Growth Hormone Inhibits the Hypophysectomy-Induced Expression of Galanin in Hypothalamic Neurons of the Toad (Bufo arenarumHensel)

General and Comparative Endocrinology 105, 323–332 (1997) Article No. GC966833

Growth Hormone Inhibits the Hypophysectomy-Induced Expression of Galanin in Hypothalamic Neurons of the Toad (Bufo arenarum Hensel) M. V. Gonza´lez Nicolini, A. A. Orezzoli, M. V. Achi, M. J. Villar,1 and J. H. Tramezzani Instituto de Neurobiologı´a, Serrano 669, 1414 Buenos Aires, Argentina Accepted September 27, 1996

The expression of the neuropeptide galanin was analyzed by immunohistochemistry in magnocellular and preoptic hypothalamic neurons of toads following hypophysectomy (HPX) and pars distalectomy (PDX). There was a marked increase in the galanin-like immunoreactive expression in magnocellular hypothalamic cells 3 days after HPX, followed by a decrease to normal levels after 7 days. No changes in the expression of galanin were detected after PDX in these neurons when compared to controls. Moreover, 7 days after HPX or PDX the number of cells expressing galanin was significantly increased in the preoptic area, where numerous intraependymal cells were intensely immunoreactive. The hypophysis grafts into the hind limb in HPX or PDX animals prevented increased galanin-like immunoreactivity in preoptic cells but not in magnocellular neurons. Similarly, PDX toads given growth hormone showed no GAL-LI in the intraependymal preoptic cells. These results suggest the presence of a regional regulation of galanin expression in the preoptic area by hypophyseal hormones, in particular growth hormone. r 1997 Academic Press Galanin (GAL) is a 29-residue amidated peptide originally isolated from porcine small intestine (Tatemoto et al., 1983). The sequence of this peptide in mammals such as rat and bovine only differs from that of pig by substitutions of 3 and 4 amino acids, 1

To whom correspondence should be addressed.

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respectively, in the C-terminal region (Crawley and Wenk, 1989). The primary structure of GAL has been determined in the European green frog and it also differs from that of pig only by 2 amino acids in the C-terminal region (Chartrel et al., 1995). The peptide is widely distributed in the central nervous system (CNS), with a particularly high concentration in the hypothalamus (Arai et al., 1990; Ch’ng et al., 1985; Gentlemann et al., 1989; Melander et al., 1986; Michener et al., 1990; Ro¨kaeus et al., 1984; Skofitsch and Jacobowitz, 1985). A widespread distribution of GAL-like immunoreactivity (-LI) has also been described in the CNS of several amphibia (Gonza´lez Nicolini et al., 1995; La´za`r et al., 1991; Olivereau and Olivereau, 1992; Wolfbauer and Skofitsch, 1989). In the toad there are numerous GAL-immunoreactive (-IR) neurons in the hypothalamus, especially within the preoptic area, the suprachiasmatic nucleus, the ventral hypothalamus, and the infundibular region (Gonza´lez Nicolini et al., 1995). Among its many physiological and pharmacological actions, GAL is a potent inhibitor of insulin release (Mc Donald et al., 1985), dopamine release from the median eminence (ME) (Nordstro¨m et al., 1987), and acetylcholine release from the ventral hippocampus (Fisone et al., 1987) and clearly influences the secretion of luteinizing hormone (Lo´pez and Negro-Vilar, 1990; Lo´pez et al., 1991), prolactin (Koshiyama et al., 1990) and growth hormone (GH) (Bauer et al., 1986; Gabriel et al., 1988;

