Phylogenetic Distribution and Function of the Hypophysiotropic Hormones of the Hypothalamus

Transcription

1 AMER. ZOOL., 18: (1978). Phylogenetic Distribution and Function of the Hypophysiotropic Hormones of the Hypothalamus IVOR M. D.JACKSON Division of Endocrinology, Department of Medicine, New England Medical Center Hospital, Tufts University School of Medicine, Boston, Massachusetts SYNOPSIS. Following the isolation, synthesis and subsequent development of specific and sensitive radioimmunoassays for the hypothalamic hormones thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH) and growth hormone release-inhibiting hormone (somatostatin), it was recognized that these peptides were not localized solely in the hypothalamus, but were widely distributed throughout the mammalian nervous system. Somatostatin occurs outside the nervous system altogether, being located in the gastrointestinal tract of vertebrates where it may have a physiologic role in the secretion of gastrointestinal hormones. TRH, also, has been located outside the nervous system, occurring in large quantities in the skin ofrana species where it may be of physiologic importance in skin function. This tripeptide is found throughout the nervous system of vertebrate and invertebrate species in situations where it has no pituitary-thyroid function. These peptides are present in brain synaptosomes and enzymatic degrading systems have been recognized for each in brain tissue. For TRH, specific receptors and synthesizing activity have been detected outside the hypothalamic-pituitary system. The anatomic location, phylogenetic distribution, neurophysiologic and behavioral effects strongly support a role for these substances in neuronal regulation, apart from control of pituitary secretion. Evolutionary studies, especially of TRH, suggest that their primary function may be as neurotransmitters. INTRODUCTION It is generally accepted that the hormones secreted by the mammalian anterior pituitary are regulated by factors synthesized and secreted by peptidergic neurons in the hypothalamus (Reichlin, 1973; Blackwell and Guillemin, 1973). The isolation and synthesis of three of these hypothalamic hypophysiotropic factors or hormones, thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH) and growth hormone release inhibiting hormone (somatostatin) have provided powerful tools for the investigation of pituitary function and have permitted the development of specific radioimmunoassays for the measurement of these substances at exceedingly low concentrations (Reichlin et al., 1976). A wholly The original research reported herein was supported by a grant from the National Institutes of Health, AM unanticipated outcome was the finding that most of neural TRH and somatostatin are located outside the hypothalamus (Reichlin et al., 1976; Jackson, 1977; Jackson, 1978 for review). Further, these hormones are present in inframammalian species, though the functional significance of these substances in such animals essentially remains to be determined. Even more surprising is the revelation that somatostatin (Arimura et al., 1975) and TRH (Jackson and Reichlin, 1977a) exist in tissues other than the nervous system. It is believed that the hypothalamic peptidergic neurons are in turn regulated by neurotransmitters largely of the monoaminergic variety, and that the peptidergic neuron acts as a "neuroendocrine transducer" converting neural information from the brain into chemical information (Wurtman, 1971). More recently it has been recognized that the chemical products of the peptidergic neurons may themselves act as neurotransmitters (Martin et al., 1975; Jackson, 1977; Jackson, 385

2 386 IVOR M. D.JACKSON 1978 for review). Still to be determined is the question whether the same kind of hormonal feed-back and/or neurotransmitter control characteristic of the hypothalamus is also operative at extrahypothalamic sites. The finding of extrahypothalamic sources of hypophysiotrophic hormones provides some support for the view (ependymal tanycyte theory) that a portion of the releasing hormones reach the primary portal plexus by trans-median eminence transport, it being postulated that the releasing hormones are secreted into the ventricular system, taken up by the lumenal processes of the tanycytes of the median eminence, and then actively transported for release at the capillary end of the cell (Knigge and Silverman, 1972). In this report I will review the more recent findings concerning the anatomic and phylogenetic distribution of TRH, LH-RH and somatostatin and discuss their extrapituitary functional significance. THYROTROPIN-RELEASING HORMONE Thyrotropin-releasing hormone (TRH) radioimmunoassay and mammalian hypothalamic TRH Since the early studies of Greer (1951), and Harris and Jacobsohn (1952), it has been