Developmental Organization ofNeurophysin Neurons in the Human Brain

JURGEN K. MAI,1* SABINE LENSING-HOHN,1 ANDRE A. ENDE,1

AND MICHAEL V. SOFRONIEW2

1Department of Neuroanatomy, H.-Heine-Universitat Dusseldorf, Dusseldorf, Germany2Department of Anatomy and MRC Cambridge Centre for Brain Repair,

University of Cambridge, United Kingdom

ABSTRACTNeurophysin (NPH) was detected immunohistochemically in 34 human brains ranging in

age from 10 weeks of gestation (wg) to 3 months postnatal. Weakly-stained NPH-immunoreactive (NPH-IR) cells were already aggregated in the lateral hypothalamus in thesupraoptic nucleus at 10 wg, the first time point examined. From this time, there was a clearand consistent chronology in the first appearance of NPH-immunoreactivity in different cellgroups progressing from the supraoptic nucleus at 10 wg to cells in the accessory NPH cellgroup at 13 wg, paraventricular nucleus at 14 wg, suprachiasmatic nucleus at 18 wg andvarious other well defined clusters in the basal forebrain at 18–20 wg. NPH-IR fibers werepresent in the hypothalamo-hypophyseal tract from 10 wg, and together with other availableevidence, our findings suggest the presence of a potentially functional hypothalamo-hypophyseal system by the end of the first trimester. NPH staining patterns and orientationsof cells suggest that NPH-IR cells originate from the region of the hypothalamic sulcus in amanner consistent with animal studies, and migrate to their settling areas before expressingNPH-immunoreactivity. In spite of the likelihood that most NPH-IR cells (with the probableexception of those in the suprachiasmatic nucleus) derive from a single primordium, the finalorganization of NPH-IR cells consists of many scattered groups, as seen in the late fetal period andmature brain. Developmental analysis provides further evidence that there is a high degree ofconservation in the topographic organization of the numerous diverse NPH-IR cell groups inhumans and other mammals, suggesting that the separation and organization of these groups maybe of functional importance. J. Comp. Neurol. 385:477–489, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: vasopressin; oxytocin; paraventricular nucleus; supraoptic nucleus; accessory NPH

cell group

The neuropeptides vasopressin (VP) and oxytocin (OT)and their associated carrier molecule neurophysin (NPH)are produced in a number of different cell groups withinand outside of the hypothalamus. These neuronal groupsshow a complex topographic and connective organization(Diericks and Vandesande, 1977; Sofroniew, 1985; Mai etal., 1993). While many neurons stain intensely for VP, OT,and NPH in the basal forebrain of untreated animals, andthe location and distribution of these cells appears to becomparable among many mammals including humans(Sofroniew et al., 1981; Sofroniew, 1983, 1985b; Mai et al.,1993), a few additional neuronal groups become visibleafter pretreatment of experimental animals with a trans-port inhibitor such as colchicine (Van Leeuwen and Caffe,1983; Sofroniew, 1985a). The diverse efferent projections ofthe various VP, OT, or NPH cell groups include bloodvessels of the neurohypophysis or hypothalamo-hypophy-seal portal system, as well as neuronal targets within

many regions of the central nervous system (CNS) rangingfrom portions of the limbic system to the brainstem andspinal cord in many mammalian species, including hu-mans (Sofroniew et al., 1981; Sofroniew, 1985b; Fliers etal., 1986; Mai et al., 1993). Accordingly, VP, OT, or NPH cellgroups may be involved in a broad array of neuro-endocrine, autonomic, circadian, and behavioral functions(Swanson and Sawchenko, 1983; Buijs, 1990; Meisenbergand Simmons, 1983). Although it is clear that certain VP,OT, or NPH cell groups have separate projections to bloodvessels or neuronal targets in the CNS, the functional signifi-cance and clinical relevance of the many VP, OT, or NPH cellgroups and their projections is not well understood.

*Correspondence to: Dr. J.K. Mai, Department Neuroanatomy, Geb.22.O3.U1, Heinrich-Heine-Universitat, P.O. Box 101070, D-40001 Dussel-dorf, Germany. E-mail: mai@uni-duesseldorf.de

Received 16 August 1996; Revised 14 March 1997; Accepted 11 April 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 385:477–489 (1997)

r 1997 WILEY-LISS, INC.

