The neurophysin-containing innervation of the forebrain of the mouse

NeuroscienceVol. 24, No. 3, pp. 931-966, 1988 Printedin Great Britain

0306422/00 53.00+ 0.00 Pergamon Press plc 0 1988 IBRO

THE NEUROPHYSIN-CONTAINING INNERVATION THE FOREBRAIN OF THE MOUSE*

OF

M. C.wm_t and J. F. MORRIS$§ TDepartment of Zoology, Institute of Life Sciences, Hebrew University of Jerusalem, Israel; IDepartment of Human Anatomy, South Parks Road, Oxford University, Oxford OX1 3QX, U.K.

Ahstrae-The oxytocinergic and vasopressinergic innervation of the forebrain of normal mice was studied immunocytochemically by use of a set of mouse monoclonal anti-neurophysins applied to serial vibratome sections. The extensive hypothalamic and extra-hypothalamic location of these neuropeptides was revealed, with, or without colchicine pretreatment. Magnocellular perikarya immunoreactive for either oxytocin-neurophysin or vasopressin-neurophysin were concentrated mainly: in the anterior commissural nucleus; in various subdivisions of the paraventricular nucleus; in a profuse array in the periventricular region; in the supraoptic nucleus including its retrochiasmatic division; in various accessory nuclei; and as a number of cells scattered throughout the preoptic and hypothalamic regions. Extensive groups of parvocelhtlar neurons, containing only vasopressin-neurophysin, were located in the suprachiasmatic nucleus including a ventromedian division, in the bed nucleus of the stria terminalis, and in the medial amygdaloid nucleus. Perikarya in the magnocellular nuclei were of generally similar size distribution and there was no evidence that distinct populations of magnocellular and parvicellular neurons, separable on the basis of size, had been labelled within these nuclei. Within the paraventricular nucleus, however, neurons in the posterior part were smaller than those located more anteriorly, and the cells containing oxytocinneurophysin were slightly smaller than those containing vasopressin-neurophysin. Within the generally similar size distribution, magnocellular neurons of the anterior commissural nucleus were the largest. During processing, shrinkage of the tissue and immunolabelled cells had occurred. The immunocytochemical procedure delineated neuronal processes, in particular dendrites, very effectively. The dendrites were shown to project for far greater distances than is generally recognized, some were of a characteristic corkscrew-like morphology, and most were oriented in a well-defined pattern. Many dendrites of paraventricular neurons passed medially then caudally towards and then along the third ventricle. Most dendrites of supraoptic neurons, in particular those containing vasopressinneurophysin, had an extensive anteroposterior course beneath the pia of the base of the brain. The axons containing oxytocin- and vasopressin-neurophysin were shown to take rather different paths from the paraventricular nucleus towards the median eminence. Other well-delineated immunoreactive processes and putative terminal plexi were found throughout the preoptic area, the septum, hypothalamus, subthalamus, thalamus, habenula, amygdala and ventral hippocampus; scattered fibres were also seen in the cortex. In general the bundles of fibres containing vasopressin-neurophysin were more dense, more extensive and of wider distribution than those containing oxytocin-neurophysin, although individual fibres were in the same calibre range. Distinguishing features of the forebrain of mice include a large population of neurophysinimmunoreactive perikarya throughout the subependymal periventricular region of the rostra1 third ventricle, an extensive anterior commissural nucleus containing both oxytocin- and vasopressinneurophysin, a distinctive bilateral accessory nucleus in the medioventral hypothalamus, a rostromedian division of the suprachiasmatic nucleus, a prominent posterior division of the paraventricular nucleus, and conspicuous neurophysin-immunoreactive plexi in the subthalamus.

The hypothalamo-neurohypophysial system (HNS), which produces oxytocin, vasopressin and their respective neurophysins was the first discovered of the many peptidergic systems that now command the attention of neuroscientists. First defined histochemically more than 35 years ago,4*5 immunochemical procedures have, in the last decade, pertnitted a vast expansion of our knowledge of the HNS and other systems and their projections throughout the brain and spinal cord. However, the HNS still serves as an archetypal model, and we have a greater understanding of the neurobiology of that, than of other peptidergic systems. The biosynthesis of oxyto& vasopressin and their respective neurophysins

*Dedicated to Professor Berta Scharrer on the occasion of her 80th birthday. $To whom correspondence should be addressed. Reprint requests should be addressed to Dr M. Castel. Abbreviations: ACN, anterior commissural nucleus; BSA, bovine serum albumen; DAB, diaminobenzidine; HNS, hypothalamo-neurohypophysial system; IR, immunoreactive; MAC, accessory nucleus (characteristic of the mouse); MSA, mouse subthalamic area; OVLT. organum vasculosum of the lamina terminalis; P!$ posterior pituitary solubilate; PVN, paraventricular nucleus of the hypothalamus; rSON, retrochiasmatic supraoptic nucleus; SON, supraoptic nucleus; TBS, Tris-buffered saline. 931

M.

938

CASTEL and

from their pro-hormones pro-pressophysin and prooxyphysin have heen extensively investigated.27 The peripheral neuroendocrine functions of oxytocin and vasopressin are well documented29 and a variety of roles in the central nervous system is currently under investigation (see Ref. 32 for review), but no extracellular neuroactive role for the neuropijysins has yet been determined. However, methodologically, oxytocin-neurophysin and vasopressin-neurophysin re!atively molecules are large peptide f - I2,ooO mol. wt) that serve as excellent markers for the smalf ue~o~ptides (N loo0 mol. wt) with which they are stoichiometricaliy associated. Until recently, the rat has been the species in which However, the HNS was most investigated. 1.2.12.24.52.61.68 abundance of mice from different biotopes, and the genetic&iy aberrant strains of mice now available means that mice, of both wiM and laboratory strains, are proving increasingly popular for the investigation of vasopressinand oxytocin-containing innerv~tion.10~‘~17~‘9-2’~~~4s~~~ However, as yet, no detailed study exists of the precise distribution of these peptides in the mouse brain. Extrapolation from studies on the rat may well be unjustified, since the organization of the system in mice may deviate somewhat from that in the rat, as has been shown also for the guinea-pig.M The present study therefore seeks to describe the oxytocinergic and vasopressinergic innervation of the forebrain of mice as delineated by presence of oxytocin- and v~opre~i~-~nrophys~n ~mmunorea~t~vities (IR), to define dist~ng~is~ng features of these systems in the mouse, and to take advantage of the excellent delineation of the cytoarchitecture, particularly of the magnocellular neurons, to examine some controversies concerning their neuronal circuitry and the functional implications implicit therein.

J. F. Mocks acrolein (Polysciences), in 0.1 M sodium cacodylate or sodium phosphate buffer, at pH 6.0 or 7.4. At the end of the fixation period, brains were kept in buffer until further processing. Brains were serially sectioned by vibratome, mainly in the

transverse r&me, at SO-lOOurn. into Tris-buffered saline (TSS). The- foildwing steps kerk interposed with copious washes in TBS: (1) acidified methanol containing 3% H,Oz for 30 min, to minimize staining of endagenous peroxidases; (2) blocking solution containing I% eggalbumen or bovine serum albumin (BSA) in TBS for 6O(fmin;(3) monocfonal a~~-neu~phy~n diluted in this blocking solution for t-3 days at 4°C; (4) rabbit anti-mouse Ig conjugated to horseradish peroxidase (Bioveda. Israel) diluted 1:200-I : 400 in blocking solution, ‘for -2-3 h; (5) diaminobenzidine (DAB; Wmg/ml) in Tris bufTer containing 3~1 30% H,O, Per

15ml DAB solution, with or without 1mM imidazol. The stained vibratome sections were spread onto slides subbed with gelatin and chrome-&m, dried at room temperature, immersed in 0.05% aqueous osmium teiroxide for 5min, dehydrated through enthanol, cleared with xylene and coverslipped with Fluoromount. Some series were stained with Cresyl Violet prior to dehydration in order to facilitate orientation. Sections were viewed and micrographs prepared using Kodak Te&nical Pan Fihn in an Oiympus light microscope with regular optics or Nomarski interference 0ptiCS.

Antibodies A set of mono&ma1 anti-neurophysins

(prepared by Ben-Barak et 01.‘) was used. These antibodies have previously been used by Whltnall et al.” for immunoperoxidase for immunogold electron microscopy. studies and by us 1u~L9 The antigen was rat posterior pituitary ~i~~iate (ps). Ps 38 is an unequivocal any-oxy~~~n-ne~ophys~ at aft dilutions; PS 4t recognizes mainly vasopr~n~nuurophysjn, but may cross-react with oxytocin-neurophysin under certain conditions; PS 45 is a high-affinity ail-purpose antineurophysin, with a distinct bias in faiour 6f v&opressinneurodvsin. Ps 38 was diluted I :5@-1:200: P!S 41 l:lO-i:iOQ PS 45 l:W-1:300. In pilot studies, a wellcharacterized poIyefoaa3 anti-vasopressin se~rn~~~~ was used. Controls inch&d absorbing the antibody saiutions with neurophysin, or substituting primary and/or secondary antibody solutions with buffer or irrelevant antisera during the labelling procedure. Some sections were stained with DAEH,O,-imidazol only.

