Innervation of the Mammalian Anterior Pituitary: A Mini ReviewGONG JU*Institute of Neurosciences, The Fourth Military Medical University, Xi’an, 710032 PR China

KEY WORDS hypothalamus; adenohypophysis; peptidergic

ABSTRACT The mammalian anterior pituitary was not known to be innervated other than by afew autonomic nerve fibers. Recent studies, however, have demonstrated otherwise. A hypothesis ofneural-humoral dual regulation of the mammalian anterior pituitary has been postulated based onthe following findings: (1) the presence of substantial amounts of nerve fibers in the anteriorpituitary of a number of mammalian species; (2) close contact of the nerve fibers with the gland cells,even forming synapses; (3) the nerve fibers originate, as least partly, from the hypothalamus; (4) thenerve fibers respond actively to changes in hormonal levels of the organism; and (5) stimulation ofthe nerve fibers changes the secretory activities of the gland cells. Microsc. Res. Tech. 39:131–137,1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONThe hypothalamic regulation of the mammalian ante-

rior pituitary is known to be transmitted via the portalsystem (Harris, 1948). The hypophysiotropic neuronsin the hypothalamus (for review, see Swanson 1987)release their hormones at the median eminence, whichare then carried down the portal vessels to the anteriorpituitary. The corticotropin-releasing hormone-contain-ing neurons are distributed in the parvocellular regionof the paraventricular nucleus; the gonadotropin-releasing hormone neurons are found in the septal andpreoptic regions. They were once believed to be locatedin the arcuate nucleus, which was later found to be notthe case. The thyrotropin-releasing hormone neuronsare distributed in the parvocellular paraventriculararea, dorsomedial hypothalamic nucleus, and an arealateral to the paraventricular nucleus. The growthhormone-releasing hormone neurons are located in thearcuate nucleus, the area surrounding the ventrome-dial hypothalamic nucleus and extending to the periven-tricular region. The somatostatin neurons are distrib-uted at the periventricular region and the dopaminergicneurons which inhibit prolactin secretion are located inthe arcuate nucleus and the periventricular region.These hypophysiotropic neurons terminate densely inthe external lamina of the median eminence. Alsopresent in large amounts in the median eminence arenerve terminals from other sources. Not all of thetransmitters or modulators released in the medianeminence may be able to get access to the portal system,but at least many do.

It is generally acknowledged that the anterior pitu-itary has only a small number of autonomic nervefibers, mostly sympathetic, probably also parasympa-thetic (Aleshin, 1964; Rasmussen, 1938; Zacharias,1942). Aside from the autonomic nerve fibers, theanterior pituitary was not known to be innervated.According to Harris (1948), ‘‘there is no convincingevidence implicating the peripheral autonomic systemwith pituitary activity’’ and the anterior pituitary couldbe considered ‘‘a gland under nervous control butlacking a nerve supply.’’ Aleshin (1964), however, re-ported that stimulation of the superior cervical gan-

glion in the rabbit inhibited thyrotropin secretion andinfluenced follicle-stimulating function. The theory thatthe anterior pituitary is not directly regulated by thenervous system has met serious challenge recently.There is a growing body of evidence suggesting other-wise.

BRIEF REVIEW OF THE STUDIESON THE INNERVATION OF THE

ANTERIOR PITUITARYThe innervation of the anterior pituitary in lower

vertebrates has been relatively well studied (for review,see Ball, 1981). The median eminence-portal system isa very early event in the history of evolution. Asprimitive as in amphioxus there appear two pairs ofpeptidergic neurosecretory nuclei in the ‘‘brain blad-der,’’ which terminate synaptically on the ventral base-ment lamella of the ‘‘bladder.’’ This foreshadows themedian eminence and neurohypophysis. In some formsof cyclostomes, a rudimentary median eminence can befound, containing both peptidergic and aminergic nerveendings on ependymal processes that lie against theconnective tissue that interposes above the anteriorpituitary cells. In amphibians a typical median emi-nence-portal system constitutes the route of communi-cation between the hypothalamus and the anteriorpituitary. The portal vascular channel for humoralregulation of the anterior pituitary by the hypothala-mus is thus established. The anterior pituitary of theamphibians, meanwhile, is not directly innervated bythe hypothalamus. This relationship, once established,remained conserved through evolution. The only knownmajor exception is the teleosts, which do not have atypical median eminence-portal system but have devel-oped direct innervation, both peptidergic and aminer-gic, from the hypothalamus. A small amount of hypotha-lamic innervation can also be found in a few relatedspecies. For instance, the anterior pituitary of Ranatemparia tadpoles, but not of the adult, is sparsely

