Peptide GEGLSS-Like Immunoreactivity in the Rat Central Nervous System

Brain Research Bulletin, Vol. 44, No. 1, pp. 91–96, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00

PII S0361-9230(97)00098-1

Peptide GEGLSS-Like Immunoreactivity in the Rat Central Nervous System ANTTI A. AARNISALO,* TUULA KARHUNEN,† SAMPSA VANHATALO* AND PERTTI PANULA*†1 *Institute of Biomedicine, Department of Anatomy, P.O. Box 9 (Siltavuorenpenger 20 A), 00014 University of Helsinki, Helsinki, Finland; and †Department of Biology, Biocity, Åbo Academy University, Artillerigatan 6, SF-20520 Åbo, Finland [Received 2 December 1996; Revised 14 February 1997; Accepted 18 March 1997] ABSTRACT: A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicinetreated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSSlike immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor. © 1997 Elsevier Science Inc.

bind to a specific high affinity binding site, which is distinct from the opiate receptors [3]. Receptor autoradiography studies show specific NPFF binding sites in the posterior pituitary, hypothalamus, and in the dorsal horn of the spinal cord [4]. Intravenously administered NPAF or NPFF elevate mean arterial pressure in rats [27]. They inhibit stimulated insulin and somatostatin secretion from rat pancreas and inhibit the colonic bead expulsion time in mice [6,26]. NPFF-immunoreactive (NPFF-ir) structures are found in the CNS of rat, mainly in the posterior pituitary, medial hypothalamus, medulla, and in the dorsal horn of the spinal cord [12,13,16]. The projectories of NPFF-containing cell populations have been elucidated [2,14]. The distribution of NPAF and NPFF immunoreactivities as detected with antisera against the whole peptides is similar, which is probably due to crossreacting antibodies and the fact that the peptides originate from the same precursor [33]. Highly specific monoclonal NPFF-antibodies have been produced [16]. With these antibodies NPFF immunoreactivity can be seen in two major neuronal cell populations in the CNS of rat, in the medial hypothalamus and in the nucleus of the solitary tract. A few NPFF-ir neurons can also be seen in the paraventricular and supraoptic hypothalamic nuclei [12]. NPFF and NPAF have a similar C-terminus (-PQRFamide). As many peptides resemble this C-terminal part of NPFF, we produced a rabbit antiserum against the N-terminal fragment peptide of NPAF (AcGEGLSS) [7,25]. HPLC studies indicate that the hypothalamus of rat contains several NPFF-NPAF-related peptides [12,17,18]. One NPFF-NPAF precursor molecule has been cloned [33], but this precursor is not expressed in all NPFF-ir brain nuclei[23]. This study was designed to investigate the possible presence of peptides that resemble bovine NPAF in the rat brain.

KEY WORDS: GEGLSS, Neuropeptide AF, Neuropeptide FF, Immunohistochemistry, Rat.

INTRODUCTION Neuropeptide AF (NPAF, A18F amide, AGEGLSSPFWSLAAPQRFamide) is an amidated octadecapeptide isolated together with neuropeptide FF (NPFF, FLFQPQRFamide, morphine-modulating peptide) from the bovine brain using antisera against the molluscan cardioexcitatory peptide FMRFamide [35]. NPFF and NPAF attenuate acute morphine-induced analgesia in both rats and mice, whereas IgG from an antiserum against NPFF potentiates this effect [10,11,12]. In addition to this antiopioid-like effect, both NPAF and NPFF have a potent long-term analgesic effect, the mechanism of which is not known [8]. NPFF and NPAF 1

MATERIALS AND METHODS Animals Fourteen adult male Wistar rats aged 2 to 3 months (250 – 280 g) were used for this study. The rats were housed in a room (21°C) having a 12-h light– dark cycle. Food and water were available ad lib. All the experiments were conducted in accordance with the NIH guide regarding the care and use of animals for experimental procedures.

To whom requests for reprints should be addressed.