Determining changes in the number of morphologicallyidentified cells or fibers immunoreactive for VP, OT, orNPH, or changes in concentrations of these peptides ortheir mRNA’s or associated receptors in postmortem hu-man brain specimens from patients with documentedpathological states, may provide information regardingthe functions of different VP, OT, or NPH neuronal groups.While information is available with respect to the patho-physiology of some of the neuroendocrine VP, OT, or NPHcell groups, little is known about the function, in eitherhealth or disease, of groups with central projections.Nevertheless, a few studies suggest that changes in thenumber of VP, OT, or NPH cells in specific groups arepresent in certain disease conditions. For example, VP-immunoreactive cells were significantly decreased in thesuprachiasmatic nucleus in a group of patients withAlzheimer’s disease (Swaab et al., 1985; Goudsmit et al.,1992; Stopa et al., 1994), while NPH was significantlydecreased in the paraventricular nucleus and internalpallidal region, but increased in the substantia nigra, in agroup of patients with schizophrenia (Mai et al., 1993).

Developmental analysis of the various VP, OT, or NPHcell groups in the human forebrain will contribute to abetter understanding of the organization of the forebrainand will provide a baseline for future comparison withother markers of normal or abnormal development, ordisease. For example, changes in structure and cell num-ber have been reported in aging, AIDS, Parkinson’s dis-ease, and depression (Purba et al., 1993, 1994; Van derWoude et al., 1995; Purba et al., 1996). This study wasundertaken to provide such an analysis by examiningNPH-IR structures in a well-characterized chronologicalseries of specimens of developing human brains. NPH is asmall (ca. 10 kD) molecular weight protein which, by itself,has no known distinct functions, but is present as part ofthe precursor protein of both VP and OT (Land et al., 1982,1983). NPH is particularly stable to degradation andfixation, and serves as a sensitive immunoreactive markerfor the presence of VP and OT precursor molecules, butdoes not discriminate neurons specifically producing ei-ther of these peptides (Sofroniew, 1985b).

MATERIAL AND METHODS

Specimens

Paraffin sections through the hypothalamus of 31 fetaland 3 postnatal human brains, ranging from 10 weeks ofgestation (wg) to 3 months of age were used for this study(Table 1). Fetuses were obtained from therapeutic andspontaneous abortions. The brains were collected andexamined by the departments of Pathology and Neuropa-thology between 1989 and 1992, observing the local Ethi-cal Committee protocols. Procedures for collecting mate-rial conformed to the ethical guidelines of the HelsinkiDeclaration regarding informed consent (British MedicalCouncil, 1964). (Human Experimentation. Code of ethics ofthe World Medical Association and statement on responsi-bility in investigations on human subjects. Br. Med. J.2:177–180). The hypophysis was not recovered in any case.The age of the fetus is given as calculated gestational age,referred to the time of implantation, and was determinedfrom the reports of the last menstruation as well asmeasurements of crown rump length, crown feet length,brain weight, and body weight (Table 1). To verify theseestimates, additional data were collected: depth of matrix

layer, differentiation of the intermediate cell layer and ofnuclei, time-related expression patterns of various otherneuroactive substances in regard to systems development.

Paraffin embedding

The postmortem delay ranged between 2 hours and 3days. Whole brains were fixed in 4% or in 10% formalinand stored in this fixative for up to 2 years. Beforeembedding in paraffin, the brains were weighted andphotographs taken from the lateral, dorsal, and basalsurfaces. Afterwards all brains were postfixed for 3–5 daysin Bouin’s solution, dehydrated, and embedded in paraffin.Serial 20-µm-thick sections were prepared from all brains,and all sections were mounted on glass slides. The postna-tal brains were cut into approximately 1-cm-thick slicesafter fixation in formalin, and were dehydrated, embeddedin paraffin and cut in 20 µm serial sections. Every sectionwas mounted onto glass slides.

Immunohistochemistry

Within the area comprising the forebrain magnocellularhypothalamo-hypophyseal system, at least every 10thsection was stained for NPH. The immunohistochemicalprocedure was carried out according to Mai et al. (1993),with the peroxidase-antiperoxidase (PAP) method of Stern-berger (1979). The primary antiserum (NPH RB 41) wasapplied in a dilution of 1:300. After rinsing in Tris-phosphate-buffered saline (TBS) for 30 minutes, the sec-tions were incubated for 2 hours with sheep or swine IgG(diluted 1:100), rinsed again in TBS and incubated inperoxidase-antiperoxidase (1:75). Diaminobenzidine tetra-hydrochloride (0.05%) in TBS and hydrogen peroxide(0.3%) were used to develop the reaction product followinga final tissue rinse in 0.1 M TBS. According to the sameprotocol we applied a polyclonal antibody against vasoac-tive intestinal peptide (Peninsula, St. Helens, England,dilution 1:75) which has been shown to label the centralportion of the suprachiasmatic nucleus (see Mai et al.,1991). All sera were diluted in TBS containing 0.1% TritonX-100 and all incubations of sections with primary antibod-ies were carried out in a humidified chamber for 24 hoursat room temperature. Immunocytochemical specificity ofthe antibody has been tested earlier (see Sofroniew, 1985b).Half of the sections were counterstained with the hematoxylinmethod of Weigert.

