-378. 12. KAPPERS, J. A. 1960. The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch. Microsk. Anat. [Histochem.] 52: 163-
215. 13. KORF, H. W., AND U. WAGNER. 1980. Evidence for a nervous connection between the brain and the pineal organ in the guinea pig. Cell Tissue Res. 209: 505-5 10.
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14. Luo, Z. R., R. L. SCHULTZ, E. F. WHITTR, AND L. VOLLRATH. 1984. The ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk. J. Pineul Res. 1: 323-337. 15. MAURER, K., H. LEITNER, AND E. S&&R. 1980. Detection and localization of brainstem lesions with auditory bminstem potentials. Pages 39 l-398 in C. BARBER, Ed., Evoked Pofenrials. Univ. Park Press, Baltimore, Maryland. 16. MCCLUNG, R. E., AND N. DAFNY. 1975. Electrophysiological properties of the pineal body. II. Single unit activity. Life Sci. 16: 62 l-628. 17. MESULAM, M. M. 1978. Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26: 106- 117. 18. M#LLER, M., AND KORF, H. W. 1983. Central innervation of the pineal organ of the Mongolian gerbil. A histochemical and lesion study. Cell Tissue Res. 230: 259-272. 19. M#LLER, M., AND H. W. KORF. 1983. The origin of central pinealopetal nerve fibers in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. Cell Tissue Res. 230: 273-287. 20. PELLEGRINO DE IRALDI, A., L. M. ZIEHER, AND E. DE ROBERTIS. 1965. Ultrastructure and pharmacological studies of nerve endings in the pineal organ. Prog. Bruin Res. 10: 389421. 2 1. REV.%, ST., P. SEMM, AND L. VOLLRATH. 1984. Electrophysiological investigations on the central innervation of the rat and guinea-pig pineal gland. J. Neural Transm. 60: 3 l-43. 22. R$NNEKLEIV, 0. K., M. J. KELLY, AND W. WU~~KE. 1980. Single unit recordings in the rat pineal gland: evidence for habenulo-pineal neuronal connections. Exp. Bruin Res. 39: 187-192. 23. R@NEKLEIV, 0. K., AND M. M#LLER. 1979. Brain-pineal nervous connections in the rat: an uhrastructural study following habenular lesion. Exp. Brain Res. 37: 55 I-562. 24. SCHNEIDER,T., P. SEMM,AND L. VOLLRATH. 1981. Ultrastructural observations on the central innervation of the guinea-pig pineal gland. Cell Tissue Rex 220: 4 I-49. 25. STOTLER, W. A. 1984. Innervation ofthe human pineal organ, a study by age groups. Anat. Rec. 20% 174. 26. WIKLUND, L. 1974. Development of serotonin-containing cells and the sympathetic innervation of the habenular region in the rat brain. Cell Tissue Res. 155: 23 l-243.
NEUROLOGY
95,207-2 15 ( 1987)
Innervation of the Pineal Gland in the Rat: An HRP Study JOHN W. PATRICK~ONAND THOMAS E. SMITH’ Department ofAnatomy, Loma Linda University School of Medicine, Loma Linda, California 92350 Received June 12, 1986; revision receivedAugust 18, 1986 Injections of horseradish peroxidase were made (a) stereotaxically and (b) directly (surgically exposed) into the pineal gland of Sprague-Dawley and Long-Evans rats weighing 30 to 500 g (20 to 130 days of age). Retrogradely labeled cells were seen in the superior cervical ganglia. Anterogradely labeled fibers were observed within the pineaf stalk, lamina intercalaris, and the medial habenular nuclei. Terminal fields were identified in lamina intercalaris and medial habenular nuclei. Labeled cells were not seen within the central nervous system. These results suggest that in the rat the pineal gland is not centrally innervated but in fact is innervated solely by the sympathetic postganghonic fibers. &I I987 Academic Press, Inc.