known that the hypothalamus exerts an important influence on the regulation of the pituitary-thyroid axis. Demonstration of the existence of a thyrotropic releasing factor (TRF), as well as its purification from hypothalamic extracts, was provided by Guillemin (1964), and subsequent reports indicated that TRH activity was present in whole hypothalamic extracts and stalk median eminences (SME) of the sheep and rat (Guillemin et al., 1965; Averill and Kennedy, 1967). Physiologic data based on electrical stimulation of different hypothalamic areas (D'Angelo and Snyder, 1963; Martin and Reichlin, 1972) and the placement of intrahypothalamic pituitary grafts (Flament-Durand, 1965) suggested that TRH synthesis might occur diffusely throughout the mammalian hypothalamus. In 1969 the chemical structure of TRH was elucidated in the laboratories of Guillemin and Schally and shown to be a tripeptide amide (P glu- His-Pro NH2), molecular weight 362, following rigorous chemical analysis of large numbers of ovine and porcine hypothalamic extracts (B0ler et al., 1969; Burgus et al., 1969). The availability of chemically pure synthetic TRH led to the development of radioimmunoassays for TRH by several groups including those of Utiger (Bassiri and Utiger, 1972), Wilber (Montoya et al., 1973), Porter (Oliver et al., 1973) and myself (Jackson and Reichlin, 1973). The conjugation of TRH to a large carrier weight protein such as bovine serum albumin (Bassiri and Utiger, 1972) or bovine thyroglobulin (Tg) (Jackson and Reichlin, 1974a) permits the generation of antibody to TRH in rabbits with a high degree of sensitivity and specificity. I utilized Tg as the carrier protein following reports that this substance augmented the immunogenicity of small peptides. The histidine of TRH is readily iodinated by the Greenwood-Hunter procedure (Chloramine T, sodium metabisulfite), and the labelled hormone is separated from iodide with gel filtration on Sephadex G-10. This label is very stable, and I have used it for immunoassay purposes for periods of up to 4 months following iodination, with little damage on storage. Delayed addition of I25 I-TRH in my hands appears to increase sensitivity and charcoal (0.1%) separation of "bound from "free" hormone is a simple procedure. The double antibody technique (Bassiri and Utiger, 1972), and polyethylene glycol after short incubation (Montoya rt al., 1973) have also been reported to produce satisfactory separation. The levels of immunoreactive (IR)-TRH in the rat hypothalamus reported from different laboratories have given values of ng (Bassiri and Utiger, 1974; Jackson and Reichlin, 1974a; Oliver, et al., 1974). I have found considerable variation in the amount of IR-TRH in different groups of rat hypothalami in different experiments. These %'ariations may relate to the size of tissue block in different experiments but might also reflect seasonal or other differ-

3 DISTRIBUTION OF HYPOTHALAMIC HORMONES 387 ences. Bassiri and Utiger (1974) have also reported variations in the levels of IR-TRH in the hypothalamus when experiments were performed at different time intervals apart. I have found immunoreactive TRH readily detectable in porcine hypothalmi (500 pg/mg tissue wet weight), hamster hypothalami (480 pg/mg tissue wet weight), and in human stalk median eminence with values up to 300 pg/mg tissue (Jackson and Reichlin, 1974). Recent studies by Okon and Koch (1976), Guansing and Murk (1976) and Kubek et al. (1977) have demonstrated substantial quantities of TRH throughout the hypothalamus and SME of humans. Abalation of the "thyrotrophic" area of the hypothalamus induces hypothyroidism in the rat, but the TRH levels in the hypothalamus of such lesioned animals were as much as 35% of the values found in the controls (Jackson and Reichlin, 19776). The persistence of significant TRH levels in the hypothalamus following lesion provides an explanation for the fact that depression of baseline thyroid function after such a procedure is never as severe as that occurring after hypophysectomy. Studies by Brownstein et al. (1974) utilizing a technique which allows discrete nuclei to be dissected from the brain of the rat, showed that TRH though present in highest concentrations within nuclei of the "thyrotrophic area" were also found in the hypothalamus outside this region. Their data is in keeping with our findings in Extrahypothalamic brain Hypothalamic pituitary complex Brain stem 5 b (4-5)< Dorsal hypothalamus 49 (41-61) a Jackson and Reichlin, b Mean concentration. c Range of values. lesioned animals, as well as our report (Jackson and Reichlin, 19746) of a gradient of TRH from dorsal hypothalamus (49 pg/mg tissue) to SME (3570 pg/mg tissue) (Table 1). TRH has been reported to show immunofluorescent staining of nerve terminals in the medial part of the