0361-9230190 $3.00 + .oO

Brain Research Bullerin,Vol. 24, PP. 191-200. c Pergamon Press plc, 1990. Printed in the U.S.A

Afferent Connections of the Rat’s Supraoptic Nucleus W. A. ANDERSON,* Department

of Anatomy,

’

J. E. BRUNI AND A. KAUFMANN

The University of Manitoba,

Winnipeg,

Manitoba,

Canada

Received 6 April 1989

ANDERSON, W. A., J. E. BRUNI AND A. KAUFhMNN. Afl erent connections of the rat’s supraoptic nucleus. BRAIN RES BULL 24(2) 191-200, 19!?0.-Neurons projecting to the supraoptic nucleus (SON) have been identified following stereotaxic injections of either horseradish peroxidase or fast blue into the SON region of adult rats. The subfomical organ, median preoptic nucleus, organum vasculosum of the lamina terminalis and medial septal nucleus were the source of the largest numbers of supraoptic-projecting neurons. Several smaller projections also originate from the ipsilateral locus coeruleus, preoptic area, lateral parolfactorial area, dorsomedial nucleus of the hypothalamus, lateral parabrachial nucleus and ventrolateral medulla. Several other areas appeared to project only to the region immediately dorsal to the SON: lateral septal nucleus, diagonal band of Broca, ventral tegmental nucleus, and the supramamillary nucleus. These areas may influence SON neurosecretory function by way of intemeurons found immediately dorsal to SON. Additional areas were identified with retrograde fluorescent label only, and these projected to the area immediately dorsal to SON and/or to SON itself. Afferents Supraoptic

Horseradish peroxidase nucleus Rat

histochemistry

Fluorescence

THE rat supraoptic nucleus (SON) consists of approximately 4400-7000 magnocellular neurosecretory cells (3 1,49), whose principle efferent projection is to the neurohypophysis. The active principals, vasopressin and oxytocin, produced by these neurons are transported by axoplasmic flow to the posterior pituitary, and released there into fenestrated capillaries. The SON is known to be involved in parturition, lactation and the homeostatic regulation of the body water content, although it is not itself an osmo-receptor centre (15,7 1). Control of the secretory function of the SON is largely regulated by other neuronal centres (13,52), despite recent evidence to suggest the SON may partially regulate its own neurosecretion (366). Electrophysiological stimulation of the subfomical organ (SFO) and ipsilateral paraventricular nucleus, ventrolateral medulla (VLM) and amygdaloid body have been shown to increase antidiuretic hormone (ADH) secretion from SON neurons (16, 18, 21, 56, 58, 63, 65), whereas an inhibitory influence on these same cells has been reported following excitation of the septum, diagonal band of Broca, olfactory system, and peripheral baroreceptors (4, 9, 14, 22, 23, 51, 75, 77). Immunohistochemical methods have identified a variety of neurotransmitters in afferents to the SON (7, 10, 26, 71, 73). While gamma-aminobutyric acid (GABA) and norepinephrine (NE) have received the most attention (6, 10, 54, 71, 74), several additional transmitters and/or neuromodulator substances including dopamine, histamine, acetylcholine, glucocorticoids and angiotensin II have recently been implicated in the control of oxytocin and vasopressin secretion from the SON (1, 6,

microscopy

Retrograde

tracing

19,25,26,41). GABA and norepinephrine have been identified in pathways which mediate information to supraoptic vasopressinergic neurons from peripheral baroreceptors and chemoreceptors, respectively (54). More recently, immunostaining and receptor binding site studies have confirmed that NE, GABA, glucocorticoid and angiotensin II receptor sites do in fact exist within the SON (19, 23, 26, 74, 76). While data about the chemical nature of the afferents responsible for SON regulation has steadily increased, substantially less information is available regarding the distribution and localization of the neurons which produce these putative neurotransmitters. At the electron microscopic level, deafferentation studies of the SON have shown that at least one-third of synaptic input to this nucleus is of extranuclear origin (31,77). More recently, anterograde tracing and electrophysiological studies have identified projections from the subfomical organ (SFO) (32, 45, 55), anteroventral third ventricle (8), VLM (33), locus coeruleus (LC) (24,59), the raphe nuclei (46), dorsomedial nucleus of the hypothalamus (DMH), medial preoptic area (MPO), bed nucleus of the stria terminalis (BST) (62), septum (30,42, 53,68, 70, 77), and olfactory system (63), and have inferred that a preferential distribution may exist to oxytocinergic and vasopressinergic portions of the SON from certain afferent cell groups (30, 45, 59-62). As yet, few studies have examined the afferent connections of the SON using retrograde tracing methods (20, 45, 56, 59) and therefore little information exists regarding the distribution or numbers of neurons which project to the SON. In fact, only a few