EXPERIMENTAL PROCEDURES

~0rFho~etry Three series of siides were used to assess the size md perikarya in the various nuciei, Twenty-twoa&& common fiowemire of the oxford shape of jmm~or~ctive University strain, both male and female, were used fct~ the Cell outlines were tracedfrom the scan of a ReichW prtxmt study. Five male albino mice, weighing 2s3Og, of Visopan projector microscope at a magnification x 815, the Hebrew University Medical School strain were also and the area, maximum diameter and form factor (degree of included for comparison. Animals were kept at room tem- circularity) of these tracings were measured by u& of a perature on a day-night light-dark cycle, and had free Reichert MOP-AM02 diaitizinn analvser. The distribution access to laboratory chow and water. Some a&m& were of the areas and their -&ivalent &r&e diameters was plotted, and the KuImo~rov-S~imov test was used to injected ~~s~~ly with a low dose of c&hi&e analyse the statistical signi&xinee of differences between (7 fig/W g body weight) 24 h prior to being killed. these distributions. From the values.for individual c&s, the mean values for each nucleus in e;tch animal were calcuFixation lated, and these animal means used to derive an overall Mice were administered intrqeritoneal lethal doses of average value for the m&us. In this way the finaf average sodium pentobarbitone and, when deeply a-, data were not biased by the assessment of differ&t numbers werepcrfused~thc~witha~~t5ogal~ofgalof.~~~ of cells in different animals.

soiution coil%3&p&n, when the liver was bIanebed, about 25omf fix&W s&ion was perWed for 5-lO tin, after which brains were removed intact and kept in the same !?ixativeat room temperature on a rotary shaker for 2-3 h. Fiitive solutions contained l-5% ghttaraldshyde, usually in combination with l-4% paraformtiebyde, in some instances including also 0.2% picric acid or 0.2s1.0%

RESULTS

Technical con.si&mt;ions All fixative

mixtures used promoted

satisfactory

939

Neurophysin in the mouse forebrain

Antibodies PS 41 and PS 45 clearly labelled vasopressin-neurophysin-containing structures, but some cross-reactivity with oxytocin-neurophysin cannot be ruled out, depending on antibody dilution.’ Cross-reactivity of monoclonal antibodies cannot be selectively absorbed, because any effective absorption treatment precipitates all the antibody molecules in solution. However, PS 41 and PS 45 both labelled cell groups which were detected in affinity-purified polyclonal anti-vasopressin used by us previously.2s In practice, cross-reactivity of the monoclonal antivasopressin-neurophysin (particularly PS 41) was not a problem in this study, as is well illustrated in the paraventricular nucleus series (Fig. 3).

immunoperoxidase labelling of both hypothalamic neurophysin-immunoextra-hypothalamic and reactive elements. Fixatives prepared at pH 6.0 provided particularly well-defined labelling. Addition of acrolein to the fixative solution, even at concentrations as low as 0.25% promoted heightened specific immunoreactivity. High concentrations of glutaraldehyde (5%) visibly enhanced immunoreactivity of specific structures but, particularly after osmication, background staining was also augmented. However, the gingerish hue of specific immunoperoxidase labelling stood out clearly against the beige tones of the background and, in practice, this facilitated orientation of the tissue in the manner of a counter-stain. In colchicine-injected mice immunolabelled neuronal processes and plexi appeared more densely stained. However, colchicine treatment was essential only for the detection of the ventromedian group of vasopressin-neurophysin-IR suprachiasmatic cells.

Mapping

Specificity of the antibodies

Of the anti-neurophysins used, the anti-oxytocinneurophysin, PS 38, unequivocally labelled oxytocinergic cells and processes, without inducing any cross-reaction with vasopressinapparent neurophysin (Figs 3A-H and 5A,B for the position of paraventricular perikarya and the orientation of axonal projections). Moreover, PS 38 labelled neither the cells of the suprachiasmatic nucleus (Fig. 3A-F), nor the extensive groups of parvocellular perikarya in the bed nucleus of the stria terminalis, nor the medial amygdaloid nucleus, all of which labelled with PS 41 and PS 45 (Figs 13 and 14). PS 38 revealed only scattered single fibres and occasionally small circumscribed plexi in the septum, amygdala, hippocampus and habenula (Fig. IA-G); all areas where PS 41 and PS 45 produced conspicuous labelling (Fig. 2A-G).

Four particularly well-labelled house mouse brains, in which alternate sections were reacted with either PS 38 or PS 45, were used for graphic mapping of the distribution of oxytocin-neurophysin and vasopressin-neurophysin (Figs 1 and 2). The coordinates of Figs 1AG and 2A-G are based on the “Stereotaxic Atlas of the Mouse Forebrain” by Slotnick and Leonard.60 Each outline includes labelling data from two-four sections adjacent to the relevant bregma coordinate. Thus, the seven pairs of outlines represent a precis of the information provided by four sets of serial brain sections, and confirmed by the remaining material. In addition, the distribution of oxytocinneurophysin-IR and vasopressin-neurophysin-IR elements in the paraventricular nucleus of the albino mouse has been depicted in greater detail in micrographs of serial 70 pm-thick vibratome sections taken at intervals of 140pm (Fig. 3A-H). The vasopressin-neurophysin-IR and oxytocinneurophysin-IR systems in the two strains of mice were essentially similar, except for a more profusely

Table 1. Projected areas &m2), calculated equivalent circle diameters @m) and maximum diameters @m) (mean + S.E.M.) of cells in the hypothalami of mice (n = 3) immunocytochemically stained by use of monoclonal antisera to either oxytocinor vasopressin-neurophysin Oxytocin-neurophysin-immunoreactive Equivalent Maximum Area circle diameter diameter

Vasopressin-neurophysin-immunoreactive Equivalent Maximum circle diameter Area diameter

Anterior commissural Suprachiasmatic Supraoptic anterior

179 + 10

15.1 f 0.3

20.3 k 0.5 (283)

133 f 8

13.0 f 0.4

(66)

170 + 13 63 * 5 124k8

14.7 f 0.5 9.0 + 0.3 12.6 k 0.5

19.4 j, 1.5 (203) 10.6 + 0.2 (537) (173)

mid posterior Total Paraventricular anterior mid posterior Total Mouse accessory Periventricular Amygdala Bed nucleus stria terminalis

139 &-10 14Okl8 136&2 134 f 12 130* 11 116&6 121+6 155 + 14 133+8

13.3 *f 0.5 0.8 13.1 *0.1 13.0 L-0.5 12.8 + 0.2 12.1 + 0.4 12.7 f 0.1 14.0 f 0.6 13.0 f 0.4 -

(158) (77) 19.0* 1.4(301) (130) (146) (202) 16.2 f 1.0 (478) 18.7 f 5.0 (35) 18.2 f 0.7 (336)

132+4 126 + 8 129k2 139 + 3 139 &-12 129&7 136*6 146k5 141+ 11 108+6 117*9

13.0 + 0.3 0.5 12.6 12.9 f 0.3 13.3 f 0.1 13.3 + 0.6 12.8 + 0.3 13.2 k 0.3 13.6 f 0.3 13.3 f 0.5 11.7f0.3 12.2 f 0.7

i::; 17.0 + 0.6 (565) (96) (187) (88) 16.7 k 0.6 (371) 19.4 f 1.2 (87) 18.7 f 0.9 (262) 15.5 f 2.0(134) 17.7 f 0.5 (223)

Nucleus

S.E.M. calculated from 3 animal means; ( ) total number of cells assessed.

940 innervated periventricular mouse.

M. CAXELand 1. F. MORRIS preoptic area in the house

Perikarya. The terms “~gu~ll~ar” and “parvocisllular” are tra~~onaliy used to denote, respectively, larger (-25 pm diameter) and smaller (- 15 I;tm diameter) neurosecretory cells, with the implication of possible functional differences. In the present study a wide range of size in immunoreactive cell bodies has become apparent (Table 1) and is compounded by shrinkage of tissue {see ~an~tative Aspects, below). The supraoptic nucleus (SON) is generally acknowledged to contain only magnocellular oxyt~ner~c and vasopre~i~~r~c neurons. We have therefore used the unimodai size distribu-

tion in the SON (see Fig. 25a) as a definition of ~‘ma~~llu~ar”, In the ensuing desc~~t~ons of our tissue, ma~o~ll~lar will be used to denote an average (equivalent circle) diameter of > 12.5 pm, and parv~eIiular an average (eq~valent circle) diameter of < 12.5 pm. The dist~bu~on of cell sizes means that these two categories overlap, but we have no evidence of bimodal dist~butions of cell size in any nucleus which would justify separation of two ceil types based on size, We do not imply correlation of cell size with function, althou~ it is highly likely that size differences reflect functional diversity. Processes. A great variety of immunoreactive lrerve fibres has been revealed. In general, fine- to mediumcslibre varicose proossses are classified as axons, while thicker, non-varicose processes are classified as

Figs t and 2.

Nettrophy&

in the mouse forebrain

Figs I and 2. Diagrams representing coronal sections of the mow forebraiu on which the oxytocin~ro~hy~n-j~~o~ve (Fig. IA-G) and the vasopressin-ne~opbysin-i~~~~c~~ (Fig. 2A-o) neurons and prossses are indicated. Large dots represent cell group% short Iiues indicate the course of c& pm pun&ate making implies possible tenniual amas. An attempt has been m&e to indicate the relative amount of immunoreactive material in the dit%ereut systems, but the marking should not be taken to Rpresent a quantitative estimation of the j~~or~ct~v~t~. The diagrams are moditied from the “St~r~~~c Atlas of t.IxeAlbino Mouse Farebrain” by Slotnick and Lexmard,60and their approximate bregma coordinates are IA tmd 29, +0.7; I)3 and 2% +0.1; 1C and 2C, -0.4 1D and 2D, -1.0; IE and ZE, --1.2; IF and 2F, -2.0, 1G and ZG, -2.4.

