Cell Tissue Res (2000) 301:369–373 DOI 10.1007/s004410000251
REGULAR ARTICLE
W. Liu · M. Møller
Innervation of the rat pineal gland by PACAP-immunoreactive nerve fibers originating in the trigeminal ganglion: a degeneration study
Received: 31 March 2000 / Accepted: 25 May 2000 / Published online: 13 July 2000 © Springer-Verlag 2000
Abstract In order to establish that the pineal gland is innervated by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibers originating in the trigeminal ganglion, ophthalmic and maxillary nerves were transected by using a subtemporal fossa approach. The number of PACAP-immunoreactive nerve fibers in the pineal gland of rats with a total transection of the nerve was compared with that of rats without surgery. In the operated rat, PACAP-immunoreactive nerve fibers in the superficial pineal decreased remarkably, indicating that the trigeminal ganglion was the origin of these nerve fibers. This research provides evidence supporting the hypothesis that PACAP-immunoreactive nerves regulate the synthesis and/or secretion of melatonin in the pineal gland. Key words Trigeminal ganglion · Ophthalmic nerve · Maxillary nerve · PACAP-immunoreactive nerve fibers · Nerve transection · Pineal gland · Rat (Wistar)
Introduction The pineal gland secretes the hormone melatonin (Reiter 1991a, 1991b), which binds to G-protein-coupled membrane-bound receptors in the pars tuberalis of the adenohypophysis and the suprachiasmatic nucleus of the hypothalamus (Vanecek 1988). Binding of melatonin to these receptors regulates, in photoperiodic animals, their reproductive function (Reiter 1991a) and entrains the suprachiasmatic nucleus, the endogenous clock of the brain, to the light-dark cycle. Melatonin secretion of the pineal gland is regulated by multiple nervous pathways W. Liu (✉) ENT Department, Pearl River Hospital, 253# Gong Ye Da Road, Guangzhou 510282, PR China e-mail:
[email protected] Tel.: 0086 20 85142776, Fax: 0086 20 85142776 M. Møller Institute of Medical Anatomy, Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark
(Møller 1992) of which the sympathetic innervation from the superior cervical ganglia is considered to be functionally the most important for pineal physiology (Klein 1992). However, the mammalian pineal also possesses a diversified peptidergic innervation. In the sympathetic nerve fibers innervating the gland, neuropeptide Y is colocalized with norepinephrine (Zhang et al. 1991). In addition, nerve fibers containing vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) are present in non-sympathetic nerve fibers in the gland (Møller 1994; Møller et al. 1996). Recently, binding sites for pituitary adenylate cyclase-activating polypeptide (PACAP) have been demonstrated in the rat pineal gland by receptor autoradiography (Masuo et al. 1992). PACAP has been shown to stimulate the synthesis of melatonin in dispersed rat pinealocytes (Simonneaux et al. 1993); such a stimulatory effect is mainly exerted through the activation of a pineal VIP1/PACAP receptor subtype(Simonneaux et al. 1998). PACAP-immunoreactive (IR) nerve fibers have been located in the pineal gland of rat (Møller et al. 1999) and sheep (W. Liu et al., unpublished). In the nerve fibers of the rat pineal gland, PACAP immunoreactivity is colocalized with substance P (SP) immunoreactivity and calcitonin gene-related peptide (CGRP) immunoreactivity (Møller et al. 1999). In the gerbil, combined retrograde in-vivo tracing and immunohistochemistry have revealed that CGRP-IR fibers in the pineal originate from the trigeminal ganglion (Shiotani et al. 1986). Furthermore, removal of the superior cervical ganglia (Møller et al. 1999), sphenopalatine ganglia, and otic ganglia (W. Liu et al., unpublished) does not obviously influence the number of PACAP-IR nerve fibers in the pineal gland. Moreover, the trigeminal ganglion contains PACAP perikarya (Mulder et al. 1994). Thus, we decided to establish whether PACAP-IR nerve fibers in the pineal gland of the rat have their origin in the trigeminal ganglion.
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Materials and methods Animals Adult male Wistar rats, weighing 250–300 g, were used. They were kept under a 12/12 h light/dark schedule (light on at 6 a.m.) with food and water ad libitum. The animals were handled according to the animal-use protocol approved by our Institutional Animal Care and Use Committee.
