The presence of vasoactive intestinal polypeptide (VIP) - Springer Link

Cell Tissue Res (1985) 241:333 340

a n d Tissue ReseatT_h 9 Springer-Verlag1985

The presence of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerve fibres and VIP-receptors in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) An immunohistochemical and receptor-autoradiographic study* M. Moiler*, J.D. Mikkelsen*, J. Fahrenkrug**, and H.-W. Korf*** * Institute of Medical Anatomy, Department B, University of Copenhagen; ** Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark; *** Department of Anatomy and Cytobiology, Justus Liebig University, Giessen, Federal Republic of Germany

Summary. By use of the indirect peroxidase-antiperoxidase immunohistochemical technique, nerve terminals exhibiting vasoactive intestinal polypeptide (VIP)-like-immunoreactivity were demonstrated in the pineal gland of the Mongolian gerbil ( Meriones unguiculatus). Incubation of the superficial pineal gland of the gerbil with 60 pM of 125I-VIP showed that the gland exhibited saturation kinetics, and about 80% of the bound ~2sI-VIP could be displaced by adding a surplus of cold VIP. Incubation of unfixed, 0.1% and 4% paraformaldehyde-fixed cryostat sections of the gerbil forebrain with 125I-VIP also exhibited saturation kinetics, and displacement was possible by adding a surplus of the cold tracer. Receptor autoradiography on cryostat sections that had been incubated for 60 rain with ~2sI-VIP showed a large number of grains over cortical areas, especially over the pyramidal layer of the hippocampus and the granular layer of the dentate gyrus. A prominent labelling of the pineal gland was also observed. The presence of VIP-like-immunoreactive nerve terminals and receptors for this molecule in the pineal gland of the Mongolian gerbil supports the biochemical studies demonstrating a stimulatory function of this molecule in the synthesis of melatonin. Key words: Pineal organ (Meriones unguiculatus) - Vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibres - VIP receptors - Autoradiography

The pineal complex of the Mongolian gerbil is richly innervated by sympathetic fibres (Nielsen and Moller 1978) the perikarya of which are located in the superior cervical ganglia (Moiler et al. 1979). In the pineal complex of the gerbil these sympathetic fibres are found both perivascularly and Send offprint requests to: Morten Moiler, M.D., Institute of Medi-

cal Anatomy, Department B, University of Copenhagen, The Panum Institute, 3, Blegdamsvej, DK-2200 Copenhagen N, Denmark * Supported in part by grants from the Deutsche Forschungsgemeinschaft to H.-W.K. (Ko 758/2; 2-3) and the Carlsberg Foundation

intraparenchymally, between the pinealocytes (Moller and van Veen 1981). In addition, lesion experiments followed by electron microscopy have, in the gerbil, shown a prominent central innervation originating in the brain (Moller and K o r f 1983 a). By means of the retrograde horseradish peroxidase technique (Mesulam 1982) the perikarya of these central fibres have been demonstrated in the medial and lateral habenular nuclei, the nucleus of the posterior commissure, the dorsal nucleus of the lateral geniculate body, and in the paraventricular nucleus (Moller and K o r f 1983 b). Electron-microscopic studies have shown that the nerve terminals of these central fibres differ with respect to ultrastructural features (Korf and Moller 1985). Some of the terminals contain numerous large (100-200nm in diameter), granular vesicles, indicating the peptidergic character of these terminals. Such peptidergic pinealopetal nerve fibres occur in several mammalian species (cf. K o r f and Moller 1984). In the present investigation we have shown the occurrence of the octacosapeptide, vasoactive intestinal polypeptide (VIP), by means of radioimmunoassay in extracts of the superficial pineal of the Mongolian gerbil; furthermore, we have located its presence to nerve endings in the gland by use of immunohistochemistry. In addition, we have demonstrated the binding of 125I-VIP to the superficial pineal gland of the gerbil. This binding exhibited saturation kinetics and could be displaced by incubation with a solution containing a surplus of non-labelled VIP. By use of autoradiography on lightly fixed cryostat sections the VIP-receptor was found to be located predominantly on the cell membrane of the pinealocytes. Materials and methods Chemica&

Synthetic porcine VIP was obtained from Peninsula Labs., San Carlos, California, and labelled with 125I by use of the chloramine-T method to a specific radioactivity of 800-1000 gCi/nmol (Fahrenkrug et al. 1977). Human serum albumin was obtained from Behringwerke, Marburg/ Lahn, F.R.G. Specific rabbit antibodies against porcine

334 VIP were provided by Dr. Fahrenkrug (Dept. of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark), lot No. 5600-6 and 5603. Swine anti-rabbit IgG (code No. Z196), and soluble complexes of horseradish peroxidase anti-horseradish peroxidase (PAP) (code No. Zl13) were obtained from Dakopatts, Copenhagen.