external layer of the median emminence (Hokfelt et al., 1975a) although no immunopositive TRH perikarya were observed. The estimate of total rat hypothalamic TRH by radioimmunoassay has given levels times those previously reported by in vivo bioassay (Reichlin et al., 1972). The discrepancy is explicable on the basis of the presence of somatostatin which inhibits TRH induced TSH rise (Vale et al., 1974a). TABLE 1. TRH distribution in rat brain. Cerebellum 2 (1-3) Ventral hypothalamus 64 (23-106) TRH (pg/mg tissue) Extrahypothalamic distribution of TRH in mammalian species The first reports that IR-TRH was present in the brain outside the confines of the hypothalamus were provided by my group (Jackson and Reichlin, 1973) and that of Oliver (Oliver et al, 1973). Significant concentrations of TRH are found in the rat extrahypothalamic brain (Jackson and Reichlin, 19746) (Table 1). Although such concentrations are small when compared with the levels in the hypothalamus, quantitatively over 70% of total brain TRH is found outside this region (Oliver et al., Diencephalon 6 (3-12) Stalk median eminence 3570 ( ) Olfactory lobe 6 (5-8) Posterior pituitary 155 ( ) Cerebral cortex 2 (1-3) Anterior pituitary 10 (8-11)

4 388 IVOR M. D.JACKSON 1974; Winokur and Utiger, 1974). In an attempt to determine the source of extrahypothalamic TRH, we have studied the effects of classical thyrotrophic area lesions which bring about a reduction in hypothalamic TRH by two-thirds (Table 2). The extrahypothalamic brain TRH content was unaffected in rats so treated providing support for the intriguing hypothesis that synthesis occurs in situ (Jackson and Reichlin, 1977*). Studies using hypothalamic deafferentation, complementary to these experiments (Brownstein, et al., 1975a) demonstrated that such procedures not only leave the levels of TRH in the extrahypothalamic brain unaltered, but cause a marked reduction in hypothalamic content, suggesting that much of hypothalamic TRH may be synthesized by cells outside this area. It is of note that quantitatively the amount of TRH in the posterior pituitary is much greater than that in the anterior pituitary (Table 2). The high concentration of TRH in the posterior pituitary relative to the anterior pituitary in normal rats (15 times as shown in the study reported in Table 1) has also been observed by others (Oliver^ al., 1974). The depletion of TRH from the posterior pituitary in the lesioned animals (Table 2) supports the concept of a third neurosecretory hypothalamo-hypophysial system extending into the neurohypophysis, as suggested by Hokfelt and his colleagues {I975a,b) on the basis of immunohistochemical staining of TRH and somatostatin positive fibers reaching into the posterior pituitary from the median eminence. Whether TRH has a role in the posterior pituitary function is uncertain, but it may be of relevance that TRH is found in extraordinarily high concentration in the pituitary complex of lower vertebrates (Jackson and Reichlin, 1974a), and there is evidence (reviewed by Sawyer, 1964) that in the bony fish the neurohypophysis may be a homologue of the median eminence in higher animals. Substantial quantities of TRH are found in the spinal cord (Jackson, unpublished; WilberetaL, 1976; KardonetaL, 1977)and immunohistochemical examination has localized TRH around the motoneurons of the spinal cord (Hokfelt et al., 1975a). Networks of TRH-positive nerve terminals have also been found in many cranial nerve nuclei (Hokfelt et al., 1975a). As in the fetal rat (Eskay et al., 1974); significant concentrations of TRH are present in the extrahypothalamic brain of the human fetus (Winters et al., 1974), TRH being detected in the cerebellum as early as 9 weeks. Interestingly, the cerebellum of an anencephalic infant contained a relatively high concentration of TRH (Winters et al., 1974). It should be noted however that the area cerebrovasculosa an area lacking in nerve cells taken from an anencephalic fetus has been reported to synthesize a TSH releasing substance in vitro (Ishikawa et al., 1976). Extrahypothalamic brain tissue from normal human adults (killed in traffic accidents) contains significant concentrations of immunoassayable TRH in the thalamus and cerebral cortex (Okon and Koch, 1976). Guansing and Murk (1976) and Kubek et al. (1977) have also identified IR-TRH in the extrahypothalamic brain tissue of humans. TABLE 2. Effect of a lesion of the "thyrotrophic area" of the hypothalamus on brain distribution of TRH in the rat.' Hypothalamus Anterior pituitary Posterior pituitary Extrahypothalamic brain Lesion b 3,625±249 15±8 2 17,040±925 Control" 9, ±5 157±32 18,929±737 Significance P < P < P < P > 0.1 "Jackson and Reichlin, b Six lesioned and seven control animals were studied. c Results (mean ± s.e.m.) are given as pg per tissue.