Bilateral trigeminal ganglionectomy Twenty rats were subjected to bilateral ophthalmic and maxillary nerve transection operation by using a subtemporal fossa approach, and another five served as controls. The rats undergoing the surgery were anesthetized with tribromethanol (i.p., 250 mg/kg) and fixed in a supine position on the surgical table. Tracheotomies were performed in the lower cervical midline, and the openings were maintained by suturing the tracheal cartilage rings to the skin. Tracheotomy was performed because airway obstruction by the retractors, which kept the subtemporal fossa open during the nerve transection operation, was inevitable. To reach the subtemporal fossa, a paramedian vertical incision was made on the ventral part of neck extending rostrally into the area medial to the jaw. The dissection was started between the mandible and omohyoid muscle and continued dorsally until the medial pterygoid muscle was disclosed. This muscle was removed in its rostral area in order to expose the pterygoid fossa constituted by the sphenoidal bone in the skull base. The bone in this fossa was then removed by using an electrical drill to expose the ophthalmic and maxillary nerves, rostral to the trigeminal ganglion (Fig. 1). These nerves, making up one single bundle, were then transected by using an electric cautery combined with a fine hook. The wounds and the tracheostomata were then closed. The animals were allowed to survive for 3 days. The dura was kept intact throughout the operation, and bleeding was slight. The feeding function of each rat was normal after surgery.
Perfusion fixation and cryostat sectioning The animals were anesthetized with tribromethanol and vascularly perfused with heparinized (15,000 IU/l) phosphate-buffered saline (pH 7.4, PBS) followed by Stephanini’s fixative (2% paraformaldehyde, 0.2% picric acid in 0.1 M sodium phosphate buffer pH 7.2) for 10 min. The brains were removed and postfixed in the same fixative for 24 h. They were then cryoprotected for 24 h in 20% sucrose, quickly frozen in crushed dry ice, and sagittally sectioned in a cryostat at a thickness of 25 µm; sections were collected on gelatinized glass slides.
Fig. 1 A portion of the bone in the pterygoid fossa (shaded area) was removed to expose the nerve. The main structures around this area include the petrotympanic fissure (PTF), tympanic bulla (TB), medial pterygoid plate (MPP), oval foramen (OF) and caudal alar foramen (CAF)
We registered the nerve transection as partial or complete according to the observation of the nerve when we removed the brain after perfusion. Immunohistochemistry The sections were processed for immunohistochemistry by using a well-characterized mouse monoclonal antiPACAP antibody (code no. Mab JHH1; Bispebjerg Hospital, Denmark; diluted 1:5; Hannibal et al. 1995) followed by a slightly modified biotinylated tyramide amplification procedure as described previously (Møller et al. 1999). Briefly, after incubation for 12–18 h in antiPACAP antibody at 4°C, the sections were washed (3×10 min) in 0.25% bovine serum albumin plus 0.1% Triton X-100 (PBS-BT), followed by incubation for 60 min at room temperature in biotinylated rabbit antimouse serum (E464, Dako, Glostrup, Denmark; diluted 1:1600). The biotinylated antiserum was further intensified by using the protocol described by Berghorn et al. (1994). The sections were washed in PBS-BT (3×5 min) and incubated for 30 min at room temperature in ABCstreptavidin horseradish peroxidase complex (Vector, Burlingame; diluted 1:150). After being washed in PBSBT (3×5 min), the sections were incubated in biotinylated tyramide (tyramide system amplification; DuPont NEN, Boston, MA; diluted 1:100 in amplification buffer), washed in PBS-BT (3×5 min), incubated in ABCstreptavidin horseradish peroxidase complex (diluted 1:150), washed in PBS-BT (2×5 min) and in 0.05 M TRIS pH 7.6 (10 min), and incubated for peroxidase activity in a solution of 0.05% 3,3’-diaminobenzidine/HCl and 0.005% H2O2 in TRIS/HCL buffer (pH 7.6) for 15 min. The reaction was terminated by washing the sections in excessive amounts of distilled water. Finally, the sections were mounted on gelatinized slides, dried, and embedded in Depex. The sections were viewed and photographed on a Zeiss Axiophot microscope. The density of PACAP-IR nerve fibers was counted in eight sagittal sections, close to the median plane, from each pineal gland. The statistical evaluation was performed by calcu-
371 Fig. 2 PACAP-immunoreactive nerve fibers in the pineal gland of the rat without (A) and with (B) ophthalmic and maxillary nerve transection. The operation resulted in the disappearance of PACAP-immunoreactive nerve fibers in the parenchyma of the superficial pineal. Bars 25 µm
lating the mean density of IR fibers for each lesioned and control animal. The group means were then compared by a Mann-Whitney unpaired one-tailed test. To test the specificity of the PACAP-antiserum, sections were routinely incubated with antibodies preabsorbed with the antigens (20 µg/ml). This treatment abolished all staining.