Animals One hundred and forty-two Mongolian gerbils of both sexes, weighing between 50-80 g, were used in this investigation. The animals were kept under a photoperiod 12L/12D, with food and water ad libitum.

Measurement of VIP by radioimmunoassay The concentrations of endogenous immunoreactive VIP in the fronto-parietal cortex, the superficial pineal gland, and the cerebellum were measured by radioimmunoassay after extraction (Fahrenkrug et al. 1977, 1978) and expressed in pmol per g tissue (wet weight).

Immunohistochemistry Bouin-Hollande fixation. Gerbils were anaesthetized with tribromethanol (1 ml of a 4% solution/100 g animal) i.p. and perfused through the left ventricle of the heart: firstly, with 500 ml phosphate-buffered saline (PBS), pH 7.4, containing 15 000 IU heparine/1, followed by 500 ml 4% paraformaldehyde in 0.| M phosphate buffer, pH 7.4, for 15 min. Both perfusion fluids were equilibrated at room temperature (20 ~ C) before use. The brains were then immediately removed from the skull and postfixed for 48 h at 4 ~ in Bouin-Hollande solution (Romeis 1968), without acetic acid but to which 6% sublimate had been added. After fixation the brains were dehydrated in a graded alcohol series and embedded in paraffin. Ten-gm thick sections were cut on a serial microtome and placed on gelatinized glass slides. The sublimate crystals were removed by treating the rehydrated sections with Lugol solution (Romeis 1968) for 5 min followed by 5 min in a 0.25% thiosulphate solution.

Paraformaldehyde fixation. After tribromethanol anaesthezia, the gerbils were perfused through the left ventricle of the heart, first with 500 ml of the PBS-heparine solution followed by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (500 ml) for 15 min at room temperature. The brains were postfixed for 2 days in the same fixative at 4 ~ C followed by infiltration with a 10% sucrose solution in the same buffer for 24 h at 4 ~ C. Ten-gm thick sections were cut on a cryostat at - 2 0 ~ C and placed on gelatinized glass slides. Immunohistochemical reactions. The sections were rinsed 2 x 10 min in PBS (pH 8.0), followed by incubation in 10% swine serum with 0.3% Triton X-100 (Sigma) added for 30 min at room temperature. This was followed by incubation for 48 h at 4~ with the specific antiserum against VIP diluted 1/400 in PBS (pH 8.0) with 0.3% Triton X-100 added. The sections were then washed 3 x 10 min in PBS with 1% Triton X-100 (pH 7.4), and incubated for 1 h in swine anti-rabbit IgG diluted 1/20 in PBS with 0.3% Triton X-100 added at room temperature. After washing for

3 x 10 min in PBS with 1% Triton X-100 (pH 7.4), the sections were finally incubated for 1 h at room temperature in PAP diluted 1/80 in PBS, pH 7.4, with 0.3% Triton X- 100 added. After 2 x 10 min washing in PBS, pH 7.4, the sections were incubated for peroxidase activity in a solution of 0.025% diaminobenzidine-tetrahydrochloride and 0.003% HzO z dissolved in Tris/HC1 buffer, pH 7.6, for about 10 min. After washing in distilled water, the sections were dehydrated in a series of alcohols and embedded in Depex |