5 DISTRIBUTION OF HYPOTHALAMIC HORMONES 389 Physiologic role of TRH in the regulation of thyroid function in inframammalian species Aves. The importance of TRH in the regulation of pituitary-thyroid function in inframammalian species is uncertain. In the chick, Ochi et al. (1972) reported that the pituitary thyroid axis was unresponsive to TRH stimulation whereas Breneman and Rathkamp (1973), Newcomer and Huang (1974) and Scanes (1974) provide evidence that thyroid function can be stimulated by exogenous TRH. I examined the hypothalamus of adult chicks for TRH and found levels slightly less than that present in rat hypothalamus (Fig. 1). Although not readily measurable in mammalian blood, TRH can be measured in the circulation of the chicken and incubation 1 of plasma at 37 C does not degrade the endogenous IR-TRH (Jackson, unpublished). Amphibia. The significance of the hypothalamus in the regulation of amphibian thyroid function has not been determined for sure (Hanke, 1976 for review). However, there is evidence for hypothalamic control over amphibian metamorphosis, since lesions of the hypothalamus (Voitkevich, 1962; Hanaoka, 1967) impair metamorphosis. Thyroid hormone injected directly into the hypothalamus of the neotenic tiger salamander induces metamorphosis (Norris and Gern, 1976), findings that are in keeping with a critical TA0?OLE SALWOM FIG. 1. Concentration of TRH (pg per mg of tissue) (mean ±s.e.m.) in the olfactory lobe (telencephalon) of a number of different vertebrates. The mean TRH concentration in the hypothalamus is given for comparison (from Jackson, 1977). role for TRH in tadpole metamorphosis as postulated by Etkin (1963). He suggested that development of the tadpole hypothalamus is under positive feedback control by thyroid hormone and that a gradually rising level of circulating thyroid hormone during prometamorphosis induces maturation of the hypothalamic tissue concerned with the synthesis of TRH. This positive feedback system results in the stimulation of thyroid activity required for metamorphosis. However Gona and Gona (1974) failed to induce metamorphosis in the tadpole with TRH administration. TRH has also been given to the neotenic Mexican axolotl (salamander), a species whose plasma does not degrade TRH in vitro, and in spite of achieving high circulating levels of the exogenous material in the blood, as measured by radioimmunoassay, metamorphosis was not induced (Taurog^a/., 1974). These findings raise the possibility that in amphibia the mammalian TRH is not a physiologic TSH releasing factor in these species. Alternative explanations are possible. The TRH may induce simultaneous release of prolacin, which in the tadpole, at least, blocks metamorphosis (Etkin and Gona, 1967). Indeed antiserum to bullfrog prolactin injected into prometamorphic tadpoles of R. catesbeiana will accelerate metamorphic climax (demons and Nicoll, 1977). However, TRH given to the red eft, a species that undergoes water drive or second metamorphosis, which is known to be specially controlled by prolactin (Grant and Grant, 1958), had no effect on metamorphosis (Gona and Gona, 1974). demons et al., (1976) have reported that TRH induces prolactin release in the bullfrog and have suggested that TRH may have functioned as a prolactin releasing factor before it became a stimulator for TSH release. TRH has also been shown to release MSH from the pituitary of R. esculenta (Vaudry et al., 1977), but whether this has any relation to thyroid function is unclear at this time. Pisces. In the lung fish, TRH in high doses was not found to have any effect in stimulating thyroid function (Gorbman and Hyder, 1973). However, evidence for

6 390 IVOR M. D. JACKSON a thyrotropin inhibitory factor from the hypothalamus (TIF) has been provided by Peter and McKeown (1975) for the goldfish and other teleost fishes, and, it should be mentioned, by Rosenkilde (1972) for some species of amphibia also. Bromage (1975) has raised the interesting possibility that TRH may function as a TIF in teleost fishes. The overall evidence suggests that the hypothalamus is of importance in the regulation of thyroid function in lower animals but that the degree of autonomy of the pituitary-thyroid axis is much greater than in mamilian species. In amphibia there is evidence for a physiologic hypothalamic thyrotropin releasing factor, but this might be different from, or in addition to, the tripeptide amide, TRH. TRH distribution in submammalian chordates In view of the reported absence of a role for TRH in the regulation of thyroid function in inframammalian species, I examined the hypothalami of a number of vertebrates including snake, frog and salmon for TRH content, and found high concentrations (Jackson and Reichlin, 1974a), with values up to 10 times that found in rat hypothalamus (Figure 1). Elevated TRH levels in amphibian hypothalamus have also been reported by Taurog^ al. (1974). Further high concentrations of TRH were also found in the extrahypothalamic brain of these vertebrates (Figure 1) and evidence for its authenticity was shown by the ability of a frog brain extract to release rat TSH in vivo (Jackson and Reichlin, 1974a). We have also shown TRH to be present in the whole brain of the larval lamprey, in