*Correspondences to: Gong Ju, Institute of Neurosciences, The Fourth MilitaryMedical University, 17 Chang Le Xi Road, 710032 PR China.

Received 1 February 1995; accepted in revised form 20 August 1995

MICROSCOPY RESEARCH AND TECHNIQUE 39:131–137 (1997)

r 1997 WILEY-LISS, INC.

innervated by hypothalamic monoaminergic neurons.Metuzals (1956), using Bielshowsky-Gros silver impreg-nation technique, stained dense secretomotor terminalnerve nets in the duck. This finding, however, has notbeen accepted without skepticism.

The innervation of the mammalian anterior pituitarywas a much-debated subject in the first half of thiscentury (for reviews see Rasmussen, 1938; Romeis,1940; Szentagothai et al., 1962). It has been claimedthat there were substantial amounts of nerve fibers inthe anterior pituitaries of the dog, cat, rabbit, and rat(Brooks and Gersh, 1938; Croll, 1929; Dandy, 1913;Hair, 1938; Pines, 1925), mostly using silver impregna-tion methods. Others, however, could find only occa-sional or a few fibers (see Rasmussen, 1938). Rasmus-sen (1938) studied the innervation of human anteriorpituitary and was able to find only a few nerve fibersrunning among the gland cells. Most of the nerve fibersin the anterior pituitary were related to the bloodvessels. Referring to previous studies, he justifiablypointed out that ‘‘many mistakes have arisen from afailure to realize the limitation of the technic used. Thetendency for reticular connective tissue to stain withcertain silver methods, and especially the much-usedBielshowsky technic has been particularly disturbing.’’Studies using silver methods continued to appear tillthe late 1950s. Using the Bielshowsky-Gros method,Metuzals described secretomotor nerve fibers in theanterior pituitaries of the horse (1954), and cat (1958).Szentagothai et al. (1962) concluded in their classicalbook Hypothalamic Control of the Anterior Pituitarythat ‘‘a careful analysis of their (of the earlier authors)descriptions and illustrations proves undoubtedly thatreticular fibers have been mistaken for nerve ele-ments. . . . Thus a true hypothalamic innervation ofadenohypophyseal tissue is probably . . . negligible inthe pars distalis. Even Metuzals who, on the basis ofreally excellent preparations (1956) was rather positivein his earlier view on the direct hypothalamic innerva-tion of the adenohypophysis (pars distalis) expressedhimself recently with more reserve (1959). For someyears we were inclined to share the views of J. Metuzals,but have recently been forced to accept the essentiallynegative conclusions of Green and Harris (1947).’’

With the advent of immunohistochemical technique,the innervation of the anterior pituitary has beenrecently reinvestigated. A small amount of nerve fibersof different chemical nature has been found in theanterior pituitary of the rat. Thus, while studying thedistribution of calcitonin gene-related peptide (CGRP)-immunoreactivity in the central nervous system, Rosen-feld et al. (1983) and Skofitsch and Jacobowitz (1985a)identified a few CGRP-immunoreactive (ir) nerve fibersin the anterior pituitary. Westlund and colleaguesreported the presence of 5-HT and somatostatin nervefibers (Westland and Childs, 1982; Westland et al.,1983). Mikkelsen et al. (1989) found substance P (SP)-irnerve fibers and speculated that they may be autonomicin nature. Gon et al. (1990), confirming the presence ofboth SP- and CGRP-ir nerve fibers in the anteriorpituitary, demonstrated colocalization of CGRP- andSP-immunoreactivities. Ju et al. (1993) later describedthe same phenomenon. An article reporting the pres-ence of a few tyrosine hydroxylase-ir nerve fibers,probably dopaminergic as assessed by the absence of

dopamine-b-hydroxylase immunoreactivity, appearednot long ago (Bayet et al., 1994). Also found present inthe nerve fibers of the anterior pituitary of the rat isGAP-43-immunoreactivity (Paden et al., 1994).