Receptor binding of 125i_VIP Biochemical studies. The animals were anaesthetized with ether and decapitated. The superficial pineal glands were immediately removed under a stereomicroscope, leaving the stalk and deep pineal attached to the brain. The superficial pineals were then incubated for different times in 25 mM Tris/HC1 buffer, pH 7.4, containing 2 mM MgC12, 1 mM mercaptoethanol and 10 g/1 human serum albumin, to which 125I-VIP was added at a final concentration of 60 pM (" total" binding). "Unspecific" binding was demonstrated by adding 1 gM unlabelled VIP to the above-described medium. After incubation the pineals were washed 3 x 5 min in the incubation medium without l zsI-VIP; the bound radioactivity was counted in a well-type gamma counter (LKB Wallac Rackgamma). 125I-VIP-binding to cryostat sections. The animals were anaesthetized with ether and decapitated. The brains were immediately removed and frozen on a CO2-expansion cooler. Ten-lam thick cryostat sections were cut at 8-12~ C on a cryostat and placed on gelatinized slides. Some of the sections were then fixed for 10 min in a solution of 0.1 M phosphate buffer, pH 7.4, containing either 0.1% or 4% paraformaldehyde as the fixing agent at room temperature. After 3 x 10 rain washing in phosphate buffer, the sections were incubated for "total" or "unspecific" binding with 125I-VIP as described above. Following incubations, the sections were washed 3 x 10 min with PBS at 4 ~ C, removed from the slides with a razor blade, placed in a counting tube, and the radioactivity counted in a gamma-counter. Receptor autoradiography Unfixed, 0.!% or 4.0% paraformaldehyde-fixed cryostat sections were incubated for "total" or "unspecific" binding for 60 min and after washing in PBS at 4 ~ C dried for several days and then dipped in an Ilford K-2 emulsion, diluted 1/1 with distilled water. Other 125I-VIP-incubated sections were covered with K-2 emulsion-coated coverslips, which were glued to the slides (Young and Kuhar 1979). After exposure times varying from 1 to 14 days, these autoradiographs were developed in Amidol and fixed in thiosulphate. Some of the sections were counterstained with 0.1% cresyl violet before dehydration and mounting in Depex|

Photograph), The immunohistochemical sections were photographed in transmitted light. DAB-interference filters (Moller et al. 1984) were used to increase contrast between the brownstained neurons and the background. The autoradiographs were photographed in transmitted light as well as in the darkfield mode.

335

Fig. 1. VIP-like-immunoreactive, perivascularly located boutons (arrows)in the superficial pineal of the Mongolian gerbil. • 1000 Fig. 2. VIP-like-immunoreactive boutons (arrows) located intraparenchymally between the pinealocytes of the superficial pineal gland

(CP) of the gerbil; Sa subarachnoidal space, x 1000

Table 1. Concentration of immunoreactive VIP in the gerbil brain

Cerebral cortex Pineal gland Cerebellum

52.6 pmol/g 14.0 pmol/g Not detectable

between the pinealocytes. The terminal diameters varied from 0.5 to 2 ~tm. VIP-immunoreactive fibres were also observed in the projections of the stria medullaris towards the deep pineal.

125i_VIP binding to entire pineals

The concentration of immunoreactive VIP in the superficial pineal, the fronto-parietal cortex and the cerebellum of the gerbil is given in Table 1.

The superficial pineal glands were incubated with lzsI-VIP in toto due to the small amount of tissue constituting this gland (0.3 mg/gland). As shown in Fig. 3 the binding was saturated after incubation for 60 min. Further incubation decreased the amount of ~zsI-VIP bound to the tissue. About 80% of the bound ~zsI-VIP could be displaced by addition of a surplus of cold VIP to the incubation solution.

Immunohistochemistry

m~I-VIP binding to unfixed and fixed cryostat sections

VIP-like-immunoreactive nerve fibres could be demonstrated in the gerbil brain after both 4% paraformaldehyde fixation and Bouin-Hollande fixation. However, the 4% paraformaldehyde fixative was superior with respect to number of immunoreactive terminals and perikarya. VIP-immunoreactive fibres were observed in the superficial pineal gland (Figs. 1, 2), the deep pineal gland, and the stalk. The terminals were predominantly located perivascularly (Fig. 1) and rarely intraparenchymally (Fig. 2)

As shown in Fig. 4 the unfixed cryostat sections of the gerbil forebrain were able to bind lzsI-VIP (total-binding). The binding was saturated after about 60 min and decreased slowly with further incubation. About 60% of the total 125I-VIP binding could be displaced by addition of a surplus of cold VIP. The "specific" binding was defined as " t o t a l " binding minus "unspecific" binding (the binding observed when surplus of cold VIP had been added). The "specific" binding increased after the sections had been

Results

Measurements of VIP by radioimmunoassay

336 cpm

cpm 3O

I

400

i

L

J j_ ~ 1 ~ -

~-JJ~

/ 15

/

i

/ I

200

I

Q

j~

20

e0

40

80

/

//

mill

30

Fig. 3, Time course of binding of 60 pmol ~2sI-VIP to pineal glands in toto (pH 7.4, 20~ C) in presence of 1 pmol unlabelled VIP ( e - - e , unspecific binding) and absence of unlabelled VIP ( o - - o , total binding). Values are the mean + S.D.