M. CASTELand J. F. MORRIS

942

Abbreviations

used in the figures

Upper case denotes nuclei and areas; lower case denotes fibre tracts AA AB ACB ACC AC ACN AC0 AHA AL AM aPVN AR AV BST ca caP cc CLA CPU dbb & f fi GP HL HM hnt hp HPC ic IC LHA LS LV 3v mAC MD

anterior amygdatoid area basal amygdaloid nucleus accumkns nucleus accessory magnocellular nucleus central amygdaloid nucleus anterior commissural nucleus cortical amygdaloid nucleus anterior hypothalamic area lateral amygdaloid nucleus medial amygdaloid nucleus paraventricular nucleus, pars anterior arcuate nucleus anteroventral thalamic nucleus bed nucleus of stria terminalis anterior commissure anterior commissure, pars posterior corpus callosum claustrum caudate/putamen diagonal band of Broca external capsule entopeduncular nucleus fomix hippccampal timbria globus pallidus lateral habenular nucleus medial habenular nucleus hypothalamo-neurohypophysial tract habenulo-interpeduncular tract hippocampus internal capsule islands of Calleja lateral hypothalamic area lateral septum lateral ventricle third ventricle mouse accessory nucleus mediodorsal thalamic nucleus

However, since dendrites thus de&d may transform distally into thickly or thinly varicose processes, this poses a dilemma of definition. Axons

dendrites.

median eminence median forebrain bundle medial preoptic area medial septum mouse subthalamic area ventromedian suprachiasmatic nucleus mamillothalamic tract neocortex optic chiasm optic tract vascular organ of lamina terminalis < periamygdaloid cortex PAM cerebral peduncle posterior hypothalamic area KI pirifotm cortex PIR dorsal premamillary nucleus PMD ventral premamillary nudeus PMV pPVN paraventricular nucleus (pars posterior) PRT pretectal area parataenial nucleus PT paraventricular thalamic nucleus PV periventricular area PVA paraventricular nucleus (pars medialis) PVN reuniens thalamic nucleus RE reticular thalamic nucleus RT rSON retrochiasmatic supraoptic nucleus subiculum S suprachiasmatic nucleus SCN subfornical organ SF0 stria medullaris ::,mON supraoptic nucleus stria terminalis St stria terminalis (pars hypothalamica) sth olfactory tract to1 triangular nucleus of septum TS olfactory tubercle tuo VMH ventromedial hypothalamic nucleus zona incerta ZI

ME mfb MPO MS MSA mSCN mt N oc

that appear punctate or tortuous, apparently encircling other neuronal elements in the neuropil, are tentatively regarded as terminal fields.

Fig. 3. Paraventricular nucleus of the albino mouse as revealed in consecutive vibratome sections labelled alternatively for oxytocin-neurophyain (GT-NP; Figs JA,C,E and G, left hand aide of plate) and vasopreasin-neurophyrdn (VP-W, Figs 3B.D.F and H; right hand side of plate). No distinction is made between magnocellular and parvoceilular perikrya at this magnification (x40). In the anterior PVN (aPVN) oxytocinergic and vasopreaakrgic elements intermingle in the lateral wings of the nucleus (A and ceIla are also found ventral to the wings of the PVN (B). In the B); periventricular (PV) w middle part of the PVN, oxytocinergic per&arya am concentrated mainly around the periphery of the lateral wings (C and E), whereas vasop ma&erg& perikya are found throughout the nucleus (D and F). In the posterior PVN (PPVN), ~xynxinergic perkarya and proceases (G) intermi&e with thick vasopressinergic dendrites (H, and see Fig. SA and B for higher magnifications). The axonal projections of the oxytocinergic neurons towards the hyp&alamo-neurohypophysial tract (hnt) leave the PVN at an angle somewhat diirent from those of the vaaopmkmrgic neurons (compare A,C,E and G with B,D,F and I-I). Most vaaqeaainergic axorta~appear to pps%dorsal to the formx (f) and project further laterally, ’ axons ah8o run below the fomix. Almost all before turning in a ventral dimctioa; some thepairedvasopr4winqicsuprachiesma’ oxytocinergic axons pass below the fornix nucleus (SCN) may alao be seen, the irmnuanrosotivity predominating in dorsomedial areas but al: occurring ventrokmlly (B and D). A,C a&l E show that oxytocin-neurophyain imnaunoreactivity is conspicuously absent in the StlprachipMlatic nuoleua and surrounding area. In the supraoptic nucleus (SON) vasopmkn-tteurophysln immunomactivity pppasrs more intense than oxytoein-neurophysin immunoreactivity throughout the series. Aomasory dusters (ACC) are generally more conspicuous in vasopressin-nettrophysin-labelled sections (B and H).

Fig. 3. 943

944

M. CASTEL and J. F. MORRIS

Distribution of oxytocin -neurophysin immunoreactivity (Fig. IA-F); and nuclei containing both oxytocin -neurophysin - and vasopressin -neurophysin immunoreactive cells Rostrally, oxytocin-neurophysin-IR perikarya appear in the mediobasal preoptic area near the vascular organ of the lamina terminalis (OVLT) (Fig. lA), these cells probably representing the most rostra1 extent of the anterior SON. Proceeding caudally, cells of the SON lie on the dorsolateral aspect of the optic tract as well as along the floor of the adjacent temporal lobe (Fig. 1B-D) up to the point where the optic tract penetrates the neuropil of the brain, between the amygdala and the base of the hypothalamus (Fig. 1E). A number of oxytocinneurophysin-IR neurons ramify over the optic tract, their dendrites projecting in several directions (Fig. 1E). Cells and processes of the retrochiasmatic division of the SON (rSON), both oxytocinneurophysin- and vasopreasin-neurophysin-IR, lie in large numbers close to the floor of the ventromedial hypothalamus, lateral to the arcuate nucleus (Figs 1E and 7A,B). In general, oxytocin-neurophysin-IR axons of the SON funnel into the median eminence where they join the hypothalamo-neurohypophysial tract. Most of the oxytocin-neurophysin-IR dendrites of the SON appear to remain within the nucleus, but some spread out laterally along the pial surface of the ventral glial lamina, where vasopressin-neurophysinIR dendrites predominate (see below). In the preoptic area, at the level of the anterior commissure, a characteristic variety of oxytocinneurophysin-IR processes, including a dense plexus of dendrites (Fig. 1A) appear to be the rostra1 projections of the impressive anterior commissural nucleus (Figs lB, 8 and 9). The magnocelhdar perikarya of the anterior commissural nucleus are multipolar, some dendrites projecting medially towards the third ventricle, and others projecting dorsolaterally and dorsomedially at various angles (Figs 1B and

8A-C) with some clearly directed towards the bed nucleus of the stria terminalis (Fig. 8A). Rostrally, the oxytocin-neurophysin-IR perikarya of the anterior commissural nucleus, located somewhat lateral to the third ventricle (Fig. 9B), appear to intermingle with thick vasopressin-neurophysin-IR dendrites (Fig. 9A) of cells presumably located more caudally. The bulk of the anterior commissural nucleus approaches closer to the third ventricle (Fig. 8A-Q but at no point nucleus (Fig. the disparity not consider

does it merge with the paraventricular 4). For this reason, and also because of of cell size and shape (Table 1) we do the anterior commissural nucleus of the

mouse as a subdivision of the paraventricular nucleus as some authors maintain for the rat67.6abut rather as an independent entity.““* In the mouse, in contrast to the rat, the periventricular area, particularly the preoptic and anterior hypothalamic regions, is characterized by a large number of oxytocin-neurophysin-IR and vasopressin-neurophysin-IR perikarya and processes (Figs 1B and llA,B). The axons of these periventricular neurons generally extend lateroventrally towards the hypothalamo-neurohypophysial tract, whereas many of their dendrites run parallel to the ventricular wall in the subependymal region (Fig. 11A). It often appears as if both cells and dendrites contact the ventricular lumen, but this is likely to be an illusion created by the relatively thick vibratome sections and the heavy staining of the processes (Fig. 1lA,B). Located in the anterior hypothalamus, midway between the paraventricular and suprachiasmatic nuclei, is a paired accessory nucleus typical of the mouse5’ (MAC) (Figs lOA,B and 12A), which contains both oxytocin-neurophysin-IR and vasopressinneurophysin-IR cells. Although this nucleus lies at about the same craniocaudal level as the circular nucleus in the rat,“,70 it is markedly different cytoarchitecturally. The magnocellular neurons in the MAC are arranged rather diffusely (Fig. 12A) and a

Fig. 4. The anterior hypothalamic area of a cokhichm-treated house mourn,,lahelled with anti-oxytocinneurophysin. 01 the left are mngno&iular neurons of the caudal part of the anterior commissural nucleus (ACN), and on the right the most rostral parvocelhdar neurons of the anterior pamventricular nucleus (aPVN); note also the row of periventricuhu (PV) neurons near the third ventrick (3V). Tire ACN appears quite distinctive from the aPVN. The tip of the dmcendmg fomix (f) is seen. Nuclei of background cells have heen “counterstained” by osmium tetroxide treatment. x 307. Fig. 5. Adjacent sections through the posterior paraventricular nucleus (pPVN), lahelled respectively for oxytocin-neurophysin and vasopmssin-neurophysm (details of Fig. 3G and H). At this level of the pPVN, parvocelhrlaroxytocinergic perikarya and thick magnoceUular vasopressmergic dendrites intermingle; very few vasopressinergic cells are seen (B). x 154. Fig. 6. Posterior PVN of a house mouse, lnhelkd with anti-neurophysin. Note the immunoreactive processes (arrows) passing dorsal to the third ventricle (3V) between the pPVN of the right and left sides. x96. Fig. 7. Neurophysin-immunoreactive cells and processes in the bilateral retrochiasmatic supraoptic nucleus (rSGN) at the base of the posterior h-us; colchichm-treated housc mourn..Most of the dendrites of these magnocelhdar neurons project towards a web of immunoreactive perikarya and processes in the ventral pial lamina (arrows). In the hackgrouud, parallel to the base of the hypothalamus, the fine axons of the hypothalamo-neurohypophysial tract (hnt) may he discerned. x 154.

Figs 8 and 9. Neurophysin immunoreactivity in the anterior commissural nucleus (ACN) of the house mouse. Fig. 8A. Overview of the anterodorsal hypothalamic area showing the ACN below the anterior comtnissure (caf, on either side of the third ventricle (3V). Some immunoreactive elements extend upward via the bed nucleus of the stria terminalis (BST) towards the inte~entricular foramen of Munroe (fm). f = fornix. x 55. Fig 83 and C. Higher ma~ifi~tion ( x 152) detail of the left (B) and right (C) ACN. Some fine, varicose axons may be seen (A); well-delineated non-varicose dendritic processes extend in several directions; note corkscrew-like dendrites (arrow) extending towards the third ventricle (3V). Fig. 9. Adjacent vibratome sections through the pre-optic area showing the rostra1 ACN stained for vasopressin-neurophysin (A) and oxytocin-neurophysin (B). In this region, thick vasopressin-neurophysinIR dendrites intermingle with oxytocin-neurophysin-IR perikarya (x313). Some of the vasopressinneurophysin-IR dendrites (arrow) have spine-like excrescences (inset x627). The third ventricle (not shown) is located to the left of the figure. The asterisk denotes a blood vessel which occurs m both sections.