Analysis

For macro- and microscopic evaluation, direct negativeprints of all NPH-immunostained sections (lumograms)were prepared at a magnification that depended uponbrain size and was between 36.5 and 313.5. Cameralucida drawings of pial and ventricular surfaces and ofrelevant histological landmarks were prepared, into whichthe topography of cells and the intensities of cell body andfiber staining were included. Landmarks apparent on thephotographs of the brain surfaces were correlated with the

TABLE 1. Characteristics of Specimens Used for This Study1

Weeks of gestation 10–15 16–20 21–24 25–birth birth–3 mo.Number of brains 7 7 7 10 3Mean CRL (cm) 6.6 14.8 20.0 29.3 36.4Mean CFL (cm) 11.6 22.5 31.0 56.2 53.8Mean body weight (g) 43.1 254.4 459.4 2281.1 3710.0Mean brain weight (g) 15.0 78.0 91.0 224.3 451.3Mean brain length (mm) 17.0 36.1 46.4 58.8 n.d.

1CFL, Crown feet length; CRL, crown rump length; n.d., not determined.

478 J.K. MAI ET AL.

surface contours of the histological sections. By this means,the angle of sectioning could be determined and was drawnon the photographs. Sections transsecting the middle ofthe anterior commissure were used as a reference pointfrom which to obtain equivalent section levels for compari-son in different brains. Since all brains were removed fromthe cranium before fixation, distortions of the tissuesometimes occurred, resulting in uneven section levels onboth hemispheres and differing cutting angles. Disruptionof brain tissue at the base of the forebrain sometimescaused artefactual opening of the third ventricle or discon-nection of the mediobasal prosencephalon. Most of theseartifacts could be compensated for, because first, serialsections were prepared from all brains; second, the dis-tance between the sections stained for NPH was kept shortand third, lumograms and camera lucida drawings en-abled a 3-D reconformation of such brain areas.

Nomenclature

The nomenclature used for the various nuclear groups ofNPH-IR cells has been adapted from previous studies ofdifferent mammalian species including humans (Ulfig etal., 1990; Armstrong, 1995; Mai et al., 1993). Cell groupsare listed in the abbreviations (Table 2).

RESULTS

Observations are described in chronological order ofspecimen age ranging from 10 wg to 3 months postnatal(Table 1), major changes at particular ages are high-lighted. Special reference is made to (i) the first appear-ance of NPH-immunoreactivity in various nuclear groupsand fiber bundles within the region of the basal forebrain(Table 2), (ii) changes in the state of differentiation ofdifferent NPH-IR cells, and (iii) the gradual maturation of thetopographic organization of the nuclear groups of NPH-IRcells.

10 weeks of gestation

NPH-immunoreactive (NPH-IR) cells and processes werepresent at 10 wg, at the first examination. At this time,

weakly stained NPH cells were found clustered in thelateral hypothalamus dorsal to the optic tract (Fig. 1). Thiscluster had no contact with the pial surface. The NPH-IRcells had a uni- or bipolar morphology and the reactionproduct appeared concentrated around their somata andoutlined their processes. These processes were most oftenoriented perpendicularly to the fibers of the optic tract,and only cells located in the immediate proximity of thetract showed processes oriented parallel to it (Fig. 1B).Numerous varicose NPH fibers were located within theoptic tract and chiasm and in the overlying hypothalamus.At this early stage densely packed fibers close to themidline and in the retrochiasmatic area already form thehypothalamo-hypophyseal tract (HHT) (Fig. 1d). In addi-tion, varicose fibers were already present posteriolaterallyto the SON extending all the way to the level of the futureventral tegmental area below the cerebral peduncle (Fig.1A). These fibers were observed only at 10–12 wg.

13 wg

At 13 wg NPH-IR cells of the SON were placed laterally(Fig. 2A) and dorsally to the optic tract in the shape of abroad flattened structure. NPH-IR SON cells that wereclose to the pial surface appeared undifferentiated withfew or no processes (Fig. 2B), whereas cells located dor-sally in the SON had a larger diameter and one to threeimmunoreactive processes oriented in a dorsomedial direc-tion. From this area a band of scattered, well-differenti-ated cells extended towards the hypothalamic sulcus (Fig.2A). This band, collectively named accessory NPH cellgroup, had a wide expansion in both the anterior-posteriorand dorso-ventral directions, reaching the telencephalicborder laterally and the ventral tegmental area caudally.At this time, the PVN showed no NPH-IR cells. The optictract formed a compact bundle, and varicose fibers werelocated dorsal to and outside the optic tract (Fig. 2C).