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Maiter et al., 1990; Murakami et al., 1987; Ottlecz et al., 1988; Tanoh et al., 1991). Additionally, GAL stimulation of cAMP production has been shown in the anterior pituitary in the frog (Chartrel et al., 1995). In rats, peripheral axotomy markely upregulates both GAL and GAL messenger ribonucleic acid (mRNA) levels in dorsal root ganglia (Ho¨kfelt et al., 1987; Villar et al., 1989; Zhang et al., 1995) and increases the transport of the peptide into both central and peripheral branches of primary sensory neurons, suggesting a role for this peptide under pathological conditions (Villar et al., 1991). In addition, the expression of GAL transiently increases in magnocellular hypothalamic neurons after hypophysectomy, suggesting perhaps that GAL is involved in the processes of degeneration and/or regeneration that occur in these neurons when subjected to injury (Meister et al., 1990; Selvais et al., 1993; Villar et al., 1990). Conversely, GAL-LI in the external layer of the median eminence was reported to be reduced after hypophysectomy (Selvais et al., 1993). The present study was conducted first to determine whether GAL expression in hypothalamic–hypophysiotropic systems of amphibia responds to injury in the same way as in the rat. Second, experiments were conducted to establish a possible regulation of GAL expression by the pituitary gland and pituitary hormones, in particular GH.

EXPERIMENTAL PROCEDURES Surgical Procedures One hundred adult male toads (Bufo arenarum Hensel), weighing 100–150 g, were purchased from a local supplier and housed in plastic cages with food and water ad libitum. Toads were subjected to one of the following surgical procedures: (1) complete pituitary ablation (HPX) (n 5 18); (2) complete ablation of the pituitary, which was autografted to the surface of the semimembranosus muscle in the hind limb (HPX 1 GRAFT) (n 5 18); (3) ablation of the pars distalis (PDX) (n 5 18); (4) removal of the pars distalis followed by its autografting (PDX 1 GRAFT) (n 5 18) as in group two; (5) ablation of the pars distalis and

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Nicolini et al.

GH replacement therapy (PDX 1 GH) (n 5 4); and (6) ablation of the pars distalis and saline administration (PDX 1 S) (n 5 4). In the experimental animals, after anesthesia with 3-aminobenzoic acid ethyl ester methanesulfonate (MS 222, Sandoz Ltd., Basel, Switzerland) an incision was made in the mucosa of the mouth and the retractor bulbi muscles of the eyes were sectioned. The parasphenoid bone was drilled off and the hypophysis exposed by removal of cartilage and meninges. Subsequently: (a) the hypophysis was separated from the median eminence (experimental groups HPX and HPX 1 GRAFT) or (b) the pars distalis was carefully separated from the neurointermediate lobe and dissected out (experimental groups PDX, PDX 1 GRAFT, PDX 1 GH, and PDX 1 S). The complete hypophysis or the pars distalis was discarded, respectively, in HPX and PDX animals. Subsequently, in HPX 1 GRAFT and PDX 1 GRAFT animals the complete hypophysis or the pars distalis was respectively autografted. Finally, the parasphenoid bone was replaced and the area sealed with gelfoam. In addition, in these grafting experiments the animals were transplanted with an extra hypophysis or pars distalis every second day. Four PDX animals were treated with bovine GH (NIH-USDA-bGH-B-1 1.9 IU/mg) 200 µg/day (Walker Farmer et al., 1977) or saline solution (PDX 1 S) was injected into the dorsal lymph sac for 7 days after ablation of the pars distalis. After 48 hr and 3, 7, and 14 days, controls and experimental animals were anesthetized and perfused through the aorta with 50 ml (28°) of 0.6% NaCl followed by 300 ml (4°) of a mixture of Formalin and picric acid [4% paraformaldehyde and 0.4% picric acid in 0.16 M sodium phosphate buffer (PBS), pH 6.9; (Zamboni and De Martino, 1967)]. The brains were dissected out and immersed in the same fixative overnight and transferred to PBS (pH 7.4) containing 10% sucrose, 0.02% Bacitracin (Sigma, St. Louis, MO), and 0.01% sodium azide (Merck, Darmstadt, Germany) for at least 48 hr. A complete series of frontal sections (20 µm thickness) was obtained in a cryostat (Microm, Zeiss, Germany) and processed by the avidin– biotin complex (ABC) technique (Hsu et al., 1981). The pituitary grafts were histologically examined following hematoxylin–eosin staining.