946

Neurophysin in the mouse forebrain striking feature is the clear orientation of numerous thick dendrites oriented towards the third ventricle (Fig. lOA,B). The paraventricular nucleus is an extensive, complex and heterogeneous nucleus in which oxytocinneurophysin-IR and vasopressin-neurophysin-IR cell bodies either intermingle or are segregated in specific positions (Figs 3-6). Immunoreactive perikarya are either magnocellular or parvocellular. For convenience we refer to anterior, middle and posterior portions of the paraventricular nucleus, each with a distinctive arrangement of oxytocin-neurophysin-IR and vasopressin-neurophysin-IR perikarya (Fig. 3A-H). Anterior PVN refers to the most rostra1 part of the nucleus, where the lateral wings have a relatively small span, which comprises predominantly oxytocintontaining cells (Figs 3A,B and 4). Middle PVN encompasses the bulk of the nucleus with well-developed lateral wings, where oxytocin- and vasopressin-containing elements intermingle, with some bias in favour of the latter (Fig. 3E-F). Posterior PVN refers to the most caudal part of the nucleus, where the dorsoventral span of the wings is more restricted but their lateral extent is greater; here cells are predominantly oxytocin-containing, intermingled with vasopressin-containing dendrites (Figs 3G,H, 5A,B and 6). A variety of schemes and divisions of the paraventricular nucleus, based on the considerable cytological and neurochemical heterogeneities in the nucleus, have been described.‘,2*s9,67@ However, the subdivisions used here consider only the positions of oxytocinergic and vasopressinergic elements within the nucleus. The posterior part of the paraventricular nucleus is another area where oxytocin-neurophysin-IR (oxytocinergic) perikarya intermingle with thick vasopressin-neurophysin-IR (vasopressinergic) dendrites, the cells of origin of which are located at a different level (Figs 3G-H and 5A,B) (cf. rostra1 anterior commissural nucleus, above). The direction taken by oxytocin-neurophysin-IR and vasopressin-neurophysin-IR axons as they pass laterally from the body of the paraventricular nucleus differs markedly. Oxytocin-neurophysin-IR axons generally pass ventrally beneath the fomix (Fig. 3A,C and E) and appear to join the hypothalamoneurohypophysial tract in rather medial position (Fig. 3C,E and G). In contrast, many of the vasopressin-neurophysin-IR axons from the paraventricular nucleus arch over the fornix in a lateral direction before turning ventrally (Fig. 3D,F and H), and some may even traverse the supraoptic nucleus before joining the hypothalamo-hypophysial tract (Fig. 3D and F). In the posterior paraventricular nucleus (Fig. 3G) there are few laterally directed axonal projections from the oxytocin-neurophysin-IR perikarya, because these neurons appear to project caudally toward the midbrain (cf. rat12~s9~6’@). Posteriorly, the left and right wings of the para-

941

ventricular nucleus are often seen to be joined by immunoreactive processes that span the dorsal tip of the third ventricle (Fig. 6), an observation of interest in view of the known functional synchrony between right and left paraventricular nuclei.50 oxytocin -neurophysin -immune Extra-hypothalamic reactive projections in the forebrain A small number of oxytocin-neurophysin-IR axons pass dorsally through the lateral and medial septum (Fig. 1A and B), forming a marked contrast to the large number of vasopressin-neurophysin-IR axons that pass to and appear to terminate in the lateral septum (Fig. 2A and B). There is, however, a moderately dense plexus of oxytocin-neurophysin-IR fibres in the triangular nucleus of the septum (Fig. 1B). Some oxytocinergic projections extended into the olfactory bulbs. Sparse oxytocinergic innervation occurs in the medial thalamic area and laterally along the reticular nucleus of the thalamus (Fig. 1D and G). Single oxytocin-neurophysin-IR fibres are found throughout the amygdala, whereas small plexi occur in the central amygdaloid nucleus and in the substantia innominata/basal nucleus of Meynert (Fig. 1E). A few oxytocin-neurophysin-IR fibres meander through the ventral hippocampal complex and the cortex (Fig. 1G). Except for the occasional “aberrant” magnocellular oxytocin-neurophysin-IR perikaryon found dorsal to the anterior commissure and the solitary oxytocin-neurophysin-IR cell often seen in the olfacoxytocintory tubercle, no extra-hypothalamic neurophysin-IR cells have been revealed in the forebrain (Fig. 1B). Localization of vasopressin -neurophysin -immune reactive cell bodies The cytoarchitecture of the anterior commissural, paraventricular, supraoptic and mouse accessory nuclei, where both oxytocin-neurophysin-IR and vasopressin-neurophysin-IR perikarya are located, have been described above. There are, however, extensive groups of parvocellular neurons in the mouse forebrain that are exclusively immunoreactive for vasopressin-neurophysin and hence, presumably, vasopressinergic. These are found in the suprachiasmatic nucleus (Figs 2C and 3B,D,F), including a ventromedian cluster (Fig. 12A and B), in the bed nucleus of the stria terminalis (Figs 2B,C and 13A,B) and in the medial nucleus of the amygdala (Figs 2E and 14A-C). Small (9.0 pm diameter; Table 1) vasopressinneurophysin-IR cells are found throughout the suprachiasmatic nucleus, but are concentrated particularly in large dorsomedial and lesser ventrolateral groups (Fig. 3B and D). In colchicine-treated house mice, a tight cluster of intensely immunoreactive vasopressin-neurophysin-IR cells was visualized ventromedially, below the third ventricle (Fig. 12A and B). This median cell group is somewhat rostra1 to the

948

M. CASTELand J. F. MORRIS

Figs 10 and

1I

Neurophysin in the mouse forebrain

bulk of the bilateral suprachiasmatic nucleus but, judging by general proximity and cytological similarity, we consider it a vasopressin-neurophysincontaining subdivision of the suprachiasmatic nucleus that has not, to our knowledge, been previously reported in this or other species. Irrespective of colchicine treatment, in most male specimens, a group of small (w 12 pm diameter; Table 1) punctately labelled vasopressin-neurophysin-IR neurons could be discerned lateral to the anterior commissural nucleus (Figs 28 and 13B). Their ellipsoidal cell bodies were slanted obliquely dorsoventrally, relative to the vertical line of the third ventricle (Figs 2B and 13B). Traced through serial transverse sections, this well-populated cell group could be seen more caudally extending from the stria terminalis (pars hypothalamica), through the main body of the bed nucleus that lies lateral to the descending limbs of the fomix, and up into the anterior thalamic area (Fig. 13A). However, their processes were extremely fme and impossible to follow beyond the nucleus of origin. Another group of exclusively vasopressinneurophysindR perikarya, located in the medial nucleus of the amygdala, is comprised of cells similar to those in the bed nucleus of the stria terminalis (Figs 2E and 14A-C). These cells, from which short delicate processes extend, are enmeshed in a plexus of medium-calibre vasopressin-neurophysin-IR fibres from \?rhich they seem to be cytologically distinct (Figs 2E and 14AX). Many areas of the amygdala are profusely innervated by vasopressin-neurophysinIR fibres of different calibre (Fig. 2C-F) which seem to originate from the hypothalamus (see below). Hence, it is difficult to assess, by immunoperoxidase staining alone, the direction of possible efferents from the parvocellular vasopressin-neurophysin-IR neurons in the medial amygdaloid nucleus. Distribution of reactive processes

vasopressin -neurophysin -immune -

Dendrites. Long dendrites with distinctive orientation and often corkscrew-like form (e.g. Fig. 8B) in

949

the anterior commissural nucleus (Figs 8A-C and 9A), the mouse accessory nucleus (Fig. 10A and B), the anterior periventricular cells (Fig. 1lA), the posterior paraventricular nucleus (Fig. 5B), and the retrochiasmatic supraoptic nucleus associated with the ventral pial lamina of the forebrain (Fig. 7A and B) have already been described. This extensive dendritic system, which is primarily vasopressinergic, certainly increases the receptive fields of magnocellular neurons far beyond their perikarya of origin (see Discussion). Vasopressincontaining processes and plexi have also been identified within the taenia tecti rostra1 to the septum and in sparse distribution throughout the olfactory lobes. Magnocellular processes. The vasopressinneurophysin-IR component of magnocellular axons in the hypothalamo-neurohypophysial tract appeared more extensive than the oxytocin-neurophysin-IR component (Fig. 3A-H). The course of these immunoreactive axons toward the median eminence and the infundibulum is a characteristic feature of hypothalamic organization in all species studied. However, in the dorsolateral area of the posterior hypothalamus, bordering on the subthalamus and amygdala, the complex organization of the vasopressin-neurophysin-IR tracts, some of which project to extra-hypothalamic destinations, is more difficult to unravel (Figs 2D,E, 3H, 17 and 18A). Vasopressin-neurophysin-IR axons in this area not only turn ventrally towards the supraoptic nucleus and the base of the brain, but also appear to proceed laterally towards the amygdala via the substantia innominata, and dorsally towards the striatum via the reticular nucleus of the thalamus, along the medial aspect of the internal capsule. This complex arrangement (Figs 17 and 18A) has been somewhat simplified in Fig. 2D. Two other areas, both beneath the thalamus, appear to be points where vasopressinneurophysin-IR fibres make distinct changes of direction. Located ventral to the zona incerta is a circular area tentatively designated as “mouse subthalamic area” which is the focus of punctate immunoreactive terminals, and the site where varicose axons deviate from the hypothalamo-neurohypophysial tract and

Fig. 10. The neurophysin-immunorective accessory hypothalamic nucleus typical of the mouse (MAC); normal house mouse. (A) Many large, conspicuous dendrites of MAC are orientated medially towards the anteroventral part of the third ventricle (3V); the fine punctate processes running parallel to the ventricular wall originate from the suprachiasmatic nucleus (SCN), cell bodies of which lie caudal to this section. OC = optic chiasma. x 160. (B) Photomontage (two different levels of focus) detail of a cluster of MAC cells; large, corkscrew-like dendrites extend towards the third ventricle (to the right of the figure); fine-calibre axons project laterally into the hypothalamus. x 330. (MAC is also depicted in Fig. 12a). Fig. 11. Periventricular (pv) neurophysin-immunoreactive cells. (A) shows immunoreactive periventricular (pv) cells in the area between the anterior paraventricular nucleus (aPVN) and the SCN (of which only the fine dorsally directed axons may be seen). Note the subcpendymal area where thick immunoreactive dendrites (arrows) run vertically and craniocaudally parallel to the third ventricle. x 160. (B) Periventricular cells (PV) in the pmoptic (PO) area rostra1 to the anterior paraventricular nucleus, labelled with antioxytccin-neurophysin. Note also the lack of punctate axons in the background, which indicates that the ascending periventricular plexus is entirely vasopressinergic. x 160.