the head end of the amphioxus (Jackson and Reichlin, 1974a; Table 3) and in the circumesophageal ganglia of the invertebrate snail (Grimm-Jorgensen et al., 1975). As the lamprey lacks TSH, and the amphioxus and snail lack a pituitary I propose that the TSH-regulating function of TRH may be a late evolutionary development representing an example of an organism acquiring a new function for a pre-existing chemical substance or hormone, analogous to the evolution of neurohypophysial hor- TABLE 3. Levels of TRH in the brain and pituitary complex of the ammocetes larva of lamprey and of amphioxus. a Species Lamprey (4) b (ammocetes) (Petromyzon marinus) Amphioxus (4) (Branchiostoma lanceolatum) Whole brain 0 38 (25-60)" "Jackson and Reichlin, 1974a. b Number of animals examined. c Pg/mg tissue wet weight. " Range of values. e Pg/whole undissected head. ' Pg/whole pituitary. 2e Pituitary complex' 145 Not examined mones. In a sense, the pituitary has "coopted" TRH as a regulatory hormone. Pineal I also found TRH to be present in the frog (Rana pipiens) pineal in high concentrations which are influenced by the degree of photoillumination; changing seasons are associated with swings in pineal TRH concentrations as much as fold (Jackson et al., 1977). Comparable findings have also been reported by Kiihn and Engelen (1976) who, with an in vivo bioassay in the rat, have demonstrated a seasonal variation in the PRL and TSH-releasing activity in the hypothalamus of the frog. The function of TRH in the frog pineal is unknown, but the circannual rhythm and effect of illumination bespeak for a role in neurotransmission and this is supported by evidence that TRH has an excitatory action on frog motoneurones (Nicoll, 1977). Blood Unlike TRH in mammals (Jackson and Reichlin, 19746), TRH circulates in the blood of Rana pipiens in high concentration ( ng/ml) and shows rapid degradation in vitro with a tl/2 of 1.8 min at 26 C and 0.95 min at 37 C (Jackson and Reichlin, 1977c). An extract of frog blood containing 100 ng IR-TRH produced a TSH

7 DISTRIBUTION OF HYPOTHALAMIC HORMONES 391 rise in the rat in vivo comparable to synthetic TRH, 100 ng, while a sample of the same frog blood, allowed first to incubate at 37 C, contained only 12 pg IR-TRH in the extract and caused no elevation in rat TSH (Jackson and Reichlin, 1977c). These studies support the authenticity of frog blood IR-TRH. Since frog brain weighs only mg, and contains approximately 100 ng TRH, it seems unlikely that brain TRH can account for the bulk of blood TRH. Skin Examination of organ distribution showed huge quantities of immunoreactive and bio-active TRH in the skin (mean 48 ng/mg protein) (Fig. 2), concentrations 3-4 times that of the hypothalamus; the retina contained lesser quantities of TRH, while thoracic or gastrointestinal organs contained no significant levels of TRH (Table 4). Since frog skin is approximately 10 g in weight I estimate frog skin to contain over Time (minutes) EXTRACT OF FROG SKIN FIG. 2. Effect of an extract of skin from the frog (Rana pipiens) on the release of TSH in the ratm vivo. The skin was extracted in 90% methanol and the dried supernatant, reconstituted in buffer, was assayed for IR-TRH content. Skin extract containing 100 ng IR-TRH, made up to 1 ml with saline, was injected IV into each of 5 Sprague-Dawley male rats, under nembutal anesthesia, and blood sampled at 2 and 5 min for TSH measurement. Each of the 5 control rats received saline alone. Results show mean ±S.E.M. rise in serum TSH. The skin extract exhibited biologic potency appropriate to its content of IR-TRH. Saline treated controls showed no TSH rise. (From Jackson and Reichlin, 1977a) TABLE 4. TRH concentration in various frog (Rana pipiens) tissues removed from a group of four animals. 3 Organ Hypothalamus Extrahypothalamic brain Spinal cord Splanchnic nerve Skin" Retina 0 Heart Lung Tongue Stomach Intestine Liver Spleen Kidney Gonad Muscle Blood TRH (pig per g of protein) 14.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± /xg/ml whole blood a The blood TRH level is given for comparison. Results shown mean ± S.E.M. (from Jackson and Reichlin, 1977a). b Protein content of skin is 15.7 ± 0.4% wet weight (n = 6). The mean skin: blood concentration gradient oftrhis91:l. c Tissue obtained from a separate group of 6 frogs. 50 fig TRH. In the frog the skin is an important organ in salt and water balance and excretion and generally in maintaining homeostasis. The specific function of skin TRH is unknown but the massive quantities of TRH present in the integument of this species suggests a role for this peptide in skin function. It appears that blood TRH is derived from the skin in this species and that frog skin is an active peptide secreting organ. This study provides evidence that the physiological role of TRH, like that of other neural peptides such as somatostatin (Arimura et al., 1975), is not restricted to the CNS (Jackson and Reichlin, 1977a). Support for the authenticity of the TRH present in the frog skin is further provided by the work of Yasuhara and Nakajima (1975) who described the occurrence of a tripeptide chemically characterized as pyroglutamylhistidyl prolineamide, in an extract of skin from the Korean frog, Bombina orientalis.