Results and discussion PACAP-IR nerve fibers were numerous in most of the pineal glands from the control animals (Fig. 2A); they approached the distal tip of the superficial pineal gland
via the conarian nerve. Generally, the fibers first entered the pineal capsule and then penetrated into the superficial pineal gland where they were found in both a perivascular and intraparenchymal position between the pinealocytes, an innervation pattern exhibited by other pinealopetal fibers with their origin outside the brain (Møller et al. 1991). Among the 20 operated rats, five had their ophthalmic and maxillary nerves totally transected. In the pineal glands of these rats, the PACAP-IR nerve fibers in the capsule or the parenchyma, including both the perivascular and the peripinealocytic fibers, were significantly less numerous than in the glands from control animals. The most striking disappearance of the nerve fibers occurred
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and to cause a relaxation of the contraction of the cerebral arteries (Uddman et al. 1993). The pineal vasculature originates from blood vessels located in the subarachnoidal space. It is therefore not surprising that the vascular system of the rat pineal is innervated from the trigeminal ganglion. This is supported by the finding that intravenous injection of PACAP in rabbits results in an increased blood flow in the pineal gland (Nilsson 1994). The finding that, in the present experiment, the PACAP-IR nerve fibers winding in the parenchyma between the pinealocytes disappear after transection of the ophthalmic and maxillary nerves, together with the physiological observation that PACAP is able to stimulate melatonin secretion of the pinealocytes (Simonneaux et al. 1993; Chik and Ho 1995; Yuwiler et al. 1995), supports the hypothesis that the sensory trigeminal nerve has a secretomotor function in the pineal. However, to what extent these nerves affect melatonin synthesis and secretion in the pineal must be studied in greater detail. Conclusion
Fig. 3 Significantly decreased number of PACAP-immunoreactive nerve fibers in rats with total transection of the ophthalmic (left) and maxillary (right) nerves in comparison with control animals
in the parenchyma where the nerve fibers could hardly be seen after trigeminal nerve transection (Figs. 2B, 3). We noticed no obvious change with respect to the PACAP-IR nerve fibers in the rats with partial nerve transection. This result provided evidence that mere tracheotomy and dissection in the subtemporal fossa had no significant impact on PACAP-IR nerve fibers in the pineal gland. We believe that our study has established that the PACAP-IR nerve fibers in the pineal gland of the rat have their origin in the trigeminal ganglion. In other studies (Møller et al. 1999; W. Liu et al., unpublished), the removal of the superior cervical ganglia, sphenopalatine ganglia and otic ganglia did not affect the appearance of the PACAP-IR nerve fibers, excluding these ganglia as their major origin, although all of these ganglia contain PACAP-IR perikarya. We had assumed that the PACAP-IR nerve fibers might arise from the ganglion along the ophthalmic nerve, then accompany the nasal ciliary nerve, and finally join the ethmoidal nerve to enter the ethmoidal foramen and reach the intracranial structures. However, after bilateral transection of the ethmoidal nerve of the rat, PACAP-IR nerve fibers in the pineal gland did not change (W. Liu et al., unpublished). The exact route that these nerve fibers take before reaching the pineal requires further study. Perikarya in the trigeminal ganglion containing CGRP and SP are known to innervate cerebral blood vessels (Suzuki and Hardebo 1991; Uddman et al. 1993)
Melatonin secretion of the pineal gland is regulated by multiple nervous pathways of which the sympathetic innervation from the superior cervical ganglia is considered to be functionally the most important for pineal physiology. However, the mammalian pineal also possesses a diversified peptidergic innervation from other sources. We have provided evidence that PACAP-IR neurons in the trigeminal ganglion innervate the superficial pineal gland. Acknowledgements The authors are grateful to Mrs. Ursula Rentzmann for her technical assistance.
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