Fig. 12. The ventr~m~ian cluster of supra~hiasmat~c nucleus neurons (mSCN) below the third ventricle (3V) is labelled for vasopressin-neurophysin in a colchicine-treated house mouse. Tite main parts of the suprachiasmatic nuclei (SCN) lie caudai to these sections (see Fig. 3B.D and F). In (A) several cells af the principle SCN are seen to the right of vmSCN, and two rnagnuceil&r perikarya (arrows) to the left. Ma~~lluiar perikarya are seen also in the paired mouse tuxeasory nucleus (MAC), which lies sin-fR punetate preoesses v&c& originate in dorsolateral TV SCN, Nate the fine vasopressin-neuroph SCM, and wiucfi prOJeCt in a dorsal direction paraliei to ti: e w&s of the t&d vent&& (3v). QC = Optic chiasma. x 144. (B) mSCN (arrows) in a house mouse, in which the third ventri& is u&&ted. ‘&e inset shows higher power detail of the c&s of vmSCN and surrounding tine V~~~~n-~urop~~~n-I~ axons. x 207. 950

Neurophysin in the mouse forebrain pass towards the thalamus (Fig. 17). More caudally, the entopeduncular nucleus, within the ventral extremity of the internal capsule, also deflects magnocellular processes, some of which may originate in a nearby hypothalamic accessory nucleus (Figs 2E and 18A) around itself. The difficulties of accurately defining structures in the subthalamic area of the mouse forebrain, and the anomalies and inconsistencies in the neuroanatomical literature, are dealt with in the Discussion. Processes of suprachiasmatic neurons. The hypothalamus is also a rich source of parvocellular vasopressinergic innervation, as may be seen from the profusion of fine-calibre, varicose axons that emerge from the suprachiasmatic nucleus (Figs 2C, 3B,D,F, lOA, 11A and 12). Some project rostrally in the direction of the OVLT and may be followed in the preoptic area between the ascending limbs of the diagonal band of Broca, where they appear to sprout collaterals at right angles to the main axon (Fig. 15A-C). This ladder-like axonal collateralization occurs in the vicinity of capillaries of the OVLT (Fig. 1X). Ascending midline preoptic fibres may continue into a modest vasopressinergic tract in the medial septum which also includes a magnocellular component, judging by the range in axon calibre (Fig. 16). In the rat this medial septal tract is said to join the septal fnnbria and enter the dorsal hippocampus, and possibly even the subfomical organ.61 The proximity of axons of an appropriate calibre suggests that the suprachiasmatic nucleus may also contribute, at least in part, to the vasopressinneurophysin-IR innervation of the vertical limb of the diagonal band of Broca (Fig. 15a and b) and, from thence, the lateral septum. In the rat, however, experimental evidence suggests that these areas are innervated mainly by the parvocellular vasopressinergic neurons of the bed nucleus of the stria terminalis22-r” (see Discussion). Within the hypothalamus, dense tracts of fine vasopressin-neurophysin-IR fibres ascend from the suprachiasmatic nucleus on either side of the third ventricle (Figs 2C and 3B,D,F). These tracts traverse the dendritic fields of the mouse accessory nucleus (Fig. lOA), the region of immunoreactive periventricular cells and the anterior paraventricular nucleus (Fig. 11A) where they appear to give off punctate terminal collaterals. Many suprachiasmatic fibres ascend from the anterior hypothalamus into the anterior thalamic area (Fig. 2C) and appear to give rise to the dense vasopressinergic terminal arborizations in the paraventricular nucleus of the thalamus and the parataenial nuclei (Fig. 19). Apparent suprachiasmatic projections also emerge from the mediodorsal aspect of the paraventricular nucleus of the hypothalamus more caudally, and form a single cone-like tract in the reuniens nucleus at the base of the medial thalamus (Figs 2D and 19). Terminal arborization appears to occur in the reuniens nucleus, and fibres also appear to ascend dorsally via the

951

rhomboid nucleus towards the paraventricular nucleus of the thalamus (Fig. 19). As the suprachiasmatic nucleus apparently innervates the above-mentioned thalamic nuclei in the vicinity of the ventricle, it would seem plausible that the same holds true for the rich vasopressinergic plexi in the lateral habenular nuclei (Fig. 20A and B) for which no other afferent tract of vasopressinergic fibres can be distinguished. This origin was contended for the rat some years ago.63 However, more recent experimental evidence suggests that the bed nucleus of the stria terminalis is responsible for the profuse vasopressin-containing innervation of the habenular complex” (see Discussion). Processes in the amygaida. Throughout its rostrocaudal extent the amygdala, including its cortical region, contains vasopressinergic fibres which range in appearance from elongated axons in passage of various calibres and tortuous dendritic receptive fields, to punctate terminal fields (Figs 2B-F and 21A,B). This wide range of vasopressin-neurophysinIR fibres appear (from their diameter) to be derived from both magnocellular and parvocellular components, for which three possible sources are available: (a) via the stria terminalis from the hypothalamus. This could convey magnocellular projections as well as fine-calibre processes from the parvocellular vasopressinergic neurons in the bed nucleus of the stria terminalis; (b) via the hypothalamic-subthalamic-amygdaloid junction (Figs 2D,E, 17 and 18A) where magnocellular projections are deflected towards the amygdala; and (c) locally from the parvocellular vasopressinneurophysin-IR neurons in the medial amygdaloid nucleus (Figs 2E and 14A-D). Processes in the ventral hippocampus. The strata of the ventral hippocampal complex, including the paraventricular area, and also the.claustrum, are innervated by vasopressin-neurophysin-IR fibres (Figs 2G, 22 and 23). Reticular collaterization may be seen in the stratum oriens (Fig. 23). This innervation appears to reach the ventral hippocampus via the amygdala and, theoretically, could have descended from the dorsal hippocampus, though we have not been able to visualize fibres taking this route in the mouse. Cerebral cortex. A few tine vasopressinneurophysin-IR fibres were consistently found in the cerebral cortex just lateral to the striatum. These occupied a rather limited dorsoventral extent but were spread out rostrocaudally (Fig. 2A-G) and could not be traced further superficially than the deepest layers of the cortex. Quantitative aspects

Table 1 gives the dimensions of oxytocinneurophysin-IR and vasopressin-neurophysin-IR neurons in the different nuclear groups of the forebrain in which they are found. These data are expressed as the mean IfrS.E.M. of the average values for each individual animal. The data for the supra-

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Figs 13 and 14. Clusters of parvocellular vasopressinergic cells in the bed nucleus of the stria terminalis (BST) (Fig. 13A and B); and in the medial amygdaloid nucleus (AM) (Fig. 14A D). Fig. 13A is from a parasaggital section through the forebrain of an albino mouse, taken at an angle that provides a panoramic view of both the heavily labelled magnocellular (mag) perikarya, dendrites of which run towards the third ventricle (3V), and the weakly labelled parvocellular perikarya found in the BST. x 88. Fig. 13B. Weaklylabelled vasopressin-neurophysin-IR cells in the BST of a normal house mouse (see Fig. 2B for orientation), x 141. The cells indicated by an arrow are magnified in the inset, x 202. Fig. 14A. Moderately labelled vasopressin-neurophysin-IR cells in the medial amygdala (AM) against a background of punctately labelled immunoreactive processes. Fig. 14B, superimposed on Fig. 14A, is taken from a sequential vibratome section, showing two immunoreactive parvocellular perikarya closely adjacent to the amygdaloid aspect of the optic tract (OT). x 202. The cells indicated by an arrow in Fig. 14A are magnified in Fig. 14C. x 404. Punctately labelled immunoreactive processes (arrows) are seen to better advantage in Fig. 14D. x 404.

952

Figs 15 and 16. Vasopressin-~~ophysinmmunoreactive innervation in the preoptic and septai arem of a cokhiinetreated house mouse. Fig. 1SA is a ~no~~~ view at the preoptic fJ%) level (x X20), showing vasopressinneurophysin-IR axons coursing via the dlagona1 band of Broca @BB), around the accumbens nucleus (ACB) and into the ventral pat? of the lateral septum (LS) near the lateral ventricle (LV). The organum vasculosum of the lamina tesminalis (OVLT), at the base af the PO, is also innervated by vasopressin-neurophys~“I~ fibres (arrow), which have been ~fied in Fig. I5B and C to show the ladder-like branching of the axons. Asterisk = blood capillaries; ca = anterior ~~i~u~; CPU = ~u~te~put~n~ nuclei of background cells have been ~‘~oun~~~n~ by osmium tetroxida treatment. Fig. 2% x311; Fig. 1X x 792. Fig. 16. Twa typm of vasopressinergic aviation in the septum caudai to that depicted in Fig. ISA. Terminal abortion in the lateral septum (LS} near the lateral ventricle fLV); and fibres of passage, seen above the triangular nucleus (TS) in the medial septum (MS). x 290.