8 392 IVOR M. D. JACKSON LUTEINIZING HORMONE-RELEASING HORMONE Distribution of LH-RH in mammalian species It seems clear that the LH-RH decapeptide isolated and synthesized in Schally's laboratory is a physiologic releasing hormone for both LH and FSH from the mammalian pituitary (Schally et al., 1973, for review). In the rat, immunoassayable and bioassayable LH-RH are present in the medial basal hypothalamus especially in the arcuate nucleus (ARC), and in the preoptic suprachiasmatic tissue (Wheaton et al., 1975). As for TRH and somatostatin, deafferentation of the rat hypothalamus causes a marked reduction in the LH-RH content of the medial basal hypothalamus (MBH) suggesting that such LH-RH arises from, or is controlled by, cells elsewhere in the brain (Brownstein et al., 1976). This view is supported by work from our group. Mice given monosodium glutamate show degeneration of > 80% of the cell bodies in the ARC, but the LH-RH content, and intensity of immunohistochemical staining is not affected. This suggests that LH-RH may not be synthesized there, but transported to the ME by axons passing through the ARC (Lechan et al., 1976). The data are consistent with the hypothesis of a dual central influence on pituitary gonadotropin secretion the LH-RH passing through the MBH controlling the tonic, and the LH-RH from the pre-optic suprachiasmatic nuclei the cyclic, secretion of LH. However the LH-RH in the ARC of the rat (and mouse) involved in the tonic discharge of LH from the anterior pituitary may in fact be synthesized in the preoptic area (Kalra, 1976). It seems likely that species differences exist, for surgical isolation of the MBH of the guinea pig causes only a slight reduction in LH-RH content of the ME (Silverman, 1976). This data implies an LH-RH synthesizing locus intrinsic to the MBH and in this regard the guinea pig resembles the monkey (Krey et al., 1975) rather than the rat. LH-RH has been detected in extracts of fetal human brain as early as 4 1/2 weeks (Winters et al., 1974). In the adult human brain, Barry (1977) has observed LH-RH positive perikarya, scattered from the septo-pre-optic region up to the retromammillary area, that give rise to two main tracts ending in the infundibulum and the lamina terminalis. The highest concentrations of LH-RH occur in the preoptic region and in the pituitary stalk, the proximal portion of which is the homologue of the rat median, eminence (Okon and Koch, 1976). Initial reports of large quantities of LH-RH in extracts of rat extrahypothalamic brain (White et al., 1974) have not been confirmed by us. Rather, we have found that LH-RH in extrahypothalamic brain of the rat amounted to only 17% of total brain LH-RH (Jackson, in preparation). We have also reported negligible levels of LH-RH in the extrahypothalamic brain of the mouse (Lechan et al., 1976). The absence of LH-RH in brain tissues outside the hypothalamus (in marked contrast to TRH) was also reported in humans (Okon and Koch, 1976). Distribution of LH-RH in lower vertebrates LH-RH is detectable in the chicken and shows immunochemical and chromatographic similarity with the mammalian decapeptide (Jeffcoatee<a/., 1974). However, previous studies by G. L. Jackson (1971) suggested that chicken LH-RH was not the same as mammalian LH-RH on the basis of differences in the chromatographic properties of biologically active fractions on ion-exchange columns. Immunohistochemical staining for LH-RH was reported by dereviers and Dubois (1974) in the median eminence of the cockerel, and by McNeill et al. (1976) in the duck where nerve fibers were observed to be projected to the portal systems of both cephalic and caudal parts of the anterior pituitary where it probably regulates both LH and FSH secretion. Exogenous LH-RH given to the cockerel induces a rapid rise in LH (Turret al.\ 1973). In amphibia, LH-RH is effective in stimulating gonadal function (Mazzi et al., 1974; Thornton and Geschwind, 1974). Studies by ourselves (Alpert et al., 1976a) m

9 DISTRIBUTION OF HYPOTHALAMIC HORMONES 393 * and others (Deery, 1974; Doer-Schott and Dubois, 1976; Goos et al., 1976) have demonstrated the presence of immuno-reactive LH-RH in amphibian hypothalamus. In frogs (Rana pipiens and Rana catesbeiana) immunoreactive LH-RH was found within neuronal perikarya in the diagonal band of Broca and in the median septal nucleus intermingled with non-immunoreactive neurons (Figure 3). These findings are comparable to those of Goos et al. (1976) who report IR-LH-RH in perikarya in front of the preoptic recess and giving rise to axons radiating to the outer zone of the ME in R. esculenta. The presence of distinct bundles of LH-RH containing fibers extending from the vicinity of the neuronal cell bodies to the ME is evidence for the existence of an LH-RH peptidergic septo-infundibular-axonal pathway in frogs (Alpert et al., 1976a). This pathway probably functions in the control of gonadal activity since transection of the.1 ">*,;f ' /? «'..