e0

min

Fig. 4. Time course of binding of 60pmol '25I-VIP to unfixed sections of gerbil forebrain (pH 7.4, 20~ C) in presence of 1 ~umol unlabelled VIP ( o - - o , unspecific binding) and absence of unlabelied VIP ( o - - o , total binding). Values are the mean ___ S.D.

cpm

i

i

1000

500

3'o

60

' '

30

60

3'0

fixed with 0.1% paraformaldehyde for 10 min prior to 12sIVIP i n c u b a t i o n and even more after fixation with 4% paraformaldehyde (Fig. 5).

Receptor autoradiography A dense labelling was observed above the granular layer of the dentate gyrus (Fig. 6) and the pyramidal layer of the hippocampus (Fig. 7). The grains were predominantly located above the neuronal cell membranes. A dense labelling was also observed above the pineal gland (Fig. 10), with a tendency to be located in relation to the cell membranes of the pinealocytes (Figs. 8, 9). The best structural preservation and grain localization was obtained on autoradiographs from brain tissue fixed with 4% paraformaldehyde prior to 125I-VIP incubation

60

min

Fig. 5. Time course of binding of 60 pmol azsI-VIP to unfixed (left curve), 0.1% paraformaldehydefixed (middle curve) and 4% paraformaldehydefixed (right curve) sections of gerbil forebrain. The fixation times were 10 min at 20~ C. The sections were incubated in a solution containing 60 pmol 125I-VIP (pH 7.4, 20~ C) in the presence of 1 pmol unlabelled VIP (e, o, unspecific binding) or in absence of unlabelled VIP ( o - - o , total binding). Values are the mean _ S.D.

(Fig. 10). Grain counts performed on paraformaldehydefixed autoradiographs, which had been exposed for 8 days, are shown in Fig. 11. The density of specific grains over the superficial pineal gland is seen to be a factor 1.6 higher than the density above the cingulate cortex.

Discussion Vasoactive intestinal polypeptide (VIP) is an octacosapeptide present in many nerve fibres located in the peripheral as well as in the central nervous system. Within the brain, VIP is predominantly located in allo- and neocortex, diencephalon, and to a minor degree in the brainstem, but has up to date not been detected in the cerebellum. VIP-immunoreactive nerve fibres have earlier been described in the pineal gland of the rabbit, pig, and cat (Udd-

Fig. 6. Dark-field photomicrograph of part of a frontal section that was incubated with 125I_VIP for 60 min. Heavy labelling of granular layer of the dentate gyrus (arrows) is seen. Exposure time : 4 days. x 75

Fig. 7. Dark-field photomicrograph of part of a frontal section that was incubated with 125I-VIP for 60 min. The grains are located over neurons in the pyramidal layer (p/) of the hippocampus. A neurone (n) in the stratum oriens is labelled. Exposure time: 4 days. • 570

337

Fig. 8. Dark-field photomicrograph of part of a section of the superficial pineal gland incubated with lzsI-VIP for 60 min; a acervulus. Exposure time: 4 days. • 970

Fig. 9. 125I-VIP receptor autoradiograph weakly counterstained with 0.1% cresyl violet of the same superficial pineal gland as shown in Fig. 9. In the transmitted light many grains are seen to be located over the pinealocytes, apparently their plasma membranes (c). Exposure time: 4 days. x 970

338

Fig. 10. Dark-field photomicrograph of a receptor autoradiograph exposed for eight days. Heavy labelling is seen over the superficial pineal gland (CP) and cingulate cortex (cortex). A smaller number of grains is located over the superficial part of the superior colliculus (Col. sup.). The coarse light-reflecting structures in the pineal are acervuli. Frontal section. Exposure time: 14 days. • 50