14 wg

At this time, the NPH-IR cells of the SON formed threemajor clusters linked together by strands of scatteredcells. The main dorsomedial cluster extended along thedorsal border of the optic chiasm and tract (Fig. 3A,B), theother two were located medially and laterally to the crestof the optic tract. Cells of the latter portion, located close tothe pial surface, extended to the telo-diencephalic borderand contained densely packed and poorly differentiatedcells, a characteristic feature until the time of birth (Figs.4F, 5B, 7D). The accessory NPH cells began to formdistinct clusters (Fig. 3C). Within the PVN the firstNPH-IR cells were found at 14 wg, and formed a looselypacked group of well differentiated cells with intenselystained processes. The medial border of NPH-IR cells inthe PVN was located some distance from the ventricle anteri-orly and became increasingly close to the ventricular border inthe caudal direction, but never reached the ependymal lining.During the consecutive 4 weeks, NPH-IR PVN cells graduallyacquired their typical juxta-ependymal location.

16 wg

At 16 wg the accessory NPH cell group, intermediatebetween the SON and PVN, was well developed andformed a continuous stream of cells diagonally betweenthe SON and dorsal PVN. These cells were frequentlyassociated with blood vessels. PVN NPH-IR cells extendedin parallel to the ventricle from the region dorsal to the

TABLE 2. First Appearance of NPH-immunoreactivity in Fibers and Cellsin the Developing Human Basal Forebrain1

Week ofgestation 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fibers in basalforebrain 1

Cells in SON 1Cells in ACG 1Cells in PVN 1Cells in ACG

associatedwith bv 1

Fibers betweenependymalcells 1

Cells in SCN 1Cells in PL/SL 1Cells adjacent to

internal cap-sule 1

Cells in BNST 1Cells in DPF 1Cells in MFB 1Cells within GPi 1

1ACG, Accessory NPH cell group; BNST, bed nucleus of stria terminalis; bv, blood vessel;DPF, dorsal parafornical nucleus; GPi, internal segment of the globus pallidus; MFB,medial forebrain bundle; PL/SL, pallidal/sublenticular complex; PVN, paraventricularnucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; 1, time of firstappearance of NPH-immunoreactivity in fibers and cells.

NPH NEURONS IN THE DEVELOPING HUMAN BRAIN 479

hypothalamic sulcus ventrally to the dorsal border of thefuture SCN. At this age it was already apparent thatNPH-IR cells in the SON were densely packed, whereasNPH-IR cells in the PVN were more loosely arranged withmany nonimmunoreactive cells scattered in between. Thischaracteristic difference in cell density was preservedthrough subsequent stages of development (Figs. 4F–I, 7D,F).

18–19 wg

By 18 wg, the distribution of NPH-IR cells had estab-lished a pattern similar to that found in the newborn, andwas characterized in most regions by intensely stainedcells and branching processes (Fig. 4). NPH-IR cells of the

dorsal PVN now showed a more advanced stage of differen-tiation as compared with the ventral division (Fig. 4G–I).Ventrally the PVN showed a precise lateral demarcation,whereas dorsally at the level of the hypothalamic sulcusstrands of cells spread laterally to merge with cells of theaccessory NPH cell group (Fig. 4A–G). During the follow-ing weeks, cells of this latter group extended in severaldirections. In particular, numerous NPH-IR cells accumu-lated along the medial perimeter of the globus pallidus(Fig. 4A–D). By 18 wg NPH-IR fibers in the vicinity of thePVN extended between ependymal cells and came intoclose contact with the third ventricle. Within the SCN, anumber of small cells began to exhibit NPH-immunoreac-

Fig. 1. A–D: Coronal sections through the retrochiasmatic area of a10 wg human brain stained for neurophysin (NPH). A: Camera lucidadrawing (inset shows entire section) showing the distribution ofNPH-IR varicose fibers and cells in the basal forebrain. Arrowsindicate fibers extending from the supraoptic nucleus (SON) to theventral tegmental area. Boxes indicate areas shown in B–D. B:NPH-IR cells and processes dorsomedial to the SON are oriented

towards the hypothalamic sulcus (arrows). Processes of cells withinthe forming SON close to the optic tract (OT) are oriented in parallel tothe pial surface (arrowhead). C: NPH-IR cells of the SON and varicosefibers (arrows) within the neighboring OT. D: NPH-IR varicose fibersin the dorsal part of the optic chiasm (OCh) and the overlying area inthe midline (arrow). 3V, third ventricle, PM, pial membrane. Scalebars 5 12.5 µm in B,C and 5 50 µm in D.