INTRODUCTION The innervation of the mammalian pineal gland has received renewed attention in recent years. In the past the gland was considered as being innervated solely by sympathetic fibers originating in the superior cervical ganglia (12). However, recent electrophysiological studies (5, 16, 2 1, 22), morphological studies (13, 18, 23, 24), and neurohistochemical studies (9, 18, 19) suggestedthat the gland may also be innervated by a direct central pathway-the epithalamoepiphyseal tract. These fibers are thought to be those found within the pineal stalk.The fibers within the stalk were at first thought of asbeing aberrant fibers exiting from the habenular and posterior commissures (12). Various attempts have been made to identify the perikaryon of these fibers in the guinea pig (13), Mongolian gerbil (19), and rat (9, 11). Abbreviations: DMB-diaminobenzidine, HRP-WGA-HRP-wheat germ agglutinin, SCG-superior cervical ganglion, TMB-tetramethylbenzidine. ’ We thank C. L. Wendtland, Linda Haines, and E. F. Reiber for the typing of the manuscript. This work was supported by the Samuel F. Crooks Chair of Anatomy Fund. 207 0014-4886/87 $3.00 Copyright Q 1987 by Academic Pms, Inc. AU rights of reproduction in any form reserved.
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Although there are species differences to be considered, these perikarya are widely distributed within the central nervous system (CNS). It has been suggested that these neurons provide an efferent pathway in the CNS for various modalities such as light, auditory, and olfactory stimuli (6, 16) that may influence pineal function. In studies using the rat model, Guerillot et al. (11) identified neurons in the habenulae, superior colliculi, amygdala, paraventricular and suprachiasmatic nuclei, preoptic area, and olfactory centers as projecting to the pineal. Dafny (9), using the same method, found labeled cells only in the medial habenular nuclei and stria medulllaris. The present study was conducted to determine the precise distribution of the neurons innervating the pineal gland and to evaluate possible strain variations. MATERIALS
AND
METHODS
A total of 85 rats (65 Sprague-Dawley and 20 Long-Evans) weighing 30 (weanlings) to 500 g representing an age range from 20 days to approximately 130 days, were used in the present study. The animals were exposed to a 12-h light: 12-h dark cycle. All animals were anesthetized with pentobarbital (30 m/kg). Stereotaxic Injections. Fifteen Sprague-Dawley rats (150 to 315 g) were prepared for placement of the micropipet within the pineal gland. This included making a small opening in the area of the lambda exposing the confluence of the sinuses. The micropipet was stereotaxically inserted in the midline 9.1 mm posterior to bregma, through the confluence of the sinuses to a depth of 2.5 mm from the overlying bone surface. A volume of 0.3 to 1 ~1 30% HRP in saline or a volume of 0.2 to 0.5 ~1 1% HRP-wheat germ agglutinin (WGA) in saline was hydraulically injected into the gland using a 1-pl Hamilton syringe. Surgical Exposure of the Pineal. In the remainder of animals, the confluence of the sinuses and the left limb of the transverse sinus were exposed and an incision made just left of the sinuses. The most distal end of the transverse sinus was cut after the placement of ligatures around the sinus. The sinus was then reflected to the right side thus exposing the pineal gland. The micropipet was visibly placed with the aid of a Zeiss dissecting microscope within the gland using a micromanipulator. Volumes of HRP and HRP-WGA as mentioned above were injected into the gland during a period of 15 to 30 min. After 12 to 72 h ( 10 to 24 h for the animals receiving the HRP-WGA), the animals were anesthetized and the head perfused via the aorta with 250 ml saline at 25°C then with 250 ml 1% paraformaldehyde and 1.25 glutaralde-
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hyde in 0.1 A4 phosphate buffer (pH 7.4) at 25°C. This was followed by 250 ml 10% sucrose in 0.1 M phosphate buffer (PH 7.4) at 4°C. The brain and superior cervical ganglia (SCG) were removed and stored in the sucrose buffer solution for 12 to 24 h at 4°C. Frozen 30qm serial sections in the coronal or sag&al planes were made and incubated using tetramethyl benzidine (TMB) as the chromogen following the protocol of Mesulam (17). The sections were then mounted on gelatin-coated slides and coverslipped. The sections were examined under light- and dark-field illumination for the presence of HRP-positive neurons. RESULTS The injection of HRP, both conjugated and nonconjugated, into the pineal gland resulted in consistent labeling of postganglionic cells in the superior cervical ganglia, fibers within the pineal stalk, and a terminal field in lamina intercalaris (deep pineal) extending into the posterior area of the medial habenular nuclei. The anatomical relationship of the pineal gland to other adjacent CNS structures are shown in Fig. 1. Pineal Stalk, Fig. 2. Labeled