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92 Surgical Procedures Five rats were treated with an intracerebroventricular injection of colchicine (Merck, 10 mg/ml). All surgical procedures were performed under appropriate anesthesia. Sodium pentobarbital (Mebunat, Orion, Finland, 30 mg/kg) was injected intraperitoneally. A local anesthetic lidocaine hydrochloride (Lidocain, Orion, Finland, 10 mg/ml) was infiltrated under the skin and under the periosteum before the intracerebroventricular injection. A trepane hole was made and colchicine was injected into the lateral ventricle. The skin was sutured and the rats were allowed to survive for 48 h before perfusion. In the retrograde tracing experiments the pituitary gland was exposed by a parapharyngeal approach [30] and the drilling hole was made to the base of the skull. Fluorogold (1 ml, in 4% saline) was injected into the posterior and intermediate lobes of the pituitary gland [9,29]. After 1 week survival time the animals were treated with an intraventricular colchicine as described above, and sacrificed after 48 h. The pituitary stalk transsections were done with a stereotaxic apparatus (distance from bregma 24.5 mm, midline). The knive was placed by the coordinates, and after this the knive was rotated in the stalk. Sham-operated animals received only the needle injection, the knive was not rotated in these cases [34]. The animals were sacrificed after 1 week of survival time [21]. Tissue Preparation All rats were anesthetized with sodium pentobarbital as above and perfused through the left ventricle with 0.9% saline (200 ml, at room temperature) followed by 200 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The brain, spinal cord, and pituitary gland were removed. Parts of the liver, adrenal glands, duodenum, and pancreas were removed for the analysis. The tissue samples were postfixed for 2 h at 4°C in the same fixative. After this they were immersed in 20% sucrose (in 0.1 M sodium phosphate buffer, pH 7.4) overnight or longer. Production and Characterization of Antibodies The peptide acetyl-Gly-Glu-Gly-Leu-Ser-Ser (acGEGLSS) was synthesized using the solid phase system (Applied Biosystems), purified with reversed phase HPLC and verified with mass spectrometry. Six milligrams of the peptide was conjugated to keyhole limpet hemocyanin with 1-ethyl-3,3(dimethylaminopropyl)carbodiimide as described earlier [24]. Three rabbits were injected intradermally to four sites with 500 mg of the conjugate and same amount of Freund’s complete adjuvant. After 5 weeks the rabbits were boosted with 300 mg of the conjugate and Freund’s incomplete adjuvant. Ten days after this injection the rabbits were tested for the first time, and further test bleeds were taken every 10 days after this. Immunohistochemistry Tissue sections were made with a cryostat and the sections (brain and spinal cord 15 mm, pituitary 10 mm, and other tissue samples 15 mm-thick sections) were collected on gelatin-coated glass slides and the slides were air dried for 1 h. Cryostat sections with mirror surfaces of two consecutive sections were taken on glass slides, to compare the neuronal cell body, fiber, and terminal distribution of NPFF and GEGLSS. Polyclonal vasopressin and oxytocin antisera (Incstar, Stillwater, USA) were used to compare the distribution of GEGLSS-ir neurons in SON and PVN. The antibody elution technique [32] was used. The glass slides were washed with phosphate-buffered saline containing 0.25% Triton X-100 (PBS-T) for 2 3 15 min. They were then incubated 24 – 48 h at 4°C with GEGLSS antiserum 50 G diluted 1:200 in PBS-T