‘Part of this work was presented at the Canadian Federation of Biological Societies Meeting held in Toronto, Ontario, Canada, June, 1985. *Requests for reprints should be addressed to Dr. W. A. Anderson, Department of Anatomy, Faculties of Medicine and Dentistry, The University Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada, R3E OW3.

191

of

192

ANDERSON. BRUNI ANDKAWMANN ABBREVIATIONS

AAA ACB AMB ARH AV BC CA Cl CPU CT DBB DMH DR DTV FLM FX HPC IP LC LHA LM LS MFB MPO MS NOT NR NST NTM NTST

[According

Anterior amygdaloid area Lateral parolfactotial area Nucleus ambiguus Arcuate nucleus Anterovenhal nucleus of the thalamus Brachium conjunctivum Anterior commissure Internal capsule Caudate nucleus-putamen Central tegmental nucleus Diagonal band of Broca Dorsomedial hypothalamic nucleus Dorsal raphe nucleus Tegmental decussation Medial longitudinal fasciculus Fomix Hippocampus Interpeduncular nucleus Locus coeruleus Lateral hypothalamic area Medial lemniscus Lateral septal nucleus Medial forebrain bundle Median preoptic area Medial septal nucleus Nucleus of the olfactory tract

NVM tlVII OC OT OVLT

Red nucleus Sensory nucleus of the trigeminal nerve Motor nucleus of the trigeminal nerve Nucleus of the spinal tract of the trigeminal

to Pellegrino

nerve

PAG PBL PBM PC PH PMV POA PT PV PVN RF SC SF0 SN SM SOL SON SUM TCS TS V VLM VMH VTN VII

Medial vestihular nucleu:, Facial nucleus Optic chiasm Optic tract Organum vasculosum of the lamina terminalis Pans Periaqueductal gray Lateral parabrachial nucleus Medial parabrachial nucleus Cerebral peduncle Posterior hypothalamic nucleus Ventral premamillary nucleus Preoptic area Paratenial nucleus of the thalamus Paraventricular nucleus of the thalamus Paraventricular nucleus of the hypothalamus Reticular formation Suprachiasmatic nucleus Subfomical organ Substantia nigra Stria medullaris thalami Nucleus of the solitary tract Supraoptic nucleus Supramamillary nucleus Corticospinal tract Nucleus triangularis septi Trigeminal nerve Ventrolateral medulla Ventromedial hypothalamic nucleus Ventral tegmental nucleus Facial nerve ____

et a/. (SO).]

of the projections identified using anterograde tracing methods have actually been confirmed to date using retrograde tracing methods. Noradrenergic projections from the VLM and LC (59), and the input from the SF0 (45) are the only extrahypothalamic projections to the SON which have been adequately identified. In view of the number of extrahypothalamic centres which have recently been reported to provide synaptic input to the SON a reevaluation of the distribution of neurons which send axons to the SON would seem appropriate. The aim of the present study, therefore, was to identify the afferent projections to the SON, using the highly sensitive retrograde tracing methods: wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and fast blue (FB). Previous studies with the exception of one recent publication (72), have used tracing methods with less sensitive chromogens (i.e., diaminobenzidine or o-dianisidine). The use of the fluorescent dye FB, and lectin bound HRP with the chromogen tetramethylbenzidine (TMB) represents a refinement in methodology that has heretofore not been applied to the study of afferent projections to the SON. These procedures have allowed us to demonstrate a more extensive distribution of neurons which project to the rat SON. Evidence is provided which suggests that several previously reported SON afferents instead terminate in areas immediately dorsal to the SON. METHOD Adult male Sprague-Dawley rats, weighing between 250-350 g, were used in this investigation. The animals were anaesthetized with a mixture of xylazine (10 mgikg b.wt.) and ketamine (90 mg/kg b.wt.) administered intramuscularly (0.23 ml/l00 g b.wt.), prior to immobilization in a Kopf stereotaxic frame (David Kopf