1

2

3

4

5

6

Fig. 11. Histogram showing grain counts over three areas of 12SlVIP receptor autoradiographs exposed for eight days (cf. Fig. 10). Columns 1-3 exhibit counts obtained from unfixed sections and columns 4-6 from sections fixed in 4% paraformaldehyde for 10 min. The superficial pineals (columns 3 and 6) showed a larger number of grains than the cingulate cortex (columns 2 and 5). The grain counts over the white matter (crus cerebri) are shown in columns 1 and 4. Abscissa represents the number of grains per 500 lam2, bars the standard deviations of the counts man et al. 1980b; Moller et al. 1981). In these animals the localization was mainly perivascular in accord with our observation in the gerbil. Immunohistochemical studies performed recently in our laboratory on the gerbil brain have shown VIP to be located in the perikarya of the suprachiasmatic and paraventricular nucleus of the hypothalamus. By use of the H R P neurone-tracing technique, we have, in the gerbil, provided evidence for a direct projection from the paraventricular nucleus to the superficial pineal gland (Meller and K o r f 1983b). Therefore, the paraventricular

nucleus might contain the perikarya from which the fibres exhibiting VIP-like immunoreactivity originate, thereby being part of the central innervation of the gerbil pineal (Korf and Moller 1984). The superior cervical ganglion as the origin of the VIPpositive fibres cannot be totally excluded. VIP-positive perikarya have been described in the sympathetic trunk (Lundberg et al. 1982) including the superior cervical ganglion of the cat and rat (H6kfelt et al. 1982). However, the VIPpositive fibres innervating the blood vessels of the choroid plexus of the cat do not originate from this site since removal of both superior cervical ganglia does not lead to degeneration of these fibres (Larsson et al. 1976). VIP-positive perikarya have also been found in parasympathetic ganglia, e.g., the pterogopalatine ganglion (Uddman et al. 1980a). However, a parasympathetic innervation of the gerbil pineal has never been documented. Recently, we obtained ultrastructural support for the presence of peptidergic fibres in the pineal complex of the Mongolian gerbil (Korf and Moiler 1984). By transmission electron microscopy, terminals were observed which, in addition to small (40-60 nm in diameter) electron-lucent transmitter vesicles, contained numerous large granular vesicles (diameter 100-200 nm). It is possible that VIP might be stored in such granular elements. This is supported by the fact that ultracentrifugation studies of rat brain have recovered VIP from the crude mitochondrial fraction, which contains synaptosomes (Besson et al. 1979). In addition, immunocytochemical studies on the peripheral (Larsson 1977; Johansson and Lundberg 1981; Probert et al. 1981) and central nervous system (Pelletier et al. 1981) have shown that VIP is located in 100-nm granular vesicles.

339 The measurements o f immunoreactive VIP in the gerbil pineal conform to the immunocytochemical findings. The a m o u n t measured in the fronto-parietal cortex o f the gerbil is smaller than the a m o u n t measured from the same cortical areas in other rodents (Lor6n et al. 1979) but greater than that measured in the pig ( F a h r e n k r u g and De Muckadell 1978). The a m o u n t o f V I P in the superficial pineal of the gerbil is m o d e r a t e but in accord with the n u m b e r o f fibres observed by immunohistochemistry a n d the n u m b e r of " p e p t i d e r g i c " terminals seen by electron microscopy. Unfixed entire pineals were selected for kinetic lzsI-VIP binding studies. The paucity o f tissue (about 0.3 m g / p e r gland) makes such studies impossible on 10-gm thick cryostat sections o f the gland. The superficial pineal gland o f the M o n g o l i a n gerbil is well suited for incubation studies in toto. The small size o f the gland ( l . 5 x 0 . 6 x 0 . 3 mm) and the large perivascular spaces facilitate penetration by diffusion in c o m p a r i s o n to normal brain tissue. W i t h a diffusion coefficient o f 10 - 6 cm2/sec and by the use of" the equation M = 2]/~-t, the concentration of V I P in the centre o f the gland will be half o f the concentration in the incubation m e d i u m after 5 min. The binding was performed at 20 ~ C to reduce the degradation o f the b o u n d lzsI-VIP observed at higher temperatures (Ottesen et al. 1982; A m i n a r off and Rosselin 1982). The pineal showed a binding kinetic with saturation after a b o u t 60 to 80 min o f incubation. This is in accord with binding studies on rat brain synaptosomes (Staun-Olsen et al. 1982) and crude s m o o t h muscle membranes from the m y o m e t r i u m o f the cat (Ottesen et al. 1982). I n c u b a t i o n o f non-fixed cryostat sections o f the gerbil forebrain also showed binding o f azsI-VIP to the sections that were saturated after incubation for 60-80 rain. A b o u t 60% o f the b o u n d 125I-VIP could be displaced by adding a surplus o f non-labelled VIP. It was o f interest that the specific binding o f these cryostat sections increased after 0.1% p a r a f o r m a l d e h y d e fixation and even more after fixation with 4% paraformaldehyde. This is possibly due to retention o f the receptor molecule, located in the cell membrane, by cross-linking the receptor with structural proteins in the m e m b r a n e itself without destroying the receptor p r o p e r t y o f the molecule. Such a function o f the fixative is k n o w n in enzyme histochemistry, where several enzymes located in the cell m e m b r a n e can only be detected after a mild fixation (H~yer and Moller 1977). These results are in discordance with earlier studies showing that fixation o f tissue, especially with glutaraldehyde, destroys the receptor binding o f tissues (Young and K u h a r 1979). The receptor a u t o r a d i o g r a m s o f the gerbil forebrain showed the grains to be located p r e d o m i n a n t l y above the cell m e m b r a n e s o f neurones and the pinealocytes. This was easily detected in the a u t o r a d i o g r a p h s developed after very short exposure times. The a m o u n t o f grains was greatest over the p y r a m i d a l cells o f the h i p p o c a m p u s and the granular cells o f the dentate gyrus. A n i m p o r t a n t question is whether VIP plays a functional role in the pineal complex o f the M o n g o l i a n gerbil. V I P has been shown to act as a neurotransmitter depolarising neurones in the cerebral cortex, hippocampus, and spinal cord (Phillis 1982). In addition, VIP can stimulate several neuroendocrine responses in the intermediate and neural lobe o f the pituitary (Besson et al. 1982). In the pineal, V I P has been shown to increase cyclic A M P a n d serotonin-N-acetyltransferase activity ( K a n e k o et al. 1980; Yuwiler 1983). It is o f considerable interest that