fibers were consistently found in the stalk when HRP was injected into the rostral portion of the gland. These fibers coursed parallel within the stalk, giving the appearance of a small “tract.” Upon passing under the habenular commissure these fibers diverged, then entered the lamina intercalaris and the posterior region of the medial habenular nuclei. There were some fibers that seemed to terminate on the choroid plexus of the ventricular recess. Lamina Intercalaris, Figs. 3 and 4. Some labeled fibers of the stalk terminated within the lamina intercalaris and were visualized as a terminal field, i.e., an aggregation of terminal boutons and others terminated in the medial habenular nucleus. The terminal field was most pronounced when the conjugated form of the tracer was used. The terminal field included the entire region of lamina inter&&. Of the labeled fibers observed coursing the entire length of the pineal stalk, the majority seemed to terminate here. The number of fibers leaving the proximal end of the pineal was always greater than that entering the lamina intercalaris. Medial Habenular Nuclei, Fig. 5. Diverging fibers were seen leaving lamina intercalaris and entering the posterior region of the medial habenular nuclei. These fibers terminated within each nucleus forming terminal fields. Labeled fibers or terminal boutons were not observed in the surrounding brain tissue. Superior Cervical Ganglia, Fig. 6. Labeled perikarya were found consistently in the rostral third of the ganglia. There appeared to be no given pattern of distribution within the region. There were a few labeled cells scattered
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FIG. 1. A cresyl violet-stained paramedian section through the proximal end of the pineal gland showing its attachment to lamina intercalaris, habenular and posterior commissures via the pineal stalk. X65. FIG. 2. Dark-field photomicrograph of the pineal stalk showing HRP-labeled fibers. Spicular artifacts (A) were seen within the recess of the third ventricle. X325. FIG. 3. Dark-field photomicrograph of a paramedian section of the pineal stalk, habenular commissure, lamina intercalaris, and posterior commissure; showing HRP-labeled fibers within the pineal stalk and lamina intercalaris. X325.
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throughout the remainder of the ganglia with the frequency decreasingcaudally. In some animals in which the injections were made stereotaxically, one ganglion seemedto be more dominant than the other, i.e., having more labeled cells.Examination of the pineal gland showed complete filling of the gland with the tracer. In animals in which the gland was surgically exposed, which included the cutting of one limb of the transverse sinus, labeled cells were found only in the contralateral ganglion. Age Projile. Failure to find labeled perikarya within the CNS prompted the idea of a possibleage-related degeneration of centrally innervating fibers. Animals ranging from weanlings (20 to 25 days) to adults weighing approximately 500 g(130+ days) yielded the sameresults,i.e., labeled postganglionic cells,fibers in the stalk, and terminal fields in the lamina intercalaris. Subjectively the number of labeled fibers in the stalk remained relatively the same in all groups. Strain. Both Sprague-Dawley and Long-Evans (hooded) rats yielded similar results.In no instance were labeled cellsfound within the CNS. DISCUSSION The encapsulation and lack of rigid attachment of the pineal makesit very difficult to penetrate. This is acutely important if the injections are made stereotaxically. It was later observed that in such an approach the pineal oftentimes rotates along its anterior-posterior axis as the micropipet is advanced to its predetermined depth. The application of the tracer would not only partially enter the gland but would spill into the ventricular recessand/ or the transverse cerebral fissure. In a more direct approach, i.e., surgical exposure of the gland (seeMethods), the micropipet and gland could be manipulated so that one could visually record the entry and ultimate injection of the tracer material. However, despite such careful manipulation, there were times when the tracer would leak at the point of entry, spilling some
PIG. 4. Dark-field photomicrograph of a coronal section of lamina intercalaris. HRP-labeled fibers were seen diverging and traversing lamina intercalaris. Note the terminal field within the lamina (arrow). X600. FIG. 5. Dark-field photomicrograph of the posterior area of the medial habcnular nucleus. Anterogradely labeled fibers were seen in terminal fields as indicated (between the arrows). X750. FIG. 6. Dark-field photomicrograph of retrogradely labeled postganglionic neurons within the superior cervical ganglia. X255. Note: All pictures are from the Sprague-Dawley strain. Abbreviations: A-spicular artifacts, H-habenular commissure, LI-lamina intercalaris, Ppineal gland, PC-posterior commissure, S-pineal stalk, V3-third ventricle.