Instruments, Tujunga, CA). Unilateral injections were made into the left SON using a dorsal approach and stereotaxic coordinates (A, 0.625; H, 0.395; L, 0.185) derived from the atlas of Pellegrino et al. (50). Injections were made using a fine glass cannula (30-50 pm o.d.), mounted on a 1 p,l Hamilton syringe. In twenty rats, 0.005 to 0.01 ~1 of a l-15% solution of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) in combination with 0.3% poly-L-omithine was pressure injected into the SON over 15-20 minutes. Following survival periods ranging from l-7 days, these animals were anaesthetized with sodium pentobarbital(65 mg/kg b.wt.) and perfused transcardially with 1.25% glutaraldehyde 1% paraformaldehyde in 0.1 M phosphate buffer. The brains were then removed from the skull, and serial frozen sections (40 pm thick) were cut, prior to processing for TMB-HRP histochemistry using the method of Mesulam (43). Retrogradely labeled neurons were mapped using a camera lucida device and darkfield illumination. Alternate sections were stained with neutral red and examined using brightfield optics. Although survival times of l-7 days were tested retrograde labeling was most intense when the animals were perfused 2-3 days postinjection. In an additional twenty-two rats, 0.03 to 0.2 ~1 of a 1.5% aqueous solution of the fluorochrome fast blue (FB) was pressure injected into the SON. Five to seven days later, these animals were reanaesthetized and perfused transcardially with 10% cacodylate buffered formalin. Immediately following perfusion, the brains were removed, stored overnight in 30% sucrose buffered formalin, and then sectioned serially at 30 pm. The sections were examined using a Leitz epifluorescent microscope at an excitation wavelength of 360 nm. The size and location of the injections, and the distribution of retrogradely labeled cells were mapped using a camera lucida device and an X-Y plotter. Alternate sections

193

RAT SON AFFERENTS

BR 784

BR 773

ER 753

FIG. 1. Camera lucida drawings of transverse sections through the hypothalamus of rats and 773 showing injection sites and maximum spread of WGA-HPP at 2 days postinjection. Solid areas represent labeled regions as seen with brightfield optics; stippled areas correspond to lightly labeled regions as seen under darktield illumination.

BR753

through the brain orientation

were stained with toluidine blue and used for and identification of nuclei. An aqueous solution of FB

was found not to diffuse as extensively in the neuropil WGA-HRP, and thus produced smaller injection sites.

as

RESULTS

Injection of WGA-HRP Into the SON Three cases representative of WGA-HRP injections into the SON are illustrated diagrammatically in Fig. 1. Injections aimed at the SON can be placed into two general groups. In the first group, representing approximately one-third of the injections (6 animals BR773, not shown BR774,778,779,785,789,790), WGA-HRP was largely confined to the SON unilaterally and extended throughout the rostrocaudal length of the nucleus (Fig. 1, BR773; Table 1, BR770, 773). Although the SON was heavily labeled with WGA-HRP the adjoining lateral hypothalamic region dorsal to the SON was also labeled to varying extents. The WGA-HRP staining found in the lateral hypothalamic area, however, often represented the most diffuse portion of the WGA-HRP injection site. While precautions were taken to limit the spread of WGAHRP into the region immediately dorsal to the SON, none of the injections in this study were confined solely to the nucleus. In the second group of WGA-HRP injections which consist of 14 animals (BR753-764, 781, 784) reaction product was localized to varying extents in the perinuclear zone dorsal to the SON throughout its rostrocaudal extent (Fig. 1, BR753, 784; Table 1, BR753, 781, 784). The distribution of WGA-HRP-positive neurons resulting from an injection limited mainly to the SON is detailed in Fig. 2 (BR770) and Table 1 (BR770 and 773) and is representative of all similar WGA-HRP injections. In both these cases, large numbers of retrogradely labeled neurons were found throughout the rostrocaudal extent of the median preoptic area (MPO), organum vasculosum of the lamina terminalis (OVLT), subfomical organ (SFO) and the ipsilateral lateral parabrachial nucleus (PBL) and the medial part of the preoptic area (POA) (Fig. 2, A, C-D and G). Small projections originating from the ipsilateral ventrolateral medulla (VLM), dorsal raphe nucleus (DR) and medial septal nucleus (MS) were also identified (Fig. 2, B, F, H-I; Table 1, BR770, 773). Since the injection of WGA-HRP into the SON involved diffusion of the enzyme beyond its borders, particularly dorsally, the distribution of afferents to the nucleus ultimately was determined using a subtractive approach. In animals BR753, 781 and 784 the WGA-HRP was confined to the region immediately dorsal to the SON (Table 1; Fig. 1, BR784, 753). When the location of retrogradely labeled cells in these animals was compared with BR770 and 773 marked differences were found. The most notable differences were that no retrogradely labeled neurons were found