the latter response is mediated via a non-beta-adrenergic receptor. This strongly indicates that V I P in the gerbil pineal is a neurotransmitter, located in an extrasympathetic nervous system, which is able to stimulate melatonin synthesis. References Aminaroff B, Rosseling (3 (1982) VIP receptors and control of cyclic AMP production. In: Said SI (ed) Vasoactive intestinal peptide. Advances in peptide hormone research series. Raven Press, New York, pp 307-322 Besson J, Rotsztejn WH, Bataille D (1982) Involvement of VIP in neuroendocrine functions. In : Said Sl (ed) Vasoactive intestinal peptide. Advances in peptide hormone research. Raven Press, New York, pp 253-262 Besson J, Rotsztejn W, Laburthe M, Epelbaum J, Beaudet A, Kordon C, Rosselin G (1979) Vasoactive intestinal peptide (VIP): Brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats. Brain Res 165:79-85 Fahrenkrug J, Schaffalitzky de Muckadell OB, Fahrenkrug A (1977) Vasoactive intestinal polypeptide (VIP) in human cerebrospinal fluid. Brain Res 124:581-584 Fahrenkrug J, Schaffalitzky de Muckadell OB (1978) Distribution of vasoactive intestinal polypeptide (VIP) in the porcine central nervous system. J Neurochem 31:1445-1451 H6kfelt T, Schultzberg M, Lundberg M, Fuxe K, Mutt V, Fahrenkrug J, Said SI (1982) Distribution of vasoactive intestinal polypeptide in the central and peripheral nervous system as revealed by immunocytochemistry. In: Said SI (ed) Vasoactive intestinal peptide. Advances in peptide hormone research. Raven Press, New York, pp 65-90 Hoyer PE, Moller M (1977) Histochemistry of 11 fl-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation. Histochem J 9 : 599-618 Johansson O, Lundberg JM (1981) Ultrastructural localization of VIP-like immunoreactivity in large dense-core vesicles of'cholinergic-type' nerve terminals in cat exocrine glands. Neuroscience 6 : 847-862 Kaneko T, Cheng P-Y, Oka H, Oda T, Yanaihara N, Yanaihara C (1980) Vasoactive intestinal polypeptide stimulates adenylate cyclase and serotonin N-acetyltransferase activities in rat pineal in vitro. Biochem Res 1 : 84-87 Korf H-W, Moiler M (1984) The innervation of the mammalian pineal gland with special reference to central pinealopetal projections. Pin Res Rev 2:41-86 Korf H-W, Moller M (1985) The central innervation of the mammalian pineal organ. Elsevier/North Holland (in press) Larsson L-I (1977) Ultrastructural localization of a new neuronal peptide (VIP). Histochemistry 54 : 173-176 Larsson L-I, Edvinsson L, Fahrenkrug J, H~kanson R, Owman CH, Schaffalitzky de Muckadell OB, Sundler F (1976) Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves. Brain R e s t 13:400-404 Lor+n I, Emson PC, Fahrenkrug J, Bj6rklund A, Alumets J, Hgtkanson R, Sundler F (1979) Distribution of vasoactive intestinal polypeptide in the rat and mouse brain. Neuroscience 4:1953-1976 Lundberg JM, AnggArd A, Fahrenkrug J, Johansson O, H6kfelt T (1982) Vasoactive intestinal polypeptide in cholinergic neurons of exocrine gland. In: Said SI (ed) Vasoactive intestinal peptide. Advances in peptide hormone research series. Raven Press, New York, pp 373-389 Mesulam M-M (1982) Tracing neural connections with horseradish peroxidase. John Wiely & Sons, Chichester, New York, Toronto, Singapore Moiler M, Korf H-W (1983a) Central innervation of the pineal organ of the Mongolian gerbil. Cell Tissue Res 230:259-272 Moller M, Korf H-W (1983b) The origin of central pinealopetal nerve fibres in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. Cell Tissue Res 230 : 273-287

