Axonal tracing of autonomic nerve fibers to the superficial temporal artery in the rat

Summary The origin of nerve fibers to the superficial temporal artery of the rat was studied by retrograde tracing with the fluorescent dye True Blue (TB). Application of TB to the rat superficial temporal artery labeled perikarya in the superior cervical ganglion, the otic ganglion, the sphenopalatine ganglion, the jugular-nodose ganglionic complex, and the trigeminal ganglion. The labeled perikarya were located in ipsilateral ganglia; a few neuronal somata were, in addition, seen in contralateral ganglia. Judging from the number of labeled nerve cell bodies the majority of fibers contributing to the perivascular innervation originate from the superior cervical, sphenopalatine and trigeminal ganglia. A moderate labeling was seen in the otic ganglion, whereas only few perikarya were labeled in the jugular-nodose ganglionic complex. Furthermore, TB-labeled perikarya were examined for the presence of neuropeptides. In the superior cervical ganglion, all TB-labeled nerve cell bodies contained neuropeptide Y. In the sphenopalatine and otic ganglia, the majority of the labeled perikarya were endowed with vasoactive intestinal polypeptide. In the trigeminal ganglion, the majority of the TB-labeled nerve cell bodies displayed calcitonin gene-related peptide, while a small population of the TB-labeled neuronal elements contained, in addition, substance P. In conclusion, these findings indicate that the majority of peptide-containing nerve fibers to the superficial temporal artery originate in ipsilateral cranial ganglia; a few fibers, however, may originate in contralateral ganglia.     

The anatomy and innervation of the mammalian pineal gland

The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells. In addition, perivascular phagocytes are present. The phagocytes share antigenic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of the peptide. Nerve fibers innervating the mammalian pineal gland originate from perikarya located in the sympathetic superior cervical ganglion and the parasympathetic sphenopalatine and otic ganglia. The sympathetic nerve fibers contain norepinephrine and neuropeptide Y as neurotransmitters. The parasympathetic nerve fibers contain vasoactive intestinal peptide and peptide histidine isoleucine. Recently, neurons in the trigeminal ganglion, containing substance P, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating peptide, have been shown to project to the mammalian pineal gland. Finally, nerve fibers originating from perikarya located in the brain containing, for example, GABA, orexin, serotonin, histamine, oxytocin, and vasopressin innervate the pineal gland directly via the pineal stalk. Biochemical studies have demonstrated numerous receptors on the pinealocyte cell membrane, which are able to bind the neurotransmitters located in the pinealopetal nerve fibers. These findings indicate that the mammalian pinealocyte can be influenced by a plethora of neurotransmitters.     

The effect of the transplanted pineal gland on the sympathetic innervation of the rat sublingual gland

We investigated the effect of the pineal on sympathetic neurons that normally innervate the sublingual gland of the rat. When the pineal gland was transplanted into the sublingual gland, it remained as a distinct mass that was innervated by sympathetic axons. Injection of the retrograde tracer, Fast Blue, into the sublingual gland labelled sympathetic neurons in the ipsilateral superior cervical ganglion (SCG). Thirty per cent of all neurons labelled retrogradely by Fast Blue injection into transplanted pineal glands were immunoreactive for both neuropeptide Y (NPY) and calbindin. This combination is characteristic of sympathetic neurons innervating the pineal gland in its normal location, but not the sympathetic vasoconstrictor neurons normally innervating the sublingual gland. This, and our previous study in which the pineal gland was shown to similarly influence the phenotype of salivary secretomotor neurons, suggests that a range of different functional classes of sympathetic neuron are able to change their phenotype in response to signals released by the pineal gland.This work was supported by Project Grant No. 145634 from the National Health and Medical Research Council of Australia     

Pineal 'synaptic' ribbons in sympathectomized rats

The melatonin metabolism in the mammalian pineal gland is under the clear influence of sympathetic fibers originating in the superior cervical ganglia (SCG). Previous studies suggested that also pineal 'synaptic' ribbons (SR) are regulated by the gland's sympathetic innervation. To gain more insight into the mechanisms involved, SR numbers were determined in adult rats which were chemically sympathectomized as newborns by injection of 6-hydroxydopamine (6-OHDA). In control animals (saline injections), a clear day/night difference in the number of SR is present. In sympathectomized animals, SR are higher in number throughout the 24-hour cycle but are not significantly elevated at night. The present results further strengthen the hypothesis that SR are involved in intercellular communication in the mammalian pineal gland.     

Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglia

Summary The distribution of tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive(IR) nerve fibers in the pineal complex was investigated in untreated rats and rats following bilateral removal of the superior cervical ganglia. In normal animals, a large number of TH- and NPY-IR nerve fibers were present in the pineal capsule, the perivascular spaces, and intraparenchymally between the pinealocytes throughout the superficial pineal and deep pineal gland. A small number of TH-IR and NPY-IR nerve fibers were found in the posterior and habenular commissures, a few fibers penetrating from the commissures into the deep pineal gland. To elucidate the origin of these fibers, the superior cervical ganglion was removed bilaterally in 10 animals, and the pineal complex was examined immunohistochemically. Two weeks after the ganglionectomy, the TH-IR and NPY-IR nerve fibers in the superficial pineal gland had almost completely disappeared. On the other hand, in the deep pineal and the pineal stalk, the TH-IR and NPY-IR fibers were still present after ganglionectomy. These data show that the deep pineal gland and the pineal stalk possess an extrasympathetic innervation by TH-IR and NPY-IR fibers. It is suggested that the extrasympathetic TH-IR and NPY-IR nerve fibers innervating the deep pineal and the pineal stalk originate from the brain.     

Ultrastructural changes in the rat pineal gland after sympathetic denervation. Quantitative study

Ultrastructural changes in the rat pineal gland were studied quantitatively 7 and 60 days after the sympathetic denervation by bilateral excission or decentralization of superior cervical ganglia. The surface occupied by pineal parenchymal cells decreased in rats of experimental groups with respect to the control group. Furthermore, profile areas of the cytoplasm, nucleus and nucleolus of the pinealocytes were also diminished. Cytoplasmic lipid droplets in the pinealocytes were markedly decreased in number and size in experimental rats. As demonstrated by the Kruskal-Wallis H test, statistically significant differences were found between rats of the control and operated groups. Rats treated by superior cervical ganglionectomy or decentralization showed morphological changes indicating a hypofunctional pineal gland, although differences were found between both groups.     

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

Summary In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.     

Principal neurons projecting to the pineal gland in close association with small intensely fluorescent cells in the superior cervical ganglion of rats

Summary The localization in the superior cervical ganglia (SCG) of small, intensely fluorescent (SIF) cells and of principal nerve (PN) cells innervating the pineal gland was examined in adult male Sprague-Dawley rats. PN cells were demonstrated by means of the retrograde neuron-tracing method using the fluorescent tracer Fluoro-Gold (FG) injected into the pineal gland. SIF cells were visualized by the formaldehyde-induced fluorescence method. Twentynine percent of the FG-labeled PN cells were found closely associated with SIF cells. In the rostral half of the ganglion, 43% of the SIF cells were situated in juxtaposition to one or several labeled neurons. The possible influence of SIF cells on the regulation of pineal metabolism is discussed with respect to their role as both local endocrine cells and interneurons.     

Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

Pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres were demonstrated in the rat pineal gland. These fibres entered the pineal gland through the conarian nerve at the distal tip of the gland. A high density of the fibres was observed in the capsule of the gland, from where the immunoreactive elements penetrated into the pineal perivascular spaces and parenchyma. The majority of PACAP-immunoreactive nerve fibres also contained calcitonin gene-related peptide (CGRP). Some PACAP-immunoreactive nerve fibres contained neuropeptide Y (NPY), but only occasionally was PACAP colocalized with vasoactive intestinal peptide (VIP). After removal of both superior cervical ganglia, a high number of PACAP-containing nerve fibres were still present in the gland. In the nervous system PACAP is present in two isoforms, PACAP-38 and PACAP-27. The concentration of PACAP-38 in the superficial pineal gland was determined by radioimmunoassay to be 20.4 pmol/g tissue at midday and 18.9 pmol/g tissue at midnight. The concentration of PACAP-27 was only about 3% of the concentration of PACAP-38. In summary, this study is the first demonstration of a PACAP-containing innervation of the rat pineal gland. The PACAP concentration in the pineal gland does not exhibit a day-night difference. The colocalization of PACAP with calcitonin gene-related peptide in the pincalopetal nerve fibres indicates that the majority of PACAP-immunoreactive nerve fibres might originate from the trigeminal ganglion.     