in the DR, MPG, OVLT, PBL, SFO, MO or VLM. Furthermore, several areas contained peroxidase-labeled cells which were not previously labeled following injections of WGA-HRP which were confined largely to the SON. These areas included the diagonal band of Broca (DBB), lateral septum (LS), supramamillary (SUM) and ventral tegmental nuclei (VTN) (Table 1). On the other hand, several regions which include the lateral parolfactorial area (ACB), dorsomedial hypothalamic nucleus (DMH), locus coeruleus (LC), periaqueductal gray (PAG) and the preoptic area (POA) contained similar numbers of retrogradely labeled neurons irrespective of whether the WGA-HRP injection site was localized in the SON or

TABLE 1 LOCATION AND DENSITY OF RETROGRADELY LABELED NEURONS FOUND FOLLOWING WGA-HRP INJECTIONS IN THE REGION OF THE RAT SUPRAOFTIC NUCLEUS

Animal 77tY

Source

773a

753b

Areas Projecting

MPO MS OVLT PBL SF0 VLM

+ ++++ ++ +++ ++ ++++ +*

DBB LS SUM VTN

+ + -

+ ++++ ++ +++ ++ ++++ +*

++* + + ++ ++++*

to SON (A) -

-

Dorsal to SON (B) -

++++ ++++ +++ +

+++ + Areas Projecting

ACB DMH LC PAG POA

784b

-

Areas Projecting -

781b

to A and/or B

+++* +

+++* + -

++ ++++*

+ -

++* + + + +++*

*Denotes regions giving rise to bilateral projections. All other projections are ipsilateral or midline in origin. (+) Denotes the relative number of retrogradely labeled neurons +>50; ++51-200; +++201-500; + + + + >500. Letter superscript denotes the type of injection: ‘an injection where the densest part of the WGA-HRP was confined largely to the SON; ‘an injection where the WGA-HRP was confined almost entirely dorsal to the SON.

ANDERSON.

BRUNl

AND

KAUFMANN

FIG. 2. A composite of camera lucida drawings of transverse sections from rostra1 (A) to caudal (I) brain levels of rat BR770. The location and extent of WGA-HRP injected into the SON (solid black areas) is shown at 2 days postinjection. Black dots represent the location and numbers (1 dot = 6 neurons) of retrogradely labeled neurons. For convenience labeled neurons in the MPO are represented by smaller sized dots.

restricted to the hypothalamus

immediately

dorsal to the SON.

Injection of Fast Blue Into the SON In FB-treated animals, the injection site typically consisted of a dense yellow core surrounded by a light blue zone of variable thickness. Only four rats (BR818, 826, 827, 834) that are representative of the different types of FB injections will be described here (Fig. 3). The FB injections have been divided into three main groups. The first group is represented by injections where the spread of FB was confined largely to the SON (BR826). In the second group, the FB labeled not only the SON but had

spread dorsally to include substantial portions of the lateral hypothalamic region (BR8 18). The third group is characterized by injections confined almost entirely to the hypothalamic region immediately adjacent to the SON (BR834, 827). In animal BR826, the injection site involved the caudal two-thirds of the SON (Fig. 4, C-E). The rostra1 third of the SON in this animal, however, was largely unstained. The injection site in animal BR8 18 was similar to BR826 except that the FB staining not only involved the complete rostrocaudal extent of the SON but had spread dorsal to the SON (Fig. 3) and as well involved the neuropil immediately caudal to the nucleus. The distribution of FB neurons resulting from the injection in animal BR826 is