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tracer onto the adjacent structures. Whenever such spillage occurred, the uptake of the tracer was more widely distributed within the CNS. These structures include those described by Dafny (9) and most of those listed by Gukrillot et al. (1 I), except the supraoptic nucleus and preoptic area. It is noteworthy that even though both investigators used basically the same method, including the samechromogen, diaminobenzidine (DMB); their results,except for the medial habenular nucleus, do not correspond. Gu&illot et al. (11) described thesecellsas “isolated and rare,” suggestinginconsistenciesin the location of labeled perikarya within the CNS. To suggestthat these cells are the origin of centrally innervating fibers would be impractical. The chromogen DMB has been shown to be lesssensitive than TMB ( 17). In the present study, however, not only was TMB used, but in addition to the regular HRP, the conjugated form-HRP-WGA was also utilized. The conjugated form of HRP has been demonstrated as having a much higher affinity to neurons than the regular form (10). The combination of both the conjugated HRP and TMB maximized the transport and ultimate visualization of labeled neurons. The termination of the labeled fibers within the lamina intercalaris suggeststhat they are sympathetic fibers similar to those described by Bjorklund et al. (1) and Wiklund (26). The labeling of these fibers also suggeststhat they do make synaptic contact (synapseenpassant) within the pineal, although it is possiblethat some of them may have taken up HRP at points of injury as they passthrough the gland. It is indeed conceivable that the degenerating fibers and terminals observed by Rdnnekleiv and Mdller (23) within the pineal following medial habenular lesion are sympathetic nerves which not only innervate the pineal but also the medial habenular nuclei. Not only are thesefibers and terminals found in known regions of sympathetic innervation within the gland, but the terminals undergo similar degenerative changesasseenin superior cervical ganglionectomy (20). During the initial segment of the present study, failure to locate labeled perikarya within the CNS prompted the idea of a possibleage-related degeneration of the central fibers sincethe animals used thus far weighed approximately 300 g (70 to 75 days of age). The study was therefore expanded to include weanlings (20 to 25 days, weighing 30 to 50 g) and older animals weighing approximately 500 g ( 130 days). The number of labeled fibers remained relatively constant throughout the agegroups. This suggeststhat the fibers of the pineal stalk that communicate synaptically with the gland do not undergo a maturation degeneration as is the possible casein man (25). Various electrophysiological studies have been made in an effort to verify the existenceof a central pathway to the rat pineal gland. These include extracellular recordings within the pineal in responseto electrical stimulation of distal CNS structures (the medial habenular nuclei, hypothalamus, and hippocampus) (2 1,22) and the recording of evoked potentials in responseto
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acoustic and photic stimulation (6, 7). The fact that the discharge patterns of pineal elements can be altered by stimulation of the medial habenular nucleus does not rule out the possibility that these elements are responding to antidromic stimulation of sympathetic fibers. These fibers communicate with the pineal elements via “synapse en passant,” then through the pineal stalk to enter the medial habenular nucleus. These elements would therefore respond as if they were conducting orthodromically as observed by Reuss et al. (2 1). The latency of evoked potentials recorded on the pineal surface has been used as a criterion