954

M. CASTELand f. F. Moarus

Fig. 17. Border zone between dorsal hypothalamus (H) and base of thalamus (T), of cokhicine-treated house mouse; vasopressin-neurophysin immunoreactivity. The rim of a “mouse subthahunic area” (mSA), ventral :o zona incerta (21). is heavily innervated by punctate vasopressincrgic elements (arrows). Axons originating from the paraventricular nucleus at the right (not seen), from the hypothalamoaeurohypophysial tract (hnt) seen arching over the fomix (f). Laterally (at the left of the figure) most axons turn ventrally but some project dorsally (double arrows) towards the “mouse subthalamic area” (MSubT) and the internal capsule (ic) (see Fig. 2D for orientation). x660.

: and paraventricular nuclei are given first for the oFItiC an te1ior, middle and posterior parts of the nuclei, and thesn for the nuclei as a whole. Figure 25 gives the diritriibution of areas and equivalent circle diameters of innmunoreactive cells in the different nuclei. In

general there was good agreement for both the distribution and average sizes of cells in the equ ivaknt nuclei of different animals (Fig. 25a shows the dktlibution of vasopressin-neurophysin-IR cells in the supraoptic nuclei of the three animals, and I;ee the

Neurophysin in the mouse forebrain

Fig. 18. V~op~in-ne~ophysin ~rn~or~cti~ty in the ~s~rolat~al hither area (PH), which is wedged between the ventral thalamus cr) and the medial amygdala (AM); col&icine-treated house mouse, (A) Vasopressinergic processe s, o~~natin~ in the h~th~~~, encircle the ~to~~Iar nucleus (BP) which is embedded in the internal capsule (icf. A diffuse chrster of ac~~sory magnoceBuIar neurons (ace] is located in the h~th~amus ventral to the entopenduneuiar nucleus (EP). The h~th~amo-neuroh~pbysi~ tract (hnt) descends towards the base of the h~~~arnus where a portion of the retrochiasmatic supraoptic nucleus (&ON) may be discerned. in the central amygdaloid nucleus (AC) a v~opr~in-neurophysin-~mmunor~ctive plexus is found (arrow). x 160. (3) The vasopreesm-neurophysin-IR plexus in the AC, and very fine immunoreactive fibres in the basal nneleus of Meynert (BM) and in the medial amygdaloid nucleus (AM) can be discerned at higher ~~~~tion in Fig. 18B. Just above and to the right of the asterisk a few parvooelhihu vasopressin-neurophysin-IR cells are seen. x 480. The nuclei of background eelis are ‘%ounter-stained” by osmium tetroxide treatment.

955

Figs 19 and 20. Vasopressin-neurophysikimmunoreactivity in the thalamus (I) of a cokhicine-treated house mouse. Fig. 19. Vasopressin-neurophysin innervation of the midthakmus; ventrally a cone of tine-caiibre immanoreactive axonal processes (arrow) emerges from the dorsal aspect of the coarse-tibred hypothalamic paraventricular nucleus (WN) and enters the thalamic reuniens nuckus (RE). These fine-caiibre processes (probably of suprachiasmatic origin) ascend dorsally via rhomboid nucleus (RH), where a few immunoreactive fibres are seen, to the rich plexi of vasopressin-neurophysin-IR fibres in the paraventricular thakmic nucleus (PV) and the paired parataenial nucleus (ET) (only the left side is shown) from which Bbres extend to the stria medullaris (SM) of the thalamus. Immunoreactive fibres also pass between the two pkxi (dotted arrows) and towards the third ventricle (3V). The nuclei of background cells are “counterstained” by osmium tetroxide. x 160. Fig. 20A. Vasopressin-neurophysin immunoreactivity in the habenular complex. The lateral habenukr nucleus is intensely labelkd, with projections to the stria medullaris (SM). Immunoreactivity is also seen along the habenulo-interpenduncular tract (arrows). x 59. Fig. 208. Detail of the left side of Ihe habenular complex. x 189.

Fig. 2 1I Vasopressin-neurophysin immunoreactivity in the posteriar amygdaloid area of a colcbicinetreated house mouse. (A) Pslnoramic view of a coronal section showing vasopressin-ncurophysin-IR fibrcs and plexi around the lateral ventriclo (LV) ( x 57). (IS) Higher magnification (x 185) detail showing the varicosity and arborization of the lab&d ftbres in the mntral amygdaloid nucleus (AC), the medial arn~gd~~ nucleus (AM), the hi~~rn~-~~gd~~o~d area (AEiij9 and the clanstrum (CL%). AB = basal ~rnygda~~ rmcjeus. The nuclei of ~~gr~u~$ c&s arc ~‘~~~~rs~~~~~~ by osmium tctroxidc,

Figs 22- 24. V~pre~in-neurophysin-i~unoreactivity in the ventral hippos complex @W) and the cortex (C), of a colchicine-treated house mouse. Fig. 22. Varicose immunorcactive fIbres atong the borders of the lateral ventricle (LV) ( x ISZ}. Pig. 23. Vasopressin-neurophysin-IR gbres arborize within the stratum oriens (So) of the ventral ~~~p~. SP = stratum pyramidale. x 171. Fig. 24. L&e&d fibres deep in the cortex (C) he parallel to the external capsule (CE). Arrows indicate immunoreaetive fibres in the caudate putamen (CPU). x 152. As in previous plates, nuclei of ba&ground cells have been “counterstained” by osmium tetroxide treatment (see Fig. 2G for gemeral orientation). 958

Neurophysin in the mouse forebrain

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Fig. 25. Size of neurons immunoreactive for vasopressin-neurophysin and oxytocin-neurophysin in the nuclei of the hypothalamus of the mouse. The areas of the neuronal perikarya were measured and the equivalent circle diameter calculated (see abscissa Fig. 25j). (a) Vasopressin-neurophysin-IR cells in the supraootic nucleus of three different animals. (b) Parvocellular vasopressin-neurophysin-IR cells of the suprachiasmatic nucleus. (c) Anterior commissural nucleus. (d) Supraoptic nucleus, all regions combined. (e) Paraveutricular nucleus, all regions combined. (f) Vasopressin-neurophysin-IR cells in the anterior, mid and posterior parts of the paraventricular nucleus. (g) Oxytocin-neurophysin-IR cells in the anterior, mid and posterior parts of the paraventricular nucleus. (h) Accessory nucleus characteristic of the mouse. (i) Periventricular nucleus. (j) Smaller cells of the bed nucleus of the stria tertninalis and the amygdala.

small SD. of the overall mean values). Colchicine administration did not noticeably affect the size of the cells. The sizes of magnocellular neurons in both the supraoptic and paraventricular nucleus was unimodally and symmetrically distributed. The cells

were smaller than expected from the literature (e.g. Ref. 61) and had an average equivalent circle diameter of between 12.7 and 13.2pm for both oxytocin-neurophysin-IR vasopressinand neurophysin-IR cells. The average maximum diameters were more difficult to measure because of the

960

M. CASTEL and J. F. MORRIS

difficulty of defining the junction between the perikaryon and a thick dendrite (e.g. Figs 10A and 1IB). This difficulty is reflected in part by the coefficients of variation @D./mean) for the maximum diameter data which were more than double (average 0.27) those for the area data (average 0.11). However, maximum diameters ranged between 17.0 pm and 19.0 pm, a value still smaller than the 25 pm commonly suggested in the literature. To determine whether this was due to species variation, the sizes of cells from the PVN of a heterozygous Brattleboro rat which had been similarly processed, were assessed by the same method. The oxytocin-neurophysin-IR ceils in the rat (n = 67) were 154 sf:44 pm2 in area, equivalent circle diameter, 14.0 + 4.0 pm 17.2 + 5.6 pm maximum diameter (means L- SD.); the vasopressin-neurophysin-IR cells were of similar size: area 170 & 54 g m*, equivalent circle diameter 14.7 + 4.7 pm, maximum diameter 19.5 + 5.3 pm. These values are only slightly larger than the sizes recorded for the mouse. It therefore seemed likely that tissue processing had caused some shrinkage. To analyse this possibility, the shrinkage of sections and of cells was assessed on another h~othalamus. The linear dimensions of dehydrated, mounted specimens had shrunk to 73 + 4% (means f SD., N = I I) of adjacent hydrated sections. Within the dehydrated sections the area of IR cells was decreased to 74% of that of their hydrated counterparts, equivalent to a reduction in equivalent circle diameter to 86%. The sizes of cells in the SON were unimodally distributed, as would be expected in a nucleus which contained only magnocellular neurons. However, there was a considerable range of area, equivalent circle diameter and maximum diameter of the cells, the smallest cells being less than half the diameter of the largest. Immunoreactive cells of the PVN were similar in size to those of the SON, indicating that only one population, the “magnocellular” neurons, had been stained. Cells of the SON did not show any significant systematic difference in size between anterior and posterior parts of the nucleus (Table 1). Oxytocinneurophysin-IR ceils of the posterior part of the PVN were significantly smaller than those found more rostrally (Table 1, Fig. 24F), but cells in the anterior and mid-parts of the PVN did not differ significantly in size. Vasopressin-neurophysin-KR cells in the posterior part of the PVN were also significantly (P < 0.01) smaller than those in the anterior, but not the mid-portion of the nucleus (Fig. 24G). Within the PVN but not the SON, oxytocin-neurophysin-IR cells were just significantly (P < 0.025) smaller than vasopressin-neurophysin-IR cells, The immunoreactive cells of the anterior commissural nucleus (excluding the small, weakfy stained va~p~ssin-neurophys~-IR cells identical to those of the bed nucleus of stria terminalis which encroach on the nucleus) were significantly larger (P < 0.001) than those of the PVN or SON, but the oxytocinneurophysin-IR and vasopressin-neurophysin-IR

cells of the anterior commissural nucleus did not differ significantly in size (Table 1, Fig. 24C). The immunoreactive cells of the profuse periventricular nucleus and mouse accessory nucleus were similar in size to those of the PVN and SON, and the oxytocin-neuroph~in-IR and vasopressinneurophysin-IR cells in them did not differ in size (Table 1, Fig. 24H and I). Immunoreactive cells in the suprachiasmatic nucleus were significantly (P < 0.001) smaller and more rounded than magnoceilular neurons, and the cells of its rostromedian division were the smallest neurophysin-IR cells seen (7.7 4 2.8 pm; mean equivalent circle diameter + S.D.). Within the amygdala and bed nucleus of the stria terminalis the rather faintly stained vasopressinneurophysin-IR cells were of similar size. They were si~ifi~antly (P < 0.001) smaller than vasopressinneurophysin-IR “magnoceiiuIar” neurons, and were significantly (P < 0.001) larger than cells of the suprachiasmatic nucleus. Within the rather loosely defined confines of the bed nucleus of stria terminalis there were also occasional scattered, more heavily stained vasopressin-neurophysin-IR cells indistinguishable from magnocellular neurons of other groups. DISCUSSION