195

RAT SON AFFERENTS

FIG. 3. Cameralucida drawingsof the hypothalamusof representativerats (BR826, 818, 834, 827) illustrating

the location and spread of FB in the SON at 5 days postinjection.

plotted on a series of transverse sections extending from the preoptic area to the spino-medullary junction (Fig. 4). In all of the animals receiving FB injections confined largely to the SON, retrogradely labeled neurons were found in regions of the brain as shown for BR826 (Fig. 4, Table 2, BR826, 818). Retrogradely labeled neurons were found in the arcuate nucleus (ARH), anteroventral nucleus of the thalamus (AV), paratenial nucleus of the thalamus (PT), lateral parolfactorial area (ACB), MPO, MS, nucleus of the olfactory tract (NOT), and the lateral part of the preoptic area (POA) (Fig. 4, A-E, G-H; Table 2). These areas, however, showed little or no retrograde label when the FB injection site was localized outside the SON (BR834, 827) (Table 2). In all four animais chosen to represent FB injections comparable numbers of retrogradely labeled neurons were found in the central tegmental nucleus (CT), DBB, DMH, hippocampus (HPC), locus coeruleus (LC), LS, posterior nucleus of the hypothalamus (PH), ventral premamillary nucleus (PMV), SFO, and VLM (Table 2). Labeling of the LC, IS, PAG, and PI3L was bilateral in all four FB cases. Injections of FB in case BR826, however, showed additional bilateral labeling in the DMH and PMV. In all areas with bilateral labeling, the number of cells on the ipsilateral side always exceeded those on the contralateral side. In addition, in each of the FB-injected rats, a small collection of retrogradely labeled neurons was consistently observed immediately dorsal to the SON on the contralateral side (Fig. 4, C-E). DISCUSSION

In recent years, a large number of tracing studies have been employed to examine the afferent connections of several of the hypothalamic nuclei (2, 11, 12, 27, 28, 35, 37, 38, 42, 57, 60, 62). A number of cell groups have been identified as SON afferents in rat using retrograde and anterograde tracing methods. However, not until the recent advent of lectin-conjugates of HRP, which do not label fibres of passage (S), has there been convincing evidence for extrahypothalamic sources of SON afferents. While the SF0 (25, 3X44,45,49) has been the region most consistently identified, several other neuronal groups including the MPO, OVLT and VLM have now been identified using these methods (8, 9, 33, 72). A comparison of the dis~bution of retrogradely labeled neurons found following WGA-HRP injections within tbe SON as apposed to injections which were localized dorsal to the SON (without involving this nucleus) suggests that the SFO, MPO, PBL, OVLT, DR, MS and VLM project exclusively to the SON. A similar comparison of I?3 injection sites and the distribution of retrogradely labeled neurons, however, suggests that several

additional areas project primarily to the SON with little or no termination in the area immediately dorsal to the SON (perinuclear zone). When compared with WGA-HRP injections in the SON which have a similar size and localization, we have found that the ARH, AV, PT, ACB, POA and NOT were also retrogradely labeled following discrete injections of FB in the SON. In light of the evidence that these pathways were not labeled following injections of FE%which were restricted to the perinuclear zone, it is our opinion that these areas probably send axons to the SON in a path which does not pass, for the most part, through areas labeled by the dorsally situated FB injections. The WGA-HRP findings have identified several other areas including the ACB, DMH, LC, POA and PAG which may project to both the SON and the area immediately dorsal, the perinudear zone, since both these areas contain similar numbers of WGA-HRP-labeled neurons when injections were located dorsal to the SON, or when the retrograde marker was confined primarily within the SON. The DIBB, LS, SUM and VTN areas, on the other hand, contained larger numbers of retrogradely labeled neurons only when the WGA-HRP injections were localized primarily dorsal to the SON suggesting that these four sources of afferents terminate primarily in the perinuclear zone immediately dorsal to SON rather than within the nucleus itself as reported by Rogers et al. (56). Afferent projections to SON from SF0 (25, 32,44,45, 55,65) and MPO (62), have been described in a number of studies using both anterograde and retrograde tracing methods. The SF0 is known to play a key role in vasopressin release from the SON during periods of increased blood hypertonicity and angiotensin levels (23, 29, 39). The MPO projection to SON is thought to play a role in maintaining body fluid homeostasis and hemodynamits (62). The VLM (59) and PBL (47,48) both receive projections from nucleus of the solitary tract, which in turn receives primary visceral afferent info~ation from the glossoph~nge~ and vagus nerves which are known to influence tbe secretion of vasopressin (6, 36, 59). Our study confirms previously reported projections from VLM (9, 33, 59, 60) to the SON, and in addition provides evidence for a projection from PBL. Recent autoradiographic evidence suggests that the projection from the VLM is destroyed by 5,7-d~ydroxy~t~ine ~tment and therefore is serotonergic (33). It seems likely that both the VLM and PBL relay visceral information to the SON by direct afferent pathways, although a monosynaptic projection from the PBL has not been previously described (33, 56, 59). The paratenial and anteroventral nuclei of the thalamus were retrogradely labeled with FB following injections which were restricted largely to the SON. Injections of FB into the region