AARNISALO ET AL. containing 1% normal swine serum. Control sections were incubated with preimmune serum instead of specific GEGLSS antiserum, or preabsorbed peptide antiserum. Both antisera were preabsorbed for 24 h in 4°C with different concentrations (0.1–50 mM) of the following peptides before application on sections: GEGLSS, NPFF, NPAF, and oxytocin. The slides were rinsed 2 3 15 min in PBS-T, pH 7.4. After this the second antiserum consisted of fluorescein–isothiocyanate-conjugated swine antirabbit immunoglobulins (DAKO, Copenhagen, Denmark) was applied at 1:40 dilution in PBS-T for 1 h at room temperature. The slides were rinsed 2 3 15 min in PBS, pH 7.4. The samples were mounted in PBS-glycerol 1:1. The sections were viewed with a Leitz Aristoplan fluorescence microscope equipped for epiillumination. The filter block I3 (excitation filters BP 450 – 490, dichromatic mirror RKP 510, suppression filter LP 520) or L4 (excitation filters BP 450 – 490, dichromatic mirror rkp 510, suppression filter 515–560) were used for the FITC fluorescence and for the fluorogold a Leitz UV filter (block A, excitation 340 –380 nm, emission max 400 – 430 nm) was used. Specificity tests with dot blots were done. Serial dilutions of peptides (Ac-GEGLSS, NPFF, NPAF and oxytocin), 1000, 100, 10, and 1 mM, were prepared and blotted (2 ml per dot) onto nitrocellulose filters (BA 85; Schleicher and Schuell, Keene, NH). Unfixed filters and filters incubated for 1 h in 4% PFA were used. To reduce the nonspecific background staining the filters were first incubated in 3% BSA in PBS for 2 h. The primary antiserum was diluted 1:500 in PBS and incubation was carried out overnight at 4°C. Incubations with peroxidase-labeled swine antirabbit IgG (DAKO, Copenhagen, Denmark) diluted 1:100 in PBS were carried out at room temperature for 1 h. The reaction with 3.39diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO), 50 mg/100 ml, and hydrogen peroxidase, 0.01% in 0.05 M Tris-HCl buffer, pH 7.6, was carried out at room temperature for 5 min. RESULTS GEGLSS-ir neurons were located in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei (Fig. 1). GEGLSS-ir neurons were also observed in the retrochiasmatic part of the SON (Fig. 1). Most GEGLSS-ir neurons were located in the magnocellular part of the PVN (Figs. 1 and 2). Colchicine treatment was necessary to demonstrate them. No GEGLSS-ir neurons were seen in the medial hypothalamic area between the VMH and DM (Fig. 1) or in the periventricular nucleus (Fig. 1). Sections from colchicine-treated animals displayed NPFF-ir neurons in the same area (Fig. 1). GEGLSS-ir fibers and terminal-like thickenings were seen in the median eminence (Fig. 1). In the posterior pituitary GEGLSS-ir terminal-like thickenings were seen (Fig. 1). There were no GEGLSS-ir structures in the intermediate or in the anterior lobe of the pituitary (Fig. 1). No GEGLSS-ir structures were observed in the telencephalic and mesencephalic structures. In pons and medulla no GEGLSS-ir neurons were seen throughout the nucleus of the solitary tract (Fig. 1), although NPFF-ir neurons were seen in consecutive sections in this nucleus (data not shown). However, GEGLSS-ir terminal-like thickenings and fibers were observed in the lateral parts of the nucleus of the solitary tract through the nucleus and also around the central canal (Fig. 1). There were both NPFF-ir and GEGLSS-ir structures around the central canal in the commissural part of NTS. However, no colocalization was observed (data not shown). No GEGLSS-ir structures were seen in the spinal cord or in the spinal ganglia (data not shown). Immunohistochemical blocking controls and specificity tests with dot blots were performed with NPFF, NPAF, oxytocin, and Ac-GEGLSS. The preimmune serum showed no immunore-

GEGLSS-LIKE-IMMUNOREACTIVITY IN THE RAT CNS

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FIG. 1. Photomicrographs showing in A GEGLSS-ir neurons in SON after ICV colchicine treatment, in B GEGLSS-ir neurons in the SOR. In C GEGLSS-ir fibers and terminal-like thickenings in the median eminence. In D GEGLSS-ir terminal-like thickenings in the posterior lobe of the pituitary. In E the area between the VMH and DM after colchicine treatment showing no GEGLSS-ir neurons. In F NPFF-ir neurons after antibody elution technique was used. Scale bar in A and B is the same (100 mm) and in C, D, E, and F the same (100 mm).