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tivity along the margins of the cell somata, but positive cellprocesses were not recognizable. By 18–19 wg, NPH-IRcells were found along the ventral margin of the anteriorlimb of the internal capsule (Figs. 4A,5A).

20–23 gw

By 20 wg, there appeared to be a distinction betweenmagnocellular and parvocellular NPH-IR cells located indorsolateral and ventromedial parts of the PVN. Duringthis time, various NPH-IR cell groups became apparent inseveral additional locations. From 20 wg distinct groups ofNPH-IR cells could be distinguished in the bed nucleus ofthe stria terminalis and dorsal parafornical region. NPH-IRcells also sweep laterally from the SON to distributewithin the medial forebrain bundle (Fig. 5A–C). Thesecells were separated from the NPH-IR cells which were

arranged within and immediately below the ansa lenticu-laris and which belong to the pallidal/sublenticular NPH-IRcell group (Fig. 5B,C). At 23 wg, the organization of theSCN began to reflect the pattern seen in the adult (Mai etal. 1991), with distinct subdivisions of a dorsal crescent ofNPH-IR cells around a central core of NPH-negative andVIP-positive cells (Fig. 6A–D). By 23 wg a few scatteredNPH-IR cells were observed within the internal segmentof the globus pallidus.

25 wg to birth

At 25 wg the NPH-IR paraventriculo-supraoptic tractwas well-developed and most cells of the accessory NPHgroup were oriented between these fibers reflecting thepattern seen shortly after birth (Fig. 7C). From 25 wg untilbirth there was a progressive increase of NPH-immunore-

Fig. 2. A–D: Coronal sections through the retrochiasmatic area of a13 wg human brain stained for NPH. A: Camera lucida drawing (inset:entire section) showing the distribution of NPH-IR varicose fibers andcells (dots) in the basal forebrain. Boxes indicate areas shown in B andC. ACG: Accessory NPH cell group. B: NPH-IR cells in the supraoptic

nucleus lateral (SON 1) and dorsal (SON d) to the optic tract (OT).Arrows labeled SON 1 and SON d indicate respective nuclei. C:NPH-IR fibers dorsal to the OT in the lateral hypothalamus. D: Detailof the area delimited by broken lines in B, but taken from aneighboring section. Scale bars 5 50 µm in B,C and 5 12.5 µm in D.

NPH NEURONS IN THE DEVELOPING HUMAN BRAIN 481

activity in these fibers reaching by 3 months an intensityfound in the adult neurohypophyseal system (Fig. 8). Thenumber of NPH-IR cells in the pallidal/sublenticular groupthat was present in any one section was now decreased dueto the expansion in size of this region and the encroache-ment of the medial perimeter of the globus pallidus (Fig.7A,B). From 25 wg the NPH-immunoreactivity of SCNcells was also remarkably higher and cells exhibitedNPH-IR processes.

Birth to third postnatal month

During the early postnatal period, there was little change inthe overall organization of NPH-IR cells, but a generalincrease in immunoreactivity predominantly of the magno-cellular hypothalamo-neurohypophyseal system occured(Fig. 8C). In addition, there was further maturation of cellphenotype. In particular, NPH-IR SON-cells located alongthe pial surface (Fig. 8A,B) soon became well differentiated.

Fig. 3. Coronal sections at the tuberal level of a 14 wg human brainstained for NPH. A: Camera lucida drawing (inset: entire section)showing the distribution of NPH-IR cells in the basal forebrain. Alldots represent cells (fibers are not marked). Boxes indicate areasshown in B–D. B: NPH-IR cells and fibers in the SON dorsal to the OT

extending towards the accessory NPH cell group. C: NPH-IR cellsscattered in the accessory NPH group showing randomly orientedprocesses. D: NPH-IR cells in the dorsal division of the supraopticnucleus (SON d). C and D: Counterstaining with hematoxylin; Scalebars 5 50 µm in B–D.