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP- immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.Key words: trigeminal ganglion, otic ganglion, superior cervical ganglion, arthritis, temporomandibular joint.     

Innervation of the superficial pineal of the rat using retrograde tracing methods

Fast blue (FB), rhodamine microspheres (RH), horseradish peroxidase (HRP), and wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were used as retrograde tracers to study the innervation of the rat superficial pineal gland (SP). One of the tracers was injected into the gland of each animal. All four retrograde tracers injected into the gland always labeled neurons in the superior cervical ganglia (SCG). No retrograde labeling was ever seen in the suprachiasmatic nuclei, paraventricular hypothalamic nuclei, lateral hypothalamus, habenular nuclei, amygdalar nuclei, or superior salivatory nuclei. Retrograde labeling was seen in the anterior hypothalamic nuclei, anterior thalamic nuclei, lateral geniculate bodies, and midbrain tectal structures when a tracer spread from the injection site to the overlying cortex, tectum, or commissures. Control studies included injection of tracer into the subarachnoid space around the SP or into structures adjacent to the SP. Only the injection of FB or WGA-HRP into the subarachnoid space labeled neurons in the SCG. This labeling was probably due to the spread of tracer to the choroid plexus. These results agree with recent work confirming the existence of a direct projection of the SCG into the interstitium around pinealocytes. The evidence does not substantiate an innervation originating in the habenular nuclei; the superior salivatory nuclei; or any other diencephalic, midbrain, pontine, or medullary structure.     

Central innervation of the pineal organ of the Mongolian gerbil

Nerve fibers connecting the brain with the pineal gland of the Mongolian gerbil (central pinealopetal fibers) were investigated by means of light and electron microscopy. Several myelinated fibers penetrate from the brain into the deep pineal gland, extend further into the pineal stalk and continue to the superficial portion of the pineal gland. In the centripetal direction these fibers were traced to the stria medullaris and to the habenular nuclei, where they turned laterad and then occupied a position immediately ventral to the optic tract. As shown in electron micrographs, lesions of the habenular area led to degeneration of myelinated fibers and nerve boutons in the deep pineal gland, the pineal stalk and the superficial pineal gland. Only boutons containing clear transmitter vesicles (devoid of a dense core) were observed to degenerate after the habenular lesions. On the other hand, removal of the superior cervical ganglia resulted in degeneration of boutons containing small (40 to 60 nm in diameter) dense-core vesicles. Several of the nerve fibers that penetrate into the deep pineal directly from the brain (central fibers) exhibited a positive reaction for acetylcholinesterase (AChE). AChE-positive perikarya were located in the projections of the stria medullaris, the lateral portions of the deep pineal, the area of the posterior commissure, and the periventricular gray of the mesencephalon. Such perikarya were found neither in the pineal stalk nor in the superficial pineal gland. These results present anatomical evidence that the pineal organ of the Mongolian gerbil receives multiple nervous inputs mediated by peripheral autonomic (i.e., sympathetic) nerve fibers, on the one hand, and by central fibers, on the other.     

Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.     

Alterations in γ-aminobutyric acid content in the rat superior cervical ganglion and pineal gland

The endogenous γ-aminobutyric acid (GABA) content of the rat pineal gland and superior cervical ganglion (SCG) was measured by high pressure liquid chromatography. It was found that GABA levels in both tissues increased after decapitation of the animals. The GABA content of tissues frozen within 20 seconds after decapitation was the same as that of tissues removed from animals killed by microwave irradiation. Amino-oxyacetic acid, a GABA-transaminase inhibitor, increased the endogenous GABA content of both of these tissues. Dimethylphenylpiperizinium or isoniazid administration did not alter GABA levels in these tissues. Isoproterenol increased the GABA content of the SCG but did not change the pineal gland GABA levels. The ability of the pineal gland to take up and accumulate ³H-GABA was significantly reduced in rats that had been ganglionectomized. A fluctuation in endogenous GABA levels in the pineal gland was seen to occur when measures were taken at different times of the day. These results tend to suggest that GABA may have some functional role in the pineal gland and the superior cervical ganglion.     