*.. - * 1 t K FIG. 3. Immunohistochemical staining for luteinizing hormone-releasing hormone (LH-RH) in the median septal region of R. pipiens. The immunopositive neuronal perikarya appear dark. (Alpert etal., 1976a) hypothalamus behind the optic chiasm (a procedure which severs the LH-RH containing fibers as well as the pre-optico hypophysial pathway,) prevents ovulation in amphibia. The existence of such a "higher" center regulating cyclical gonadotropin activity located outside the hypothalamus has been postulated by Dierickx (1967) in the frog. The latter investigator also postulated the presence of a tonic gonadotropin regulating center in the ventral hypothalamus, but neither ourselves nor Goos et al. (1976) found LH-RH positive cells in such an area. The only published data providing measurements of the total amount of LH-RH in the amphibian hypothalamus is that of ourselves (Alpert et al. 1976a), who found 3.3 ng/hypothalamus in the frog, Rana pipiens, and of Deery (1974), who reported 0.9 ng in the toad, Xenopus laevis. Our studies demonstrated that 16% of total brain LH-RH was located outside the hypothalamus (a value similar to that obtained in mammals) within the telencephalon-septum-optic chiasm regions. Since we extracted these tissues with 90% methanol the absolute levels need to be re-examined after acetic acid extraction. Carp or trout hypothalamic extract stimulates LH secretion from ovine pituitaries in vitro (Breton et al., 1972). Exogenous mammalian LH-RH stimulates gonadotropin secretion in the fish, but extracts from teleost hypothalamus produce different profiles of teleost gonadotropin secretion than the LH-RH decapeptide (Breton and Weil, 1973). However Deery (1974) was unable to find IR-LH-RH in the extracts of hypothalamus from dogfish or goldfish. Since the sensitivity of his RIA was 80 pg, it is possible that significant levels were undetected. Recently, in the trout fish, Goos and Murathanoglu (1977), by immunohistochemical staining, have localized LH-RH to perikarya in the forebrain and their axons. However no fluorescence was observed in the nucleus preopticus or nuclear lateralis tuberis areas that previously have been correlated with reproductive activity in the fish (Peter, 1973). It still remains to be settled whether another gonadotropin releasing hormone

10 394 IVOR M. D. JACKSON is present in the fish (or indeed in other vertebrates). Distribution tissues SOMATOSTATIN of somatostatin in mammalian Nervous system. Somatostatin, a tetradecapeptide isolated from ovine hypothalamus (Brazeau et al., 1973), initially shown to inhibit growth hormone secretion from the anterior pituitary, was subsequently shown to inhibit TSH secretion as well as gastrointestinal hormone, pancreatic endocrine and exocrine secretion (Vale et al., 1974a; Gomez-Pan and Hall, 1977; for review). Both by bioassay (Vale et al., 19746) and radioimmunoassay (Brownstein et al., 19756) somatostatin has been found widely distributed throughout the mammalian extrahypothalamic brain, including the pineal gland. Using an immunoperoxidase technique, somatostatin has been localized in the circumventricular organs, in addition to the external zone of the median eminence (ME) (Pelletier et al., 1975). Somatostatinergic neurons have been detected immunohistochemically in the anterior periventricular hypothalamus and in the pre-optic area, in part outside the confines of the hypothalamus (Alpert^ al., 19766). It is likely that these neurons inhibit the release of GH, and probably also TSH, from the anterior pituitary. Hypothalamic deafferentation caudal to the optic chasm has been shown to markedly reduce the immunoassayable and immunohistochemical content of somatostatin in the medial basal hypothalamus (Jackson, 1977) suggesting that, like TRH, extrahypothalamic neural somatostatin is synthesized in situ. With the indirect immunofluorescence technique somatostatin has been detected in some neuronal cell bodies in spinal dorsal root ganglia, as well as in fibers in the substantia gelatinosa of the spinal cord (Hokfelt et al, 19756). Gastro-intestinal tract. Somatostatin is present in mammalian stomach and pancreas {A.r\muT3.etaL, 1975) where it is localized in the argyrophilic D (Aj) cells (Hokfelt et al., 19756) and in nerves in different layers of the small and large intestine (Hokfelt et al., 19756). The distribution of somatostatin in the gastrointestinal tract corresponds with its site of action in inhibiting glucagon, insulin, gastrin and HC1 secretions (Gomez-Pan and Hall, 1977). It seems likely that somatostatin is formed in situ in the gastrointestinal tract and may have a physiologic role in the regulation of many gut hormones. Somatostatin in submairunalian species. A survey of the phylogenetic distribution of somatostatin in a number of different vertebrates has been reported by Vale et al. (1976). The highest concentration of somatostatin in the rat was found in the hypothalamus although the gastrointestinal