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Fig. 4. Coronal sections at the tuberal level of a 18 wg human brainstained for NPH immunoreactivity. A: Camera lucida drawing (inset:entire section) showing the distribution of NPH-IR cells in the basalforebrain. All dots represent cells (fibers are not indicated). Themarked boxes correspond to the areas represented in the accompany-ing photographs. B: Survey of NPH-IR cells in the SON and ACG,including the parafornical nucleus (PF) and pallidal/sublenticularcomplex of NPH cells (PL/SL). C: Detail of B showing NPH-IR cells inthe pallidal portion of the PL/SL. D: Detail of B showing NPH-IR cells

in the sublenticular portion of the PL/SL. E: Detail of B showingNPH-IR cells of the ACG. F: Detail of B showing NPH-IR cells in theSON. Note the undifferentiated state of most neurons. G: Survey ofNPH-IR cells in the paraventricular nucleus (PVN). Note the decreasein cell density from dorsal to ventral. Arrows indicate cells spreadinglaterally from the hypothalamic sulcus (HS). Fx: Fornix. H,I: Detailsof G showing respectively, NPH-IR cells in the dorsal and ventral PVN.Scale bars 5 200 µm in B, 50 µm in C–E, 12.5 µm in F, 200 µm in G,50 µm in H,I.

DISCUSSION

These findings show that NPH is expressed early in thedevelopment of the human basal forebrain and can be usedto follow the maturation of a variety of different cellgroups. The general topographic distribution of NPH-IRcells and fibers observed in this study showed a remark-able degree of consistency from specimen to specimen, andis in agreement with the few reports on NPH and itsrelated peptides during human development. While theNPH immunoreactivity does not distinguish between VPand OT neurons, it provides a sensitive marker for theanalysis of the basic organization of this system andappears to allow prior detection of the precursor proteinsthan does staining for the peptides themselves (see below).The findings are of relevance to certain aspects of hypotha-lamic development, the potential role of neurohypophysealpeptides in the development of the fetus, the consistency ofthe organization of NPH-IR cells in vertebrates, and thepotential involvement of NPH-IR cells in human diseases.

Chronology of developmentof NPH-IR structures

The presence of NPH-immunoreactivity in cells andfibers at 10 wg is in agreement with Fellmann et al. (1979)who described NPH- and VP-IR SON cells and axons, andwith Burford and Robinson (1982) who detected VP byradioimmunoassay in hypothalamic and hypophysealpreparations between 11–12 wg. The time of appearance ofNPH-immunoreactivity in the SON roughly coincides withthe time that this nucleus becomes structurally identifi-able (see Limire et al., 1979) as well as with the arrival ofthe optic tract fibers in this area (Cooper, 1945). It ispossible that the arrival of the optic tract fibers provides acue for the settlement of the migrating SON cells at thistime and provides guidance for the developing HHT. Thisrelationship may explain why varicose fibers at this timeare found amongst the optic tract fibers passing towardsthe hypophysis.

Fig. 5. Coronal sections at the tuberal level of 21 and 23 wg humanbrains stained for NPH immunoreactivity. A: Camera lucida drawing(inset: entire section) showing the distribution of NPH-IR cells in thebasal forebrain at 23 wg. All dots represent cells (fibers are notindicated). Boxes indicate areas shown in D and E. B: Survey ofNPH-IR cells in the dorsal and lateral supraoptic nucleus (SON d andSON 1), the medial forebrain bundle (MFB) and PL/SL (arrows) at 21

wg. Unlabeled arrow points to the lateral margin of the dorsalextension of the SON d·GP: Globus pallidus. C: Survey of the accessoryNPH cell group with clustering of NPH cells in the PL/SL (arrows) andassociated with the MFB (arrow). D,E: Higher magnifications ofNPH-IR cells in the PL/SL, counterstained with hematoxylin. Scalebars 5 200 µm in B, C, 12.5 µm in D,E.

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Fig. 6. Coronal sections at the level of the suprachiasmatic nucleus(SCN) of a 23 wg human brain stained for NPH immunoreactivity orVIP immunoreactivity. A: Schematic drawing showing the organiza-tion of NPH-IR cell groups in the hypothalamus at the level of the opticchiasm (OCh). Note that (mostly parvocellular) NPH-IR neurons aredistributed between the paraventricular nucleus (PVN), supraopticnucleus (SON) and SCN. Dark shading indicates areas of densecollections of NPH-cells. In areas with light shading, NPH-cells areconsistently found, however in low numbers. B: Survey of NPH-IR

cells in the anteroventral PVN and the SCN. Only the dorsal andventral SCN (SCN d and SCN v) contain NPH-positive cells, whereasits central division (SCN c) stays free of NPH immunoreactivity (*:artifact). C: Detail of B showing NPH-IR cells forming SCN d. D:VIP-IR cells in SCNc of a section neighboring C. Arrows in C and Dindicate corresponding points of the ependymal surface of the thirdventricle. The sections shown on this plate are slightly counterstainedwith hematoxylin. Scale bars 5 200 µm in B, 50 µm in C, D.