action. In blocking controls, preabsorption with 20 mM GEGLSSpeptide (1–50 mM) abolished GEGLSS-ir structures totally (Table 1). The octapeptide NPFF (1–50 mM) did not diminish the staining (Table 1). The octadecapeptide NPAF (1–50 mM), the N-terminus of which is structurally similar with GEGLSS, decreased the

intensity of GEGLSS immunoreaction, but did not abolish it totally (Table 1). Oxytocin (1–10 mM) had no effect on the GEGLSSimmunoreactivity (Table 1). When GEGLSS-peptide was applied to a nitrocellulose filter (1000, 100, 10, and 1 mM), 2 ml per spot corresponding to 2000, 200, 20, and 2 pmol/spot and detected with

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FIG. 2. In A GEGLSS-ir neuronal cell bodies in the SON and in B arg-vasopressin-ir neuronal cell bodies. Filled arrows indicate colocalization. Also neurons containing only arg-vasopressin or GEGLSS were seen (open arrows). In C GEGLSS-ir neuronal cell bodies in the SON and in D oxytocin-ir neuronal cell bodies. Filled arrows indicate colocalization. Also neurons containing only GEGLSS-ir or oxytocin-ir neuronal cell bodies were observed (closed arrows). In E GEGLSS-ir neuronal cell bodies and in F fluorogold filled neurons in the PVN. Colocalization is indicated with closed arrows. In G GEGLSS-ir structures are totally abolished from the posterior pituitary after the stalk transsection and in H GEGLSS-ir structures in a sham-operated animal. Scale bar in A, B, C, and D is the same (100 mm) and in E, F, G, and H the same (100 mm).

GEGLSS-LIKE-IMMUNOREACTIVITY IN THE RAT CNS TABLE 1 SUMMARY OF THE PREABSORPTION TESTS

GEGLSS NPFF NPAF oxytocin

1 mM

10 mM

20 mM

50 mM

11 11 11 11

1 11 11 11

2 11 1 11

2 11 1

The effect of the peptide to the GEGLSS immunoreactivity is indicated as 11 (no effect), 1 (weak effect) and 2 (the GEGLSS immunoreaction is abolished).

the GEGLSS-antiserum (1:200), a strong reaction was seen with the two higher concentrations, whereas the lower concentrations yielded no immunoreaction, and other peptides tested showed no immunoreaction (Table 2). Even in colchicine-pretreated brains it was impossible to compare the possible co-localization of NPFF-ir and GEGLSS-ir neurons in the SOR, SON, or PVN due to the low number of NPFF-ir neurons. GEGLSS immunoreactivity was partially colocalized with vasopressin-ir and oxytocin-ir neuronal cell bodies in SON and PVN (Fig. 2). A retrograde tracer, fluorogold, was injected to the posterior pituitary and to the intermediate lobe of the pituitary gland. Three cases were analyzed. Areas that were known to contain GEGLSS-ir neurons were studied (SON, SOR, and PVN). Double-labeled neurons were found in the SON and also in the SOR (Fig. 2). The PVN contained also double-labeled neurons (Fig. 2). GEGLSS-ir neurons without tracer or neurons containing only the tracer were also seen in all the studied areas (Fig. 2). Three pituitary stalk transsection cases were analyzed. GEGLSS-ir structures were totally abolished from the posterior pituitary after transsection (Fig. 2), whereas GEGLSS-ir structures were found in the sham-operated animals. Some peripheral tissues, known to contain brain-gut peptides, were also examined for the presence of GEGLSS immunoreactivity. GEGLSS-ir structures were not seen in the adrenals, duodenum, or pancreas (data not shown). DISCUSSION Our results suggest that an GEGLSS-like immunoreactive peptide is present in neurons of SON, SOR, and PVN, but undetectable in the other known NPFF-containing neuronal cell populations. However, GEGLSS-ir fibers and terminals were observed in the nucleus of the solitary tract. Colchicine treatment was neces-