HYPOTHESIS OF NEURAL-HUMORAL DUALREGULATION OF THE MAMMALIAN

ANTERIOR PITUITARYIt started with the discovery of substantial amounts

of SP-ir nerve fibers in the anterior pituitary of ma-caque monkeys in this laboratory (Ju and Liu, 1989a;Liu and Ju, 1988). Tortuous varicose fibers were foundin patches and were distributed mainly in the dorso-posterior quarter of the median part of the gland (Fig.1). Although fibers could be found along the bloodvessels, the great majority of them weaved their waybetween the gland cells. At the stalk region nerve fiberswere found running down the stalk between the portalvessels. Further studies have demonstrated consider-able amounts of SP- and CGRP-ir nerve fibers in theanterior pituitaries of human, monkey, dog, and rat. Inthe pituitaries of human, monkey, and dog, some gala-nin (GAL)-ir nerve fibers were also identified (Ju et al.,1991a,b). The morphology of the different peptidergicnerve fibers is similar, but the amount of varicositiesvaries somewhat in different animal species. In the dogand rat, the nerve fibers also appear in patches but aredistributed throughout the gland, rather then largelyconfined to a certain part of it, as in the case of themonkey.

A crucial issue is the nature of the nerve fibers,vascular or glandular. Double immunostaining tech-nique has demonstrated in the monkey that the varicosi-ties of the SP-ir nerve fibers may come very close to anddirectly abut upon corticotrophs and somatotrophs (Juand Liu, 1989b,c; Fig. 2), and, indeed, on every cell typeof the anterior pituitary. A similar relationship hasbeen found in the dog. An electron microscopic study inthe rat by Kurosumi and Kobayashi (1980) has demon-strated bundles of nonmyelinated nerve fibers andterminals in the pericapillary connective tissue space orintercellular space among the glandular cells. Somato-trophs were most frequently attached by the nerveterminals and the corticotrophs were next in frequency.No synapses were found. Theret and Tamboise (1963)described close contact between vegetative neuritesand somatotrophs and follicular cells in the rat. Theyclaimed to have observed true synapses in the anteriorpituitary. Unfortunately, no convincing illustrationshad been produced. Their figure 7 for ‘‘pseudosynapses’’appears to me more like puncta adhaerens, and theirfigure 6 rather like non-specific staining of the mem-branes. Unsicker (1971) studied the anterior pituitaryof various mammals. They were unable to find electronmicroscopical evidence for a single axon. The closerelationship between the nerve fibers and the anteriorpituitary cells has been verified in our immunoelectronmicroscopic studies in the dog (Ju and Zhang, 1990,1992): SP- and CGRP-ir nerve fibers and varicositiescan be found in direct contact, nothing intervening,with every cell type of the anterior pituitary, includingthe folliculo-stellate cell. Moreover, the relationshipbetween the nervous elements and the gland cells is soclose that interdigitation between them is often seen.Of pivotal importance is the discovery of synaptic

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contacts, both SP- and CGRP-ir, with the gland cells.They are mostly asymmetrical, and can be found mainly,if not exclusively, on corticotrophs and somatotrophs(Figs. 3,4). They carry all the morphological characteris-tics of central synapses, with presynaptic and usuallyfairly prominent postsynaptic membranes, accumula-

tion of small clear synaptic vesicles at the presynapticside, a widened synaptic cleft with electron densematerial in it, and, sometimes, subsynaptic dense bod-ies. Large dense-core vesicles, as a rule, are seenscattered in the varicosities away from the presynapticcluster of small clear vesicles and often gather near the

Fig. 1. SP-ir varicous nerve fibers in the anterior pituitary of amacaque monkey. Also shown are a few SP-ir cells. 3360. Reproducedfrom Ju and Liu, 1989a, with permission of the publisher.