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ABC Method The sections were mounted onto chrome alum– gelatin-precoated glass slides, allowed to dry for at least 1 hr, and rinsed twice in PBS. The sections were incubated for between 18 and 48 hr in a humid chamber at 4° with rabbit GAL antisera (against porcine or rat GAL, Peninsula Laboratories, Belmont, CA) diluted 1:2000 in PBS containing 0.2% (w/v) bovine serum albumin, 0.03% Triton X-100, and 0.1% (w/v) sodium azide. The slides were rinsed twice in PBS and incubated at 25° for 30 min in biotinylated goat anti-rabbit secondary antibodies (1:200, Vector Laboratories, Burlingame, CA), rinsed twice in PBS, and incubated in an ABC (Elite kit, Vector Laboratories) for 1 hr at 25°. Peroxidase activity was demonstrated by reaction with 3,38-diaminobenzidine using glucose oxidase and nickel salts for enhancement of the reaction product (Shu et al., 1988). After dehydration the sections were mounted in Permount medium under a coverslip.

Quantification The numbers of GAL-IR cells in the preoptic area, the suprachiasmatic nucleus, the ventral hypothalamus, and the infundibular region were counted in seven representative animals for each of the following experimental designs: (a) CONTROL, (b) HPX; and (c) HPX 1 GRAFT. The boundaries of each area were established using parallel series of sections stained with the Nissl technique (see Neary and Northcutt, 1983; Gonza´lez Nicolini et al., 1995). The numbers of GAL-immunopositive neurons in serial sections of each area were counted, and the mean was expressed as a single value for the final statistical analysis. A double-blind analysis of sections was followed by two independent researchers. A one-way ANOVA followed by a Student’s t test (two-tailed unpaired) assessed statistical differences in the number of GAL-IR cell profiles across the groups. Data are presented as the means 6 SEM. The criteria level for significance was set at P , 0.001.

sorbed with GAL peptide (1026 M; Peninsula Laboratories) were performed. Also, sections were incubated with only primary or secondary antibodies and processed for ABC. All sections were studied in a Nikon Microphot FXA microscope. Agfapan APX 25 (Agfa Gevaert AG, Leverkusen, Germany) was used for bright-field photography.

RESULTS GAL-IR structures were never observed in any of the immunoabsortion control experiments. Microscopic examination revealed large intact grafts with welldifferentiated cells in close contact with the semimembranosus muscle through a neoformed vascular plexus. The largest vessels were located in that part of the gland facing the muscle.

Effect of HPX on Magnocellular GAL-IR Expression In control animals only occasional lightly immunostained cells were observed in the magnocellular region of the hypothalamus of the toad (Fig. 1A). In HPX animals there was a gradual increase in the intensity of the immunostaining in magnocellular cells after 48 hr and 3 days. Here, several neurons displayed an intense GAL-LI (Figs. 1B and 1C). After 7 days of HPX virtually all GAL-IR magnocellular cells had disappeared, to resemble the immunostaining of the control group. In HPX animals with an hypophysis grafted into the hind limb, GAL-LI in magnocellular neurons resembled those of HPX animals (Fig. 1D). The temporal changes in GAL-IR increase and disappearance were also similar.

Effect of PDX on Magnocellular GAL-IR Expression Magnocellular neurons showed no changes in GAL-IR expression at any times following PDX.

Controls

Effect of HPX and Grafting on Preoptic GAL-IR Expression

For control purposes, parallel incubations of sections with GAL antisera (dilutions as above) preab-

In control toads GAL-IR somata were present along the entire rostrocaudal extension of the medial preop-

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FIG. 1. Photomicrographs showing immunostained sections with GAL-LI in the magnocellular region of a control toad (A), HPX animals with 48 hr (B) and 3 days (C) of survival, and a grafted HPX toad with 3 days of survival (D). Note that whereas in the control (A) no immunostained neurons are present, in the HPX (B–D) toads several GAL-IR neurons can be observed. Bars, 125 µm.