NPH NEURONS IN THE DEVELOPING HUMAN BRAIN 485

Figure 7

Whereas NPH-immunoreactivity occurred around 10 wgin the SON, it was first demonstrable at 13 wg in theaccessory NPH cell group, at 14 wg in the PVN and from18–20 wg in other cell groups or in clusters considered as

part of the accessory group (Table 2). This differential timecourse of expression of NPH in the different divisions of thehypothalamo-hypophyseal system appears characteristicfor various mammals including the rat (Buijs et al., 1980),mouse (Silverman et al., 1980), and brazilian opossum(Iqbal et al., 1995).

The presence of NPH in cells in the SON, accessory NPHcell group and PVN and fibers in the HHT from 10–14 wg,of VP detected by radioimmunoassay from 11–12 wg(Burford and Robinson, 1982), and of granular vesicles inthe posterior lobe of the hypophysis from 8.5 wg (Okadoand Yokota, 1980) suggests that a hypothalamo-neurohy-pophyseal system is already potentially functional by theend of the first trimester of human development. Theimportance of this system for the future development ofthe fetus is not yet clear. We first observed NPH immunore-activity in the SCN at week 18, several weeks earlier than

Fig. 7. Coronal sections at the tuberal level of a 24 wg human brainstained for NPH immunoreactivity. A: Camera lucida drawing (inset:entire section) showing the distribution of NPH-IR cells in the basalforebrain. All dots represent cells (fibers are not indicated). Boxes indicateareas shown in B–G. B: NPH-IR cell in the pallidal portion of the thepallidal/sublenticular complex of NPH cells. C: Well differentiated positivecells of the ACG located along blood vessels. D,E: Poorly differentiatedNPH-IR cells in the lateral SON. F,G: NPH-IR cells in the dorsal andventral portions of the PVN, respectively. Arrows (in G) indicate largeNPH-IR cells in the parafornical cell group (PF); note the difference in sizebetween cells of the PVN and PF. Scale bars 5 12.5 µm in B, E, 50 µm in C,D, F, G.

Fig. 8. Coronal sections through the posterior tuberal area of an 8 day(A,B) and a 3 month postnatal human brain stained for NPH immunoreac-tivity. A: Survey of NPH-IR cells in the SON, MFB and PL/SL (smallarrows). Note strands of cells extending along the pial surface towards theneighboring telencephalon (large arrow). B: Poorly differentiated NPH-IR

cells in the lateral SON. C: Survey showing the relation of NPH-IR cellsand fibers of the hypothalamo-hypophyseal system with the hypophysealstalk. AC, anterior commissure; BNST, bed nucleus of the stria terminalis;SON t, tuberal division of the supraoptic nucleus. Scale bars 5 200 µm inA,50 µm in B, 1 mm in C.

NPH NEURONS IN THE DEVELOPING HUMAN BRAIN 487

the reported first expression of VP in the SCN at week 23(Swaab et al., 1990). The earlier detection of NPH immuno-reactivity may reflect the greater stability and higherimmunoreactivity of NPH in the staining procedure.

Birth, migration, and peptideexpression of NPH-IR cells

Although this study directly cannot address the ques-tion of the birth and migration of NPH-IR cells, theobservations are consistent with descriptions from animalstudies which show that most cells of the magnocellularhypothalamic system derive from the region of the hypotha-lamic sulcus and migrate laterally and ventrally (Altmanand Bayer, 1978). In such a case, the potential fate of thechemical identity of these neurons would in part berestricted already at the time of their birth and migration(Altman and Bayer, 1978). In contrast, our findings do notsupport the suggestion of Diepen (1962) that the humanSON develops from a primordium around the evaginationof the optic vesicle and that the PVN develops from adifferent region located more medially. Thus, most humanNPH-IR cells, like those in the rat, most likely originatefrom a single primordium in the region of the hypotha-lamic sulcus, and migrate to their settling areas beforeexpressing NPH. Moreover, if the observation of the SONcells can be generalized, it appears that NPH becomesexpressed at about the time that migrating neurons havereached their settling area. The indirect evidence support-ing this is that: (i) at 10 wg, when the first NPH-IR cellsare found in the SON, bands of NPH-negative cells resem-bling migratory cells are present between the hypotha-lamic sulcus and the SON; in addition, epidermal growthfactor receptor positive radial glial cells extend from thehypothalamic sulcus into the lateral hypothalamus inhumans (Vogeley et al., 1993); (ii) at the time of 10 wg mostNPH-IR cells just dorsal to the SON are bipolar and areoriented along a diagonal trajectory between the SON andhypothalamic sulcus, whereas NPH-IR cells within theSON are oriented in parallel to the optic tract fibers, (iii)the time course of expression of NPH in the differentdivisions of the hypothalamo-hypophyseal system pro-ceeds from the SON to the accessory NPH cell group andthen to the PVN, following a sequence reciprocal to theproposed route of migration. Such a pattern of migration anddifferentiation has been observed for other structures in theCNS (Bayer andAltman, 1995; Kahle, 1969).