Fig. 2. Close contact of the SP-ir varicosities with a somatotroph inthe anterior pituitary of a macaque monkey. Oil, 31,600. Reproducedfrom Ju and Liu, 1989c, with permission of the publisher.

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plasmic membrane facing the extracellular spaces (Fig.4). The formation of synapses proves the motor natureof the nerve fibers, which apparently can in some wayregulate the activities of the gland cells. Being foundonly on corticotrophs and somatotrophs, it was specu-

lated that there may be two phases of hormone secre-tion during stress, an immediate neural phase followedlater by the humoral phase. Typical synapses have alsobeen demonstrated in the anterior pituitary of the rat,most frequently on lactotrophs (Liu et al., 1996). The

Fig. 3. An SP-ir terminal forming asymmetrical synapse (arrow)with a corticotroph of the anterior pituitary of a dog. 378,000.Reproduced from Ju and Zhang, 1990, with permission of the pub-lisher.

Fig. 4. ACGRP-ir terminal forming asymmetrical synapse(arrow) witha corticotroph of the anterior pituitary of a dog. Note the large densevesicles clustering near the extracelluar space (arrowhead). 336,000.Reproduced from Ju and Zhang, 1992, with permission of the publisher.

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discovery of synapses provides concrete support for thehypothesis of neural-humoral dual regulation of themammalian anterior pituitary.

The origin of the nerve fibers in the anterior pituitaryhas not been unequivocally determined. There areseveral possible sources: (1) Autonomic. The paucity intyrosine hydroxylase immunoreactivity in the anteriorpituitary (Bayet et al., 1994) speaks against the sympa-thetic system as the major source. (2) Trigeminal. Thetrigeminal ganglion is known to innervate the menin-ges. Therefore, it will not be surprising to find somesensory fibers in the anterior pituitary. However, periph-eral synapses are not an innate property of sensoryfibers. (3) Hypothalamic. A preliminary fluorogold retro-grade tracing study in the rat in this laboratory (unpub-lished data) has shown that they may be located in thehypothalamic periventricular region.

An interesting group of studies is the series ofinvestigations on changes of the anterior pituitarynerve fibers following hormone manipulations. It hasbeen demonstrated that following adrenalectomy in therat, the number of the CGRP-ir nerve fibers increasessignificantly (Ju et al., 1994), which implies an activeresponse of the nerve fibers to changes of hormonallevels of the organism. The increase in immunoreactivenerve fibers could be due either to an increase inpeptide content in the preexisting unstained nervefibers so as to render them detectable with immunohis-tochemical technique, or due to an actual increase inthe number of nerve fibers. A recent study with anti-body against GAP-43 (Growth Associated Protein, amembrane phosphoprotein specific to the nervous sys-tem and supposed to be related to axonal growth andsynaptic remolding, Benowitz and Perrone-Bizzozero,1991) has shown that there is a marked increase inGAP-43 immunoreactivity following adrenalectomy inthe rat, indicating an active axonal sprouting (Lu et al.,1995).

It is not clear at this moment what particular func-tions the nerve fibers in the anterior pituitary have.Studies in the rat by stimulating the nerve fibers of theanterior pituitary in vitro have demonstrated that thenerve fibers can change the secretory activities of thegland. Thus, when the nerve fibers were stimulated byelectric field (30 mA, 10 Hz) the ACTH secretion wasfound to be increased (Zhao et al., 1995) while prolactinsecretion was inhibited (unpublished data). The effectscould be blocked by TTX. SP- and CGRP-immunoreac-tivities have been demonstrated in the nerve fibers ofthe anterior pituitary in the rat. Furthermore, theoccurrence of a large amount of small clear synapticvesicles in the varicosities of the nerve fibers of theanterior pituitary (Ju and Zhang, 1990, 1992) impliesthe presence of classical transmitters as well. There isevidence that there may be differential release at thenerve terminals dependent on impulse frequency andfiring patterns (Edwards et al., 1982; Lundberg et al.,1982). Therefore, it is not at all clear exactly whatsubstance(s) had been released with the particularparameter of electric field stimulation used. SP, CGRP,and GAL have profound influence on the secretion ofthe anterior pituitary, mainly through the hypothala-mus (for review see Aronin et al., 1986; Bartfai et al.,1993; Fahim et al., 1990). The presence of large amountsof SP-, CGRP-, and GAL-ir terminals in the median