95 sary to demonstrate NPAF- and NPFF-ir neurons [12]. It was also necessary to use colchicine-treated brains to demonstrate GEGLSS-ir neurons in the rat CNS. In colchicine-treated rats both GEGLSS-ir cell bodies, fibers and terminal-like thickenings could be seen. No GEGLSS-ir neurons were seen in the medial hypothalamus, where the largests NPFF-containing neuronal cell population is located (Aarnisalo et al., unpublished). Also, no GEGLSS-ir neuronal cell bodies were seen in the periventricular nucleus, which is known to contain NPFF-ir neuronal cell bodies. GEGLSS-ir neuronal cell bodies were located in the PVN and SON, which are also known to contain few NPFF-NPAF-ir neurons ([12], Aarnisalo and Panula, unpublished). There were many more GEGLSS-ir neurons in the SON and PVN than NPFFNPAF-ir neurons. This suggests that the GEGLSS immunoreactivity identifies a subpopulation of neurons in an area that also contains NPFF-NPAF-ir peptide neurons. Both AVP- and OT-ir neurons displayed GEGLSS immunoreactivity. NPFF-NPAF-ir structures are found in the posterior pituitary [12,15] and neurohypophyseal NPFF originates from the PVN and SON [2,20]. This study suggests that GEGLSS-ir neurons in SON and PVN also project to the posterior pituitary. Pituitary stalk transsection also caused a depletion of GEGLSS-ir structures in the posterior pituitary. It seems that GEGLSS is transported from the hypothalamus to the posterior pituitary. The function of NPFF, NPAF, or related peptides in the posterior pituitary is not known with certainty. Previous studies show that NPFF is coreleased with AVP in response to hyperosmotic stimuli [19]. It has also been suggested that NPFF regulates the function of pituicytes [5]. NPFF releases prolactin both in vivo and in vitro and fullfills the criteria of a prolactin-releasing factor originating from the posterior pituitary [1]. It is possible that these GEGLSS-ir neurons project to the nucleus of the solitary tract, as vasopressin-containing and NPFFcontaining neurons do [2,28,31]. We observed no GEGLSS-ir structures in the spinal cord, where NPFF is located in the dorsal horn, in the superficial laminae [13,16]. A new NPFF-NPAF-like active peptide SLAAPQRF-NH2, originating from the rat NPFF-NPAF precursor, has been characterized and isolated from brain and spinal cord of rat [34]. Receptor binding studies show that SLAAPQRF-NH2 binds to the NPFF receptor with a very similar affinity as NPFF [34]. The GEGLSSsequence is not present in the rat NPFF-NPAF precursor cloned recently [33]. Our results give further support to the concept that rat hypothalamus contains several NPFF-NPAF related peptides, of which NPFF and SLAAPQRF-NH2 are the only known biologically active ones [12,17,18,34]. Further studies will aim at identification of the molecular forms and origin of rat GEGLSS-like peptides, and their relations to NPFF and NPAF. ACKNOWLEDGEMENTS

The authors express their gratitude to Ms. Paula Hasenson for her skillful technical assistance. This study was supported by the Sigrid Juselius Foundation, the Finnish Cultural Foundation, and the Medical Research Council of the Academy of Finland.

TABLE 2 SUMMARY OF THE DOT BLOT ANALYSIS GEGLSS

1000 mM 100 mM 10 mM 1 mM

11 1 2 2

NPAF

2 2 2 2

NPFF

2 2 2 2

OT

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2 2 2 2

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Different concentrations of GEGLSS, NPAF, NPFF, and OT (oxytocin) peptides were applied on the nitrocellulose filter (1000, 100, 10, and 1 mM). GEGLSS immunoreactivity is indicated as 11 (strong reaction), 1 (weak reaction), 2 (no reaction).

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