340 Moller M, van Veen Th (1981) Fluorescence histochemistry of the pineal gland. In : Reiter RJ (ed) The pineal gland, Vol I. Anatomy and biochemistry. CRC Press, Boca Raton, Florida, pp 69-93 Moiler M, Nielsen JT, van Veen Th (1979) Effect of superior cervical ganglionectomy on monoamine content in the epithalamic area of the Mongolian gerbil (Meriones unguiculatus): a fluorescence histochemical study. Cell Tissue Res 201 : 1-9 Moller M, Fahrenkrug J, Ottesen B (1981) The presence of vasoactive intestinal peptide (VIP) in nerve fibres connecting the brain and the pineal gland of the cat. Neurosci Lett Suppl 7:411 Moller M, Glistrup OV, Olsen W (1984) Contrast enhancement of the brownish peroxidase-activated 3,3'-diaminobenzidine tetrahydrochloride reaction product in black and white photomicrography by the use of interference filters. J Histochem Cytochem 52 : 37-42 Nielsen JT, Moiler M (1978) lnnervation of the pineal gland in the Mongolian gerbil (Meriones unguiculatus): a fluorescence microscopical study. Cell Tissue Res 187:235-250 Ottesen B, Staun-Olsen P, Gammeltoft S, Fahrenkrug J (1982) Receptors for vasoactive intestinal polypeptide on crude smooth muscle membranes from porcine uterus. Endocrinology 110:2037 2043 Pelletier G, Leclerc R, Puviani R, Polak JM (1981) Electron immunocytochemistry of vasoactive intestinal peptide (VIP) in the rat brain. Brain Res 210:356-360 Phillis JW (1982) Neuronal excitation by vasoactive intestinal poly-

peptide. In: Said S1 (ed) Vasoactive intestinal peptide. Adv Horm Res Series. Raven Press, New York, pp 299 305 Probert L, De Mey J, Polak JM (1981) Distinct subpopulations of enteric p-type neurones contain substance P and vasoactive intestinal polypeptide. Nature 294: 470-71 Romeis B (1968) Mikroskopische Technik 16th ed. R Oldenburg, Miinchen Wien, pp 1-757 Staun-Olsen P, Ottesen B, Bartels PD, Nielsen MH, Gammeltoft S, Fahrenkrug J (1982) Receptors for vasoactive intestinal polypeptide on isolated synaptosomes from rat cerebral cortex. Heterogeneity of binding and desensitization of receptors. J Neurochem 39 : 1242-1251 Uddman R, Alumets J, Ehinger B, H~kanson R, Lor6n I, Sundler F (1980a) Vasoactive intestinal peptide nerves in ocular and orbital structures of the cat. Invest Ophthalm Vis Sci 19:878-885 Uddman R, Alumets J, H~kanson R, Lor6n I, Sundler F (1980b) Vasoactive intestinal peptide (VIP) occurs in nerve of the pineal gland. Experientia 36:1119-1120 Young WS III, Kuhar MJ (1979) A new method for receptor autoradiography: (3H)opioid receptors in rat brain. Brain Res 179:255 270 Yuwiler A (1983) Vasoactive intestinal peptide stimulation of pineal serotonin-N-acetyltransferase activity: General characteristics. J Neurochem 41:146-153 Accepted March 4, 1985