tract contains the greatest amount of somatostatin. Somatostatin was found in the brain and pancreas of the frog, catfish, torpedo and hagfish. In studies performed in this laboratory we found somatostatin to be present in the skin of the frog (Rana pipiens) (Jackson, unpublished). The significance and function of somatostatin in chordates requires further study. Recently a gene for somatostatin was chemically synthesized and fused to plasmid elements in E. Coli. These organisms were subsequently able to synthesize the polypeptide in vitro (Itakurae/ al., 1977). FUNCTION OF THE EXTRAHYPOTHALAMIC DIS- TRIBUTION OF THE RELEASING HORMONES The widespread distribution of TRH, LH-RH and somatostatin in brain and/or neural tissue remote from the hypothalamus suggests that these substances could have a role in neuronal function apart from anterior pituitary regulation. The evidence supporting such a view is summarized as follows: Anatomic and phylogenetic distribution The large quantities of TRH and somatostatin found in the mammalian extrahypothalamic brain are independent of hypothalarnic secretion as determined b)

11 DISTRIBUTION OF HYPOTHALAMIC HORMONES 395 studies utilizing surgical ablation or lesions isolating the hypothalamus from the rest of the brain (Jackson and Reichlin, 19776; Brovvnstein et al., 1975a). The location of TRH in several cranial nerve nuclei of the brain stem and motor nuclei of the spinal cord (Hokfelt et al., 1975a) and of somatostatin in dorsal root ganglia (Hokfelt et al., 19756), as well as the subcellular location of these substances in synaptosomes (Bennett etal., 1975; Styne etal., 1977) suggest these peptides might function as neurotransmitters. The presence of TRH in neuronal tissues of vertebrate and invertebrate species (Jackson and Reichlin, 1974a; Taurog et al., 1974; Grimm-j0rgensen et al., 1975) in which TRH clearly has no role in the regulation of any pituitary-thyroid axis that might be present, lends credence to this hypothesis. Specific synthesizing and degrading systems and receptors in brain tissue for hypothalamic peptides Grimm-j0rgensen and McKelvy (1974) have demonstrated the in vitro synthesis of TRH by hypothalamic and forebrain fragments from adult newts (Triturus viridescens) and active enzyme degrading systems for all three hypophysiotropic principles have been demonstrated in extrahypothalamic brain tissue of the rat (Griffiths, 1976). Further, high affinity binding sites for TRH in the synaptic membrane fraction of rat extrahypothalamic brain tissue have been demonstrated (Burt and Snyder, 1975). These workers have shown that such receptors have properties similar to those of pituitary membranes. Neurophysiologic studies The hypothalamic hormones have been shown to have a profound effect on the electrical activity of single neurons. Renaud et al. (1975) applied TRH, LH-RH and somatostatin directly to central neurons microiontophoretically and reported a marked depressant action on the activity of neurons at several levels of the central nervous system. Nicoll (1977) reported an excitatory action of TRH on R. pipiens spinal motoneurons in contrast to its inhibitory action on supraspinal neurons described above, suggesting that TRH might act as an excitatory transmitter on one cell type and as an inhibitory transmitter on another. Behavioral and CNS effects The hypophysiotrophic hormones have marked central nervous system (CNS) effects unrelated to their role in the regulation of pituitary function. Hypophysectomized mice pretreated by pargyline, a monoamine oxidase inhibitor, show enhancement of the motor activity induced by L Dopa when TRH is concomitantly administered (Plotnikoff et al., 1972). The tripeptide enhances cerebral norepinephrine turnover (Keller et al., 1974) and has profound effects on thermoregulation (Metcalf, 1974). It is of note that somatostatin shows prolongation of barbiturate anesthesia and shortening of strychnine seizure activity in the rat, effects that are opposite those produced by TRH (Brown and Vale, 1975). These behavioral effects are consistant with their anatomic location TRH being immunohistochemically detected around motor neurons and having predominantly motor activity whereas somatostatin present in sensory neurons appears to act primarily as a sensory depressant. LH-RH has been shown to stimulate sexual activity in rats in which gonadal function is held constant (Moss and McCann, 1973). CONCLUSIONS It is now clear that the regulation of anterior pituitary secretion is only one aspect of the function of the hypophysiotropic hormones. The anatomic, subcellular, and phylogenetic distribution of these peptides, the presence of specific brain receptors and active neuronal synthesizing and degrading systems, along with neurophysiologic and behavior studies provide powe "ful support for the view that these substances might operate as neurotransmitters. The evidence, at least for

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