Structure and innervation of the pineal gland of the rabbit,Oryctolagus cuniculus (L.)

In the rabbit pineal gland two types of postganglionic nerve endings were found which are characterized by the presence of small dense-core vesicles or small clear vesicles. Pharmacological and cytochemical experiments showed then to be noradrenergic and cholinergic, respectively. Both types were often present in the same nerve bundle, occasionally in close opposition. Intrapineal neurons were only rarely observed. They showed cholinergic synapses on their perikaryon and dendrites as well as noradrenergic axo-dendritic close contacts. Bilateral extirpation of the superior cervical ganglia revealed the postganglionic sympathetic origin of the pineal noradrenergic nerve fibres. Moreover, it appeared that these ganglia are hardly, if at all, involved in the pathway of pineal cholinergic innervation. The results obtained from lesions of both facial nerves, taken together with the results reported in the literature, led to the conclusion that the postganglionic cholinergic nerve fibers in the pineal are of parasympathetic origin. A model for the sympathetic and parasympathetic pineal innervation is proposed.     

Effect of superior cervical ganglionectomy on monoamine content in the epithalamic area of the Mongolian gerbil (Meriones unguiculatus): A fluorescence histochemical study

Summary Extirpation of the superior cervical ganglion was performed in a series of Mongolian gerbils. One or two weeks after the ganglionectomy the animals were injected with a monoamine oxidase inhibitor. Subsequently perfusion fixation was performed using the glyoxylic acid-paraformal-dehydemagnesium method (Lorén et al., 1976) for fluorescence histochemical investigation of the monoamines of the pineal complex. In the ganglionectomized animals all of the blue-fluorescent sympathetic fibers in the pineal complex (superficial pineal gland, deep pineal gland and the pineal stalk) completely disappeared. The yellow indolamine fluorescence of the cells in the superficial pineal and the deep pineal, as well as in the pineal stalk, was markedly reduced after ganglionectomy. No change in the morphology or number of sympathetic fibers in the medial habenular nucleus was observed. These results indicate that the presence of sympathetic nerve fibers with perikarya in the superior cervical ganglion is necessary for maintaining a high indolamine content in all three parts of the pineal complex. In addition, the results also indicate that the deep pineal gland is a functional part of the pineal complex. The presence of a functionally active deep pineal, bordering the pineal recess, suggests that part of the pineal hormones might be secreted into the cerebrospinal fluid.This work was supported by the Carlsberg Foundation, the Swedish Natural Science Research Council, grant no. 2126-100, and the Danish Medical Research Council, grant no. 512-7134     

Indications for the presence of two populations of serotonin-containing pinealocytes in the pineal complex of the golden hamster (Mesocricetus auratus)

Summary Serotonin-like immunoreactivity was investigated in the pineal complex of the golden hamster by use of the indirect immunohistochemical technique. The superficial and deep portions of the pineal gland, and also the pineal stalk exhibited an intense cellular immunoreaction for serotonin. In addition, perivascular serotonin-immunoreactive nerve fibers were observed. Some serotonin-immunoreactive processes of the pinealocytes terminated on the surface of the ventricular lumen in the pineal and suprapineal recesses, indicating a receptive or secretory function of these cells. Several serotonin-immunoreactive processes connected the deep pineal with the habenular area. One week after bilateral removal of both superior cervical ganglia the serotonin immunoreaction of the entire pineal complex was greatly decreased. However, some cells in the pineal complex, of which several exhibited a neuron-like morphology, remained intensively stained after ganglionectomy. This indicates that the indoleamine content of some cells in the pineal complex of the golden hamster is independent of the sympathetic innervation.Supported by a Grant from the Italian Society for Veterinary Sciences     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.