FOLLOWINGPAGE FIG. 4. A composite of camera lucida drawings of transverse sections throughthe rostra-caudalextent of rat brain (BR826) showing the injection site (solid black area) and location of retrogradely labeled neurons, 6 days after injection of FB into the SON (each dot = 8 FB-labeled neurons),

ANDERSON. BRUNI AND

KAliF,MANh

CAUDAL

L

K

J

H

G

ROSTRAL

197

RAT SON AFFERENTS

TABLE 2 LOCATION AND RELATIVE DENSITY OF RETROGRADELY LABELED NEURONS FOLLOWING FAST BLUE (FB) INJECTIONS IN THE REGION

OF THE RAT SUPRAOF’IICNUCLEUS Animal 827c

834c

alab

826”

Source

Identified Using WGA-HFU’

Areas Projecting to SON (A) ARH AV P-r ACB MPO MS NOT OVLT POA

+ + ++ ++ +++t +++ +++ +++t +++

++* ++

++t+ +++ ++++ ++++ +++ ++++ ++++*

-

-

+ + -

+ + + + + +

0

0 0 0 0

Areas Projecting Dorsal to SON (B) DTV VLM

-

+ +

++

+

t

+

0

Areas Projecting to A and/or B AAA CT DBB DMH HPC LC LS PAG PBL PH PMV SF0 VMH VTN

++ ++ +++ ++* ++ ++ +++* ++* +++* + ++* +++ + ++

++++*

++

+++ ++++ +++* ++ +++* ++++* +++* ++++* +* +++* ++++ +++* +++*

++ ++ t+ ++* +++* ++* ++* + ++ +++ + ++

+t +t +++ ++ +++* +++* ++* +* ++ +

0 0 0

0

0

*Denotes regions giving rise to bilateral projections. Black dots denote areas retrogradely labeled following WGA-HRP injections in the SON. (+) denotes the relative number of retrogradely labeled neurons +>SO; ++51-200; +++201-500; ++++>500. Letter superscript denotes the type of injection: ‘an injection of FB which was confined largely to the SON; ‘an injection of PB which labeled not only the SON but spread dorsally; ‘an injection of FB which was confined almost entirely to the region dorsally adjacent to the SON.

dorsal to the SON in this study, however, have failed to retrogradely label the PT or AV areas suggesting that projections from these areas are in fact to the SON directly. Tribollet et al. (72), however, found labeled neurons in the PT, AV and PV only after HRP injections into the SON using the dorsal approach, but never when the ventral approach was used. These authors felt that the label found in these thalamic nuclei was likely the result of the retrograde marker being picked up by axons located along the injection tract dorsal to the SON, rather than from terminals in the SON. The present findings do not support their conclusions since the injection of a more sensitive retrograde marker, FB, into regions immediately dorsal to the SON have failed to retrogradely label neurons in the PT or AV areas. Only when the injection of FB was centred in the SON have we been able to report retrogradely labeled neurons in these regions. Several areas like the SFO, PBL and VLM which had been