eminence (Bartfai et al., 1993; Hokfelt et al., 1978;Skofitsch and Jacobowitz, 1985a,b; Tsuruo et al., 1983)implies their possible regulatory roles in the anteriorpituitary. In vitro studies also proved that they can actdirectly on the anterior pituitary cells. Thus, incubationof hemipituitaries obtained from ovariectomized rats inmedium containing SP resulted in enhanced release ofprolactin (for review see Aronin et al., 1986). Mat-sumura et al. (1982) reported a stimulatory effect of SPon b-endorphin release. Most of the in vivo studies,however, gave negative results. SP has been found tohave no direct effect on the release of growth hormone(Arisawa et al., 1989b, Vijayan and McCann, 1979,1980), gonadotropin (Vijayan and McCann, 1979), thy-rotropin (Arisawa et al., 1989a; Vijayan and McCann,1980), and basal secretion of ACTH (Jones et al., 1978).Nevertheless, SP has been found to influence the effectsof other hormones on the anterior pituitary, thus play-ing a regulatory role. Thus, SP has been found to inhibitcorticotropin-releasing hormone or vasopressin-in-duced ACTH release (Jones et al., 1978; Nicholson etal., 1984), and to inhibit the stimulatory effect of LHRHon the release of LH and FSH (Kerdelhue et al., 1978).Fahim et al. (1990) showed that at low concentration,CGRP inhibited growth hormone release from dis-persed, overnight cultured anterior pituitary cells, whileat high concentration it increased growth hormonerelease; CGRP also enhanced prolactin release. Tat-suno et al. (1991) found that CGRP significantly stimu-lated interleukin-6 production from cultured dispersedanterior pituitary cells. The presence of binding sites ofthese peptides also favors their direct action on thepituitary cells (Kerdelhue et al., 1985; Larsen et al.,1989; Wimmalawansa et al., 1987). The major source ofthese peptides in the anterior pituitary is apparentlyfrom the median eminence. The SP, CGRP, and GALcontaining cells in the anterior pituitary (Gon et al.,1990; Ju and Liu, 1989a, 1990; Steel et al., 1989)probably can also release their peptides and exert theireffects via paracrine action. Direct influences of thesepeptides on anterior pituitary cells and the presence ofthe related binding sites or receptors, therefore, do notin themselves prove the existence of direct neuralregulation of this gland. Nevertheless, they do offer thematerial basis for neural activities. It does not meanthat the peptides released at the nerve terminalsshould necessarily have the same effects as demon-strated in vitro. Yet, without the evidence for thephysiological effects of these peptides on the anteriorpituitary and particularly the presence of their bindingsites or receptors, the possible roles of the peptidergicinnervation can hardly be substantiated.

The study on the innervation of the anterior pituitaryis still in its infancy. Much remains to be elucidated.Whatever the functions of the nerve fibers turn out tobe, the notion that the anterior pituitary is not directlyregulated by neural elements cannot remain the same.

SUMMARYAlthough we do not know yet what particular func-

tions the innervation of the anterior pituitary has andhow important this regulation may be, with the evi-dence presented here it seems fairly clear that directneural regulation does exist and it is justified to postu-late the hypothesis of neural-humoral dual regulation

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of the mammalian anterior pituitary. Moreover, giventhe active axonal sprouting in the anterior pituitaryfollowing adrenalectomy, it is unlikely that the directinnervation of the anterior pituitary plays a negligiblerole.

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137INNERVATION OF ANTERIOR PITUITARY