Why the mowe? The basic organization of the oxytocinergic and vasopressinergic innervation of the normal mouse forebrain has been .described in detail for the first time. This provides a starting point for experimental studies in wild and laboratory mice, including genetically aberrant strains and mice from dtEerent biotopes. In principle, though not in detail, the neurophysincontaining innervation of the mouse forebrain resembles that of the rat,“T6’ though that of the mouse appears more strikingly profuse. This profuseness relights important features of the system that appear to be relevant to many species {see below). The relatively small size of the mouse brain, in which several levels of the immunoperoxidase-lab&d system may be viewed simultaneously in a single vibratome section, contributes to -the appearance of profuseness and to the appreciation of the relations~~ between the different components. In species with larger brains many more se$ions are required to reconstruct the cytoarchitecture of diffuse immunoreactive entities. In like manner, the brain of the diminutive, albeit exotic tree-shrew has served as fortuitous material for reviewing the principles of the oxytocinergic and vasop~~iner~~ systems in mammais.78*79 Immunolocaiization of neurophysins and its impiications for localization of oxytocin and vasopressin This study has used specific monoclonal antibodies

Neuro~hysin in the mouse forebrain directed against oxytocin- and vasopressin-associated neurophysins with a view to obtaining information about oxytocin- and vasopressin-containing neural elements. The results strongly indicate that oxytocinneurophysin-IR and vasopressin-neurophysin-IR are, indeed, found where respectively oxytocinergic and vasopressinergic cells and processes would be expected.24*6’~7**83 This implies that, throughout the forebrain, oxytocin and vasopressin are produced from precursors that also contain neurophysins immunolo~cally indistinguishable from those in magnocellular neurons.27 The finding also permits us to equate oxytocin-neurophysin-IR with oxytocincontaining, and vasopressin-neurophysin-IR with vasopressin-containing, and these terms will be used mterchangeably in the discussion. Use of these terms is not, however, intended to indicate the availability of proof that the hormonal peptides are neuroactive in all these locations, although this seems likely; also, the possibility that the neurophysins are neuroactive when released has yet to be demonstrated. ClassiJication of immunoreactive neurons: sire and tissue shrinkage The dimensions of neurophysin-IR neurons in these aldehyde-fixed, vibratome-sectioned, immunocytochemically stained tissues of the mouse are smaller than those generally reported. Comparison of our results with those reported for the rat by Sofronie~’ indicates that, in mouse-derived tissues, the cells in all nuclei are smaller, their mean equivalent circle diameters being 59 t 2% (mean f S.E.M.; comparison of 10 nuclear groups) of the values reported for the rat. Species variation can, however, only account for a small part of this difference, because tissue from a rat processed in the same way as that from mice revealed cells of similar size in the two species. The cells measured by Sofroniew (25 cells per nucleus) were from tissue fixed in Bouin’s fluid. Such fixation is reported3’ to result in a 2&25% decrease in the size of SON-containing blocks and the area of SON perikarya, compared to freeze-dried tissues. Prefixation in aldehydes, which leaves tissues osmotically active’ would also allow some shrinkage of the fixed tissue during immunocytochemical processing in the somewhat hypertonic media. It would, therefore, appear that the method of processing plays a critical role in determining the final size of neurons in mounted slides. The corollary of this is that comparison of the sizes of cells between different studies will only be valid if the tissues were identically processed. Comparison of the size of cells in different nuclei within tissue should, however, be valid. The immun~yt~hemical procedure we have used produces “Go&i-like” impregnations62 of the neurons which appears to stain entire cell bodies. Even within the SON, which is said to be an homogenous population of magnocellular neurons, a wide range of cell sizes was detected. Only coronal sections were

961

assessed so that varied orientation of the cells could have contributed to this. The essential similarity of size of the PVN neurons and their unimodal distribution indicates that our method has not stained a separate population of ‘~pa~~llular” vasopressincontaining cells in the PVN that is particularly prominent in adrenalectomized animals.56 Our method is clearly very sensitive because it stains vasopressinergic cells in the amygdala and bed nucleus of the stria terminalis even without prior colchicine tr~tment of the animals. The failure to stain a distinct group of parvocellular PVN neurons implies that such cells produce vasopressin in extremely low amounts in the absence of stimulation by adrenalectomy. The size distributions of the neurons in PVN and SON also questions the assumption that cells can readily be separated into groups of characteristic size. Sofroniew@ gives an indication of this in referring to the posterior group of PVN neurons as “mediocellular”. Our data suggest that both the PVN and SON contain neurons of a wide range of sizes and that features other than size will be needed to separate mo~holo~~ally distinct categories of cells. If the tissue shrinkage that occurred during processing had caused more shrinkage of the neuropil than of the immunoreactive neurons, such shrinkage could have caused the corkscrew-like appearance of the medially-directed dendrites of many of the PVN and mouse accessory nucleus neurons. However, given that the cells have shrunk as much as the tissue blocks and that other immunoreactive processes do not have such a configuration, this corkscrew-like configuration appears to be a real cytoarchitectural feature of such dendrites, which are very prominent in the mouse. Periventricular neurons The rostra1 periventricular region of the house and laboratory mice investigated is richly endowed with a variety of oxytocinergic and vasopressinergic cells which lie imm~iately beneath the ependyma of the third ventricle in no particular ~lationship to “established” nuclei. This feature, previously reported for wild desert mice,” distinguishes the mouse from the rat, in which this cell group is less well developed. In the guinea-pig, enhanced labelling of a distinctive group of vasopressin-confining periventricular cells has been reported to occur during fever, concomitant with increased vasopressin immunoreactivity in the lateral septum.“.82 This finding can be linked with the contention that vasopressin is a physiological antipyretic with identified septal receptors,33 and suggests a functional connection between the subependymal area of the third ventricle and the lateral septum. However, the vasopressinergic innervation of the septum is currently thought to derive from cells in the bed nucleus of the stria terminalis,****’ though other areas, including the paraventricular nucleus and suprachiasmatic nucleus49*63have also been impli-

962

M. CASTELand J. F. MORRS

cated, The septal plexi of vasopressinergic fibres could have multiple origins, including the periventricular cells. The profuse periventricular vasopressinergic cells of the mouse suggests the testable h~othesis that mice are efficient the~oregulators when challenged with fever and that these periventricular cells play a role in this regulation. In the guinea-pig, oxytocinergic cells are aligned along the ventricular walls in linear formation, and are considered an unique subdivision of the paraventricular nucleus.@ Although oxytocinergic periventricular cells are plentiful in the mouse, linear formation is not markedly apparent. Moreover, it is unlikely that the periventricular oxytocinergic cells are involved in the innervation of the septum, which has a paucity of oxytocin-containing elements in both rats and mice. The accessory nucleus of the mouse In all strains of mice studied to date a distinctive bilateral cluster of cells, located in the hypoth&amus midway between the paraventricular nucleus and the base of the brain, and which we term the “mouse accessory nucleus”, is apparent. Silverman and PickardB report that each such cluster contains about 50 magnocellular neurons, both oxytocinergic and vasopressinergic. The nucleus lies at approximately the same dorsoventral level as the circular nucleus in the rak” but differs from it in many other respects. First, MAC lies somewhat closer to the third ventricle than does the circular nucleus. Second, the diffuse arrangement of cells ’ of the MAC differs considerably from the tightly packed arrangement of cells of the circuiar nucleus, and cells of the MAC are not arranged in tube-like formation around a bfood vestselM Thirdly, cells of the MAC exhibit a profusion of long dendrites extending towards the third ventricle whereas those of the circular nucleus have a dearth of identifiable dendrites.7o Roth MAC and the circular nucleus are well vascularixed and, in fact, the intimate association of nucleus circularis cells with a blood vessel led Hatton”*70 to hypothesize an osmoreceptor function for its cells. The osmosensitivity of cells of the nucleus circularis has yet to be tested, but both they and cells of the MAC could well share the osmosensitivity demonstrated for magnocellular neurons of the supraoptic nucleus.s@s The prominent orientation of dendrites of the MAC towards the third ventricle might subserve interaction with some as yet unidentified afferent input (for example, they lie in the ascending projection of varicose vasopressinergic fibres derived from the supra&iasmatic nucleus). Equally, their orientation might serve to bring them close to the ependyma of the anteroventral third ventricle. This area is known to be important in osmoregulation,6 and the ependyma is unlikely to provide a barrier to changes in osmotic pressure since horseradish peroxidase’ and neuro-