tic nucleus, occupied the periventricular gray, and were aligned in parallel to the walls of the third ventricle (Fig. 2A). A gradual and significant increase was observed in the staining intensity of preoptic cells from 5 to 14 days after HPX (Figs. 2B and 2C). Seven days after HPX a twofold increase in the number of GAL-IR cells (n 5 7; P , 0.001 by two-tailed t test) was detected (Fig. 3A). Here, numerous intraependymal preoptic cells were intensely immunoreactive (Figs. 2B and 2C). When HPX animals were autografted (experimental group HPX 1 GRAFT) no GAL-LI was observed in the intraependymal preoptic cells. Thus, at all times tested, HPX grafted animals had a comparable immunostaining to that of the control group in the preoptic area (Figs. 2D and 3A).

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No qualitative or quantitative differences in GAL-LI were seen in any of the other three hypothalamic areas (suprachiasmatic nucleus, ventral hypothalamus, and infundibular region) examined in any of the experimental groups compared to controls (Figs. 3B–3D).

Effect of PDX and Autografting on Preoptic GAL-IR Expression After ablation of the pars distalis of the hypophysis there was a progressive increase in the intensity of GAL-LI in preoptic cells similar to that seen in the HPX animals (Fig. 4A). When these animals received pars

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FIG. 2. Photomicrographs showing immunostained sections with GAL-LI in the preoptic area of a control toad (A), HPX animals with 7 days (B) and 14 days (C) of survival, and a grafted HPX toad with 7 days of survival (D). The control (A) and grafted HPX (D) toads show few GAL-IR neurons, whereas in HPX toads (B, C) a dense population of immunostained cells (arrows) is present close to the ependyma. Bars, 125 µm in A, B, and D; 38 µm in C.

distalis grafts the number of GAL-IR cells was similar to that of the control animals (Fig. 4B).

DISCUSSION Magnocellular Neurons

Effect of PDX and GH Replacement Therapy on Preoptic GAL-IR Expression After GH administration to PDX toads for 7 days, intraependymal preoptic cells had no GAL-LI (Fig. 4D), with an immunostaining similar to control animals. In contrast, PDX animals treated with saline solution (PDX 1 S) had an increased number of preoptic cells stained for GAL (Fig. 4C), as in the HPX and PDX groups.

The present experiments showed that, in the toad, the expression of GAL in magnocellular hypothalamic neurons changed after section of their fibers projecting to the neural lobe. Thus, the lesion induced an increase in GAL-LI which peaked after 3 days, followed by a decrease detected at 7 days. These changes are akin to those described in the rat (Villar et al., 1990). However, in the toad the period of GAL-IR expression in magnocellular

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FIG. 3. Effect of HPX (black columns) and HPX followed by grafting (shadowed columns) on the expression of GAL-IR neurons compared to controls (white columns) in the preoptic area (A), the suprachiasmatic nucleus (B), the ventral hypothalamus (C), and the infundibular region (D) of the toad. The survival time in the experiment was 7 days. The numbers of immunostained neurons are expressed as the means 6 SEM. Statistical analysis was done by one-way ANOVA, followed by a Student’s t test. ***P , 0.001, n 5 7.

cells was shorter since, by 7 days, the number of immunostained cells was comparable to that of controls. As previously reported in rats (Villar et al., 1990, 1994), the changes observed in GAL-IR expression may be related to the degeneration and subsequent regeneration processes that occur after hypophysectomy, which are known to be very active in the neurosecretory system (Dellmann et al., 1987; Stutinsky, 1955; Villar et al., 1994). Hypophysial grafts did not reverse the increase in GAL-LI, suggesting that in this neuronal system the expression of GAL does not depend upon endocrine regulation. GAL and GAL mRNA, as well as other neuropeptides, are known to be downregulated by nerve growth factor (NGF) in primary sensory neurons in sciatic nerve-transected animals, using immunohistochemistry and in situ hybridization, respectively (Verge et al., 1995). Whether GAL in magnocellular cells is also

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regulated by NGF or other trophic factors is currently under study. Also, the significance of the observed accumulation of GAL in magnocellular hypothalamic neurons with their axons transected requires further clarification.