in postulating the existence of a direct central innervation of the pineal. In order to utilize such latencies, it is very important to be able to recognize the waveforms generated by the various known generators within that given pathway (15). Based on the latency considered, the recording parameters should be such as to visualize the early field potentials. These waveforms are not visible in the recordings reported by Dafny (5-8). In addition, the waveform that is used to determine the latency has to be generated within the pineal. Although the configuration of the waveform basically remains the same, it has not been demonstrated that the pineal is the generator. It is also possible that the evoked potential assigned to the pineal could be generated by either the superior or inferior colliculus in response to photic or auditory stimulus, respectively. In addition, the evoked potential attributed to the superior cervical ganglia is in conflict with the studies of Brooks et al. (4). They demonstrated that photic or electrical stimulation of the visual pathway inhibits sympathetic discharge to the pineal gland. The evidence further suggests that the inhibition occurs centrally and not in the SCG (3). The present study does suggest however, that not all fibers of the pineal stalk have synaptic communication with the pineal and those that have are “efferents” of the pineal. In a recent ultrastructural study, Lou et al. (14) showed that the number of fibers contained within the stalk decreased from its proximal end to its distal pineal connection. It is possible that these fibers may loop on themselves in the stalk and proximal pineal, returning to the epithalamic regions as suggested by Rappers ( 12). If bouton terminaux exist for these fibers within the pineal, the presence of HRP at the terminals would be picked up and transported to the perikarya of each fiber. This is borne out with respect to the sympathetic fibers. In all injections made within the pineal parenchyma, labeled cells were observed in the SCG. Postganglionic neurons in the SCG that innervate the pineal are dispersed throughout the ganglia, the majority in the rostral one-third. A topographic pattern of organization of these neurons as it relates to the pineal gland could not be discerned. However the localization of these neurons in the SCGcontralateral to the ligated transverse sinus, suggests that the projection of the SCG to the pineal gland is ipsilateral and travels in close proximity to the
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transverse sinus where it enters the gland as the nervi conarii, concurring with similar observations made by Bowers et al. (2). Although our study has identified fibers leaving the pineal by way of the stalk to innervate the lamina intercalaris and the medial habenular nuclei, it is yet to be determined if these fibers represent all the sympathetic fibers that are present in the stalk. In conclusion, although there is ample evidence that the pineal stalk in the rat contains nerve fibers, this study suggeststhat not all these fibers synapse in the pineal gland. The fibers that do communicate with the gland also terminate in the lamina intercalaris (deep pineal) and medial habenular nuclei. It further suggeststhat these fibers are postganglionic sympathetics whose somasare situated in the SCG. REFERENCES I. BJ~RKLUND, A., C. OWMAN, AND K. A. WEST. 1972. Peripheral sympathetic innervation and serotonin cells in the habenular region of the rat brain. Z. Zellforsch. Microsk. Anat. [Histochem.] 127~570-579. 2. BOWERS, C. W., L. M. DAHM, AND R. E. ZIGMOND. 1984. The number and distribution of sympathetic neurons that innervate the rat pineal gland. Neuroscience 13:87-96. 3. BOWERS, C. W., AND R. E. ZIGMOND. 