active substances can pass across it. Elucidation of the function of MAC cells must await study of their physiology, and an analysis of the connections made by their dendrites might provide useful pointers towards the appropriate questions to ask. The suprachiasmatic nucleus and its rostromedian division The suprachiasmatic nucleus of mammals is well known as an important circadian oscillator42 responsible, among many other roles, for the circadian rhythm of vasopressin concentrations in the cerebrospinal fluid. 55 Indeed, it is proposed either that suprachiasmatic neurons themselves rhythmically release vasopressin into the cerebrospinal fluid, or that they induce other vasopressinergic cells so to do. In all species so far studied, about 20% of the closely-packed small cells of the bilateral suprachiasmatic nuclei produce vasopressin and its related neurophysin;63,71 the mouse is no exception.59*63However, we have found that pretreatment of mice with colchicine reveals additional vasopressinneurophysin-contai~ng cells in a small cluster of neurons lying rostromedial to the main paired nuclei, between the ventricle and the optic chiasm. Despite a reasonably exhaustive search of the literature 11,23*59~61,63~71~72~78 we could find no mention of these cells: which we term the rostromedian division of the su~ac~asrna~ nucleus, with the possible exception of the early i~unofluore~n~ studies of Watkins26*75 illustrate vasopressinwhich immunoreactivity in a similar location in the rat, but do not specify whether the cells are magno- or parvocellular. A few magnocellular perikarya are often found rostrally, as an extension of the anterior supraoptic nucleus, but these are far fewer in number and larger in size than the dense cluster of small cells that comprise the median suprachiasmatic cluster. A “medial prechiasmatic gland” in the same area has been described,” but the “glandular” cells involved were apparently ependymal. A specialized ependyma in this region could be involved in transport of vasopressin from the rostromedian cluster of cells into the cerebrospinal fluid of the third ventricle, but whether or not this occurs must await further experimentation. It is generally held that rostrally projecting vasopre~inef~c axons from the sup~c~~matic nucleus pass through the OVLT on their way to the dorsal preoptic area, but do not terminate within the circumventricular organ. 6’v76 In the mouse, however, fine calibre vasopressin-neurophysin-IR fibres sprout rung-like collaterals, indicative of a terminal field, in the vicinity of the OVLT. The OVLT, like the anteroventral part of the third ventricle, is a critical area for osmoregulation,5’ so that the presence of terminals originating from the suprachiasmatic nucleus in this circumventricular organ would offer an anatomical substrate for osmo-circadian integration.

Neurophysin in the mouse forebrain The “new” vasopressin-containing nuclei Van Leeuwen and Caffe’4*73have recently described large groups of parvocellular vasopressin-containing neurons in the bed nucleus of the stria terminalis and in the medial amygdaloid nucleus of colchicinetreated rats, the immunolabelling being more intense in males than in females.” In the present study, these extensive cell groups were readily revealed in mice with or without colchicine pre-treatment. The vasopressin-neurophysin immunoreactivity of these “new” perikarya was always considerably lower than that of magnocellular or suprachiasmatic perikarya, and varied unpredictably so that no clear-cut effect of colchicine pretreatment could be established. This variability of labelling could reflect fluctuations in vasopressin content of the cells related to sexual” and/or seasonal cycles, as has been recently reported for the European Hamster.13 De Vries and his colleagues favour a sex-dependent role for the neurons in the bed nucleus of the stria terminalis and possibly also the medial amygdaloid nucleus.23*“*74On the basis of experimental manipulations in rats, involving lesion and tracer techniques as well as castration and testosterone therapy, these authors have inferred extensive projections from both nuclei to other brain sites (see the map in De Vries et aL”). Some of these effects of castration have been confirmed for both normal and hypogonadal (hpg) mice3’ (and C. Mayes and H. M. Charlton, personal communication). We have visualized, in the mouse both the “new” cell groups and the extensive terminal fields. However, we were unable to substantiate connections between the two from serial sections. The immunoreactive cells of the bed nucleus of the stria terminalis and of the medial amygdala have short, fine processes that can rarely be followed beyond the nucleus of origin, whereas some of the proposed projection sites display extravagant preterminal and terminal immunoreactivity. This incongruity could be explained by some feature of maturation of the pro-hormone during axonal transport, with less neurophysin exposed for immunolabelling within the cell body than along processes or in terminals. However, if this were the case, the maturation would have to differ from that in magnocellular neurons in which labelling is roughly equal from the cell body and through the axon. The incongruity might also be explained if far fewer vasopressin- and vasopressinneurophysin-containing granules accumulate in the cell bodies and proximal parts of the axon than is the case for magnocellular neurons. Elucidation of the origin of the dense vasopressinergic terminal fields, and of the projections of the “new” vasopressinergic neurons must await further experimentation. Extra-hypothalamic

vasopressinergic projections

The hypothalamus of common laboratory rodents is reasonably well mapped within the neuroanatomical literature, and there are no gross inconsist-

963

encies between the various stereotaxic atlases of the mouse41*58*60 and rat”*4*47brain. However, there are many discrepancies in the interpretation of the substructure of both the thalamus and the amygdaloidhippocampal complex. These could arise from technicalities such as the precise angle at which the brains were sectioned, from staining or other preparative procedures, or from differences in personal interpretation of inconspicuous boundaries. In most cases the presentation of complex areas such as the interface between the hypothalamus, subthalamus and amygdala reveals relatively little agreed topographical detail. This deficiency creates a problem of interpretation, because this interface is a particularly rich, complex and heterogeneous area of vasopressinergic innervation in the mouse forebrain. Not only are immunoreactive cells, fibres of passage and terminal fields confined together within a circumscribed area, but also the fibres project in many different directions against a background of nonimmunoreactive structures that cannot, in all cases, be unambiguously identified. A case in point is the circular structure, ventral to the zona incerta, which we have called the “mouse subthalamic area” (MSA; Fig. 17), surrounded by a presumptive terminal field of vasopressinergic fibres, and around which vasopressin-neurophysin-IR fibres are deflected towards the thalamus. It is uncertain whether this subthalamic area is analagous to the identified subthalamic nucleus (body of Luys) in the mouse and rat, which is generally shown located medial to the internal capsule and continuous caudally with the substantia nigra. The entopeduncular nucleus is also referred to by some authors (see Ref. 11) as a subthalamic nucleus, but no atlas indicates anything worthy of name in the area we have designated “MU”. Sidman et al.% indicate the general vicinity as tegmental areas H, and H,, (the fields of Forel) but in other atlases the fields of Fore1 are placed dorsal to the zona incerta. For the moment, these limitations restrict any meaningful interpretation that can be placed on the vasopressinergic innervation of this area. General considerations In this study, and a recent publication on the rat by De Vries et aI.,” neurophysin-immunoreactive processes and varicosities have been found in great distribution, both inside and outside the hypothalamus, indicating that vasopressin or some other component of vasopressin-containing granules may thus be neuroactive in more terminal fields than was previously considered likely. Of particular interest is the ubiquitous presence of apparently extrinsic vasopressinergic varicosities within nuclei comprising immunoreactive perikarya. Recently, it has been shown that magnocellular axons of passage in the median eminence release vasopressin in a manner distinct from the neurohaemal release that occurs at the portal plexus.3’ This finding has far-reaching impli-

964

M. CASTELand J. F. MORRIS

cations for peptidergic innervation in general, and may indicate many of the multitude of vasopressinand oxytocin-containing varicosities that were seen in the brain could release these neuropeptides to exert neurotransmitter or neuromodulatory functions. Our study also reveals dendritic fields in the mouse in considerable detail and shows that the dendrites project much further from the cell bodies than we previously appreciated. Functionally, this means that terminal fields far away from the perikarya may interact with the dendrites, and also that large areas of dendritic membrane are exposed to cerebrospinal fluid as they lie beneath the ependyma of the third ventncte or beneath the limiting glial membrane of the base of the brain.3 The chicken69 and pigeon” show neurophysin-immunoreactive dendrites of comparable magnitude, particularly in the periventricular zone. Judged from the intensity of their immunoreactivity, such dendrites contain large amounts of peptide. Dendritic transport could, therefore, convey large amounts of vasopressin, oxytocin and neurophysin in directions that have not previously been considered, when consideration has been restricted to axonal transport. The release of peptides from the dendrites has still to be demonstrated experimentally, but it could contribute to the large amounts of vasopressin, oxytocin and neurophysin found in the cerebrospinal fluid53 and to other areas of the brain where neuromodulatory roles could be exerted. Preparative techniques may well be an important factor in the degree to which dendrites are revealed. In our study the use of 5% glutaraldehyde was particularly advantageous. De Vries et al. used similarly high concentrations of glutaraldehyde in their recent impressive study of vasopressinergic innervation in the rat brain24 and Kosaka et a1.3s have recently recommended 5% glutaraldehyde for immunocytochemistry of neurotransmitter-synthesizing

enzymes in the central nervous system. Thus it appears that glutaraldehyde may have been erroneously maligned in the past as being deleterious to the preservation of antigenicity. Finally, this study reveals that two strains of laboratory mice, as also the desert-living Acomys cahirinus and Acomys russatus,‘5.‘6 have more profuse neurophysin-containing nervous systems than have yet been demonstrated in the rat. All these strains of mice have proved to be remarkably resilient to severe chronic osmotic stress, with regard both to recovery of body weight and also to replenishment of vasopressin stores within the magnocellular HNS. Furthermore, mice with hereditary nephrogenic diabetes insipidus, in which the output of urine can be even more copious than in rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain), nevertheless show large amounts of granules and hormone in both vasopressin- and oxytocin-containing neurosecretory cell~,~ whereas the Battleboro rats produce no vasopressin and have greatly depleted stores of oxytocin. 43 Whether these considerable species-related differences in response to osmotic stress can be attributed to the special features of the neurophysin-containing innervation revealed in this study remains to be determined.

acknowledge with warm thanks the set of monoclonal anti-neurophysins from Dr Yakov

Acknowledgements-We

Ben-Barak and Dr Harold Gainer. The excellent tech&al assistance of Mrs Shula Cohen, the photographicservices of the Department of Human Anatomy, Oxford, and the assistance of David Pow with the measurement of tissue shrinkage were also greatly appreciated. Research was supported by U.S.A./Israel BSF grant 84-80268 to M.C. and by grant AG43/140 and MRC G608/263 to J.F.M. The Reichert MOP-AM02 was provided by the Royal Society (to J.F.M.) M.C. also thanks the Lord Marks Foundation for sabbatical stipend.

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