The origin of the NPH-IR cells of the SCN is less clear. Inthe rat, SCN cells originate in a ventral to dorsal gradientfrom a neuroepithelial site in the midline recess of the floorof the optic invagination of the third ventricle (Bayer andAltman, 1995). Thus, NPH-IR cells in the SCN may notarise from the hypothalamic sulcus and may represent adevelopmentally distinct group of neurons from otherNPH-IR cells analysed in this study. It should be notedthat in this study we describe only those NPH-IR neuronswhich are stainable in untreated individuals, and are notable to comment on the possible distribution or origin inhumans of cells equivalent to the NPH-IR parvocellulargroups which become visible in the amygdala, BNST, locuscoeruleus, and posterior hypothalamus of rodents treatedwith the transport inhibitor colchicine (Sofroniew, 1985a,b).

Consistency of topographic organizationof NPH-IR cells among mammals

In spite of the likelihood that most NPH-IR cells derivefrom a single primordium, the final organization of NPH-IR

cells consists of many scattered groups as seen in the latefetal period and mature brain. The causes of this wide-spread distribution are not known, but in addition to cellmigration may include secondary processes such as themovement of neighboring structures. The existence ofnumerous different cell groups increases the possibility ofthe establishment of differential fiber connections and theacquisition of different functions. This view would attachan importance to the widespread distribution of NPH-IRcells, in contrast to the commonly held view of the SONand PVN as essentially one system, and the accessoryNPH cell group with its associated clusters as ‘‘ectopic’’SON or PVN cells.

One way of considering the importance of this organiza-tion is to examine its conservation in different species. TheSON and PVN are separated in all mammals and mostvertebrates studied thus far, suggesting a difference offundamental importance. The general pattern of the acces-sory NPH cell group inclusive of its associated clusters isremarkably similar among many mammals including hu-mans (Sofroniew, 1985a). VP/NPH cells are present withinthe SCN in most if not all mammalian orders (Sofroniewand Weindl, 1980). The high degree of conservation of thedistribution of these main NPH-IR cells throughout mam-mals suggests that other NPH-IR cell groups may also behomologous but their location and configuration may besomewhat different in different species due to externalinfluences. That this may be the case is suggested bycomparing the location of NPH-IR cell groups in thevicinity of the internal capsule and globus pallidus whichare particularly susceptible to external pressure influenc-ing their final location. These groups show remarkableconsistency between rodents and humans, particularly ifone compares the distribution in the rat with that in thehuman at 18 wg. At this time in the human (Fig. 4A), as inthe developing (Altman and Bayer, 1978) and adult rat(Sofroniew, 1985b; Fig. 4), a laterally placed cluster ofaccessory cells is apparent which has a close relationshipwith the internal capsule in both species. However, duringsubsequent human development the globus pallidus ap-pears to encroach heavily upon the lateral hypothalamus(Richter, 1965) scattering these cells along its outer perim-eter and within its capsule. Our studies on the matureNPH system in humans show that these cells are aconsistent feature (Mai et al., 1993).

The high degree of conservation of the organisation ofNPH-IR cell groups among mammals including humanssuggests a precisely regulated developmental program,and functional differences amongst the groups which are offundamental importance. Some differences in the roles ofvarious NPH immunoreactivity groups have already beenidentified. Separate groups of NPH-IR cells project to theposterior pituitary, portal vessels, or brain stem andsubserve different functions (Sofroniew, 1985b). More de-tailed studies of the connectivity and functions of NPH-IRgroups may eventually identify the vulnerability andinvolvement of specific groups in certain disease processes.For example, NPH-immunoreactivity was significantlydecreased in the PVN but unchanged in the SON in agroup of patients with schizophrenia (Mai et al., 1993).

ACKNOWLEDGMENTS

The authors are grateful to Dipl. Biol. Th. Vob whoassisted the morphometric evaluations and contributedconsiderably to this paper. The excellent technical assis-

488 J.K. MAI ET AL.

tance of Mrs. V. Holler and M. Kazimirek is greatlyappreciated. Photographic assistance by N. Jansen isgratefully acknowledged.

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