1982. The influence ofthe frequency and pattern of sympathetic nerve activity on serotonin N-acetyltransferase in the rat pineal. J. Physiol. (London) 330: 279-296. 4. BROOKS, C. McC., T. ISHIKAWA, AND K. KOIZUMI. 1975. Autonomic system control of the pineal gland and the role of this complex in the integration of body function. Bruin Res. 87: 181-190. 5. DAFNY, N., R. M&LUNG, AND S. J. STRADA. 1975. Neurophysiological properties of the pineal body. 1. Field potentials. L@ Sci. 16:61l-620. 6. DAFNY, N. 1977. Electrophysiological evidence of photic, acoustic, and central input to the pineal body and hypothalamus. Exp. Neurol. 55: 449-457. 7. DAFNY, N. 1980. Two photic pathways contribute to pineal evoked responses. Life Sci. 26:
737-742. 8. DAFNY, N. 1980. Photic input to rat pineal gland conveyed by both sympathetic and central alferents. J. Neural Transm. 48: 203-208. 9. DAFNY, N. 1983. Evidence that the rat has neuronal connections via the pineal stalk. Exp. Neurol. 79: 858-86 1. 10. GONATAS, N. K., C. HARPER, T. MIZUTANI, AND J. 0. GONATAS. 1979. Superior sensitivity of conjugates of horseradish with wheat germ agglutinin for studies of retrograde axonal transport. J. Histochem. Cytochem. 27: 728-734. 1I. GU~?RILLOT,C., A. PFISTER,J. MILLER, AND C. DA LAGE. 1982. Recherche de l’origine des fibres nerveuses extraorthosympathiques innervant l’epiphyse du rat (etude du transport retrograde de la peroxydase). Reprod. Nutr. Dev. 22: 37 1-378. 12. KAPPERS, J. A. 1960. The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch. Microsk. Anat. [Histochem.] 52: 163-
215. 13. KORF, H. W., AND U. WAGNER. 1980. Evidence for a nervous connection between the brain and the pineal organ in the guinea pig. Cell Tissue Res. 209: 505-5 10.
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14. Luo, Z. R., R. L. SCHULTZ, E. F. WHITTR, AND L. VOLLRATH. 1984. The ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk. J. Pineul Res. 1: 323-337. 15. MAURER, K., H. LEITNER, AND E. S&&R. 1980. Detection and localization of brainstem lesions with auditory bminstem potentials. Pages 39 l-398 in C. BARBER, Ed., Evoked Pofenrials. Univ. Park Press, Baltimore, Maryland. 16. MCCLUNG, R. E., AND N. DAFNY. 1975. Electrophysiological properties of the pineal body. II. Single unit activity. Life Sci. 16: 62 l-628. 17. MESULAM, M. M. 1978. Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26: 106- 117. 18. M#LLER, M., AND KORF, H. W. 1983. Central innervation of the pineal organ of the Mongolian gerbil. A histochemical and lesion study. Cell Tissue Res. 230: 259-272. 19. M#LLER, M., AND H. W. KORF. 1983. The origin of central pinealopetal nerve fibers in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. Cell Tissue Res. 230: 273-287. 20. PELLEGRINO DE IRALDI, A., L. M. ZIEHER, AND E. DE ROBERTIS. 1965. Ultrastructure and pharmacological studies of nerve endings in the pineal organ. Prog. Bruin Res. 10: 389421. 2 1. REV.%, ST., P. SEMM, AND L. VOLLRATH. 1984. Electrophysiological investigations on the central innervation of the rat and guinea-pig pineal gland. J. Neural Transm. 60: 3 l-43. 22. R$NNEKLEIV, 0. K., M. J. KELLY, AND W. WU~~KE. 1980. Single unit recordings in the rat pineal gland: evidence for habenulo-pineal neuronal connections. Exp. Bruin Res. 39: 187-192. 23. R@NEKLEIV, 0. K., AND M. M#LLER. 1979. Brain-pineal nervous connections in the rat: an uhrastructural study following habenular lesion. Exp. Brain Res. 37: 55 I-562. 24. SCHNEIDER,T., P. SEMM,AND L. VOLLRATH. 1981. Ultrastructural observations on the central innervation of the guinea-pig pineal gland. Cell Tissue Rex 220: 4 I-49. 25. STOTLER, W. A. 1984. Innervation ofthe human pineal organ, a study by age groups. Anat. Rec. 20% 174. 26. WIKLUND, L. 1974. Development of serotonin-containing cells and the sympathetic innervation of the habenular region in the rat brain. Cell Tissue Res. 155: 23 l-243.