Preoptic Neurons After HPX or PDX, the number of GAL-IR cells nearly doubled compared with that of control animals. Thus, it seems that the lack of pituitary hormones enhances GAL-IR expression as well as the number of neurons containing the peptide in the preoptic area. The observation that GH prevented this increase reinforces the idea that GAL in preoptic cells is regulated by pituitary hormones, in particular GH. Experiments using toad sp. GH could give further insight into this

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FIG. 4. Photomicrographs showing immunostained sections with GAL-LI in the preoptic area of toads subjected to PDX (A), PDX followed by grafting (B), PDX followed by injection with saline (C), and PDX followed by injection with growth hormone (D). Note that the population of immunoreactive GAL cells present in the preoptic area of PDX and PDX 1 S toads (arrows in A and C) is absent in PDX (B) and growth hormone-injected PDX (D) toads. Bars, 60 µm in A and B; 125 µm in C and D.

peptide–hormone interaction, since marked species specificities have been suggested for amphibian GHs (Olivereau et al., 1993). Hormones other than GH may also contribute to this regulation. Previous studies (Hooi et al., 1990; Ottlecz et al., 1988) have established that GAL acts as an important member of the hypothalamic–hypophysiotropic hormone family of peptides, showing that GAL terminals in the ME are functionally linked to the portal capillaries (Arai and Callas, 1991; Lo´pez et al., 1990, 1991). Furthermore, there are several studies showing that GAL modulates anterior pituitary hormone secretion (Bauer et al., 1986; Gabriel et al., 1988; Hooi et al., 1990; Koshiyama et al., 1990; Lo´pez and Negro-Vilar, 1990;

Lo´pez et al., 1991; Maiter et al., 1990; Murakami et al., 1987; Ottlecz et al., 1988; Tanoh et al., 1991), supporting the possible existence of a feedback mechanism by pituitary hormones over GAL as well. Indeed, there are other examples of hypothalamic peptide expression regulated by hormones; neuropeptide Y is upregulated by steroids (Sahu et al., 1992) and GAL downregulated by corticosterone in adrenalectomized animals (Hedlund et al., 1994). In the rat the reduction in GAL-LI in the external layers of the ME described after HPX (Selvais et al., 1993) was reversed by hormonal replacement with a combination of thyroxine, bovine growth hormone, cortisol, and b-estradiol (Selvais et al., 1994), giving further support to an endocrine regulation of hypothalamic peptide expression.

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Interestingly, iv infusion of GAL enhanced GH release in humans (Bauer et al., 1986; Davis et al., 1987; Hulting et al., 1991; Loche et al., 1989) and the present results indicate a possible inhibitory control of GH over GAL-IR preoptic neurons in the toad. Other lines of evidence such as the cholinergic blockade of the GAL stimulation of GH secretion in humans (Maiter et al., 1990) suggest that GAL-ergic stimulation of GH release involves GH releasing hormone and other indirect hypothalamic mechanisms (Murakami et al., 1987). Alternatively, since there is a substantial density of GAL-IR nerve terminals in the ME (Lo´pez et al., 1991; Melander et al., 1986), GAL may be released into the pituitary portal circulation (Lo´pez et al., 1990) and act directly at a pituitary level. Thus, GAL is dramatically increased in preoptic neurons with an increase in the number of GALexpressing cells in PDX animals. It may be that the elevation of GAL in these cells occurs as a result of the lack of normal feedback regulation by circulating levels of GH in PDX animals. Replacement with GH prevented the increase of GAL-LI in preoptic cells, which is consistent with this hypothesis. However, a broad spectrum of possible interactions among GAL, GH releasing hormone, GH, as well as somatostatin and noradrenaline neurons exists and GH should not be viewed as an exclusive influence.

ACKNOWLEDGMENTS This work was supported by the Fundacio´n Instituto de Neurobiologı´a, Fundacio´n Antorchas and the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de la Repu´blica Argentina. The authors express their gratitude to Mr. Angel Fusaro for expert help with photography and to the National Hormone and Pituitary Distribution Program, NIH, for providing GH.

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