shown to project exclusively to the SON following WGA-HRP injection were also found to be retrogradely labeled following injections of FB dorsal to the SON. Contrary to autoradiographic findings (33&t), we have not found evidence for projections from the SFO, PBL or VLM to the lateral hypothalamic region immediately dorsal to the SON using retrograde WGA-HRP tracing methods. It should be noted that the numbers of retrogradely labeled neurons found in the SFO, PBL and VLM were similar to those found following discrete injections of FB in the SON alone. We are of the opinion that these results can best be explained on the basis of labeling fibres of passage. It seems more likely that these axons may have taken up FB as a result of passing through the injection site, en route to the SON. Considering, however, that projections to the lateral hypothalamus dorsal to the SON exist for each of these areas, our negative WGA-HRP results may simply indicate that these pathways are not of sufficient density to demonstrate with WGA-HRP methods. Unfortunately, these discrepancies can not be satisfactorily resolved using light microscopic methods and rather require ultrastructural degeneration studies to confirm direct connections between the SFO, PBL and VLM and the lateral hypothalamus. Since fluorochromes are thought to be more effective retrograde markers than HRP, it was not surprising that several additional areas were found to be retrogradely labeled as a result of injecting FB into the SON region. More specifically, the FB experiments have identified as many as thirteen additional sources of afferents (AAA, ARH, AV, CT, DBB, DTV, HPC, NOT, PH, PMV, PT, VMH and VTN) to the SON and perinuclear region which were not identified in the present study following similar sized injections of WGA-HRP. Furthermore in this study we have reported that the number of retrogradely labeled FB neurons found in each of these areas was similar irrespective of whether the FB injection site was localized within the SON or immediately dorsal in the perinuclear zone. These results allow for several interpretations: one being that each of these areas provides a similar sized projection to both the SON and the region immediately dorsal to it. A second interpretation, as discussed above, might be that pathways to the SON from these areas unavoidably pass through injections placed dorsal to the SON. Since FB is notorious for labeling fibres of passage (64), one can not eliminate the possibility that dorsal injections may label fibres which pass through the injection site en route to the SON or other targets. Moreover, we can not exclude the possibility that these added areas of retrograde labeling represent uptake by fibres passing through the SON without synapsing. The latter suggestion, however, is regarded as unlikely since the number of extrinsic axons which pass through the SON is considered small (31). Rather, it is our contention that many of these afferents do in fact supply a direct input to the SON since the smallest of our FB injections into the SON (BR826) involved only minimal spread into the perinuclear zone and yet has shown moderate to heavy amounts of retrograde label in each of these areas. The fact that larger numbers of FB-labeled neurons have been reported following injections which spread more dorsal into the perinuclear zone while still involving the SON suggests that the perinuclear zone as well receives inputs from these same regions. Although our injections of both FB and WGA-HRP into the SON have been extremely small, we have not been able to entirely exclude labeling the perinuclear zone in any single case and therefore we are unable to eliminate this region as a possible site of termination for some of the additional afferents which we have reported. A case in point is the VLM projection to SON. The fact that a FB projection from the VLM was found following injections in the dorsal perinuclear zone and not following injections which were confined almost solely to the SON suggests that the VLM projection to the SON actually terminates outside the SON rather

.ANDERSON. BRUNI AND KAUF‘MANN

than within this nucleus as suggested initially from the WGA-HRP data (see Table 1). It is, therefore, more likely that the VLM projection to SON terminates near the outer boundary of this nucleus since this projection was also retrogradely labeled following small WGA-HRP injections in the SON. The fact that our smallest injections of FB in the SON (which were more restricted to the SON than our most discrete WGA-HRP injections) have not labeled a projection from the VLM while FB injections dorsal to the SON consistently labeled a small projection from the VLM seems to support this conclusion. These findings, however, do not exclude the possibility that VLM projects exclusively to the SON and that dorsal injections of FB label fibers of passage to the SON while the discrete SON injections of FB made in this study have not been able to retrogradely label a VLM projection to SON because of some unknown technical problem. Because FB is considered to be a more sensitive retrograde marker, the latter hypothesis is considered highly unlikely. Confirmation of the precise localization of many of these afferents will now require ultrastructural observations. Animals injected unilaterally with FB also showed labeled neurons in the lateral hypothalamic region immediately dorsal to the ipsi- and contralateral SON. Neurons exhibiting Golgi-like staining following small injections of HRP into the SON, have

previously been demonstrated ipsilaterally in this area (70,721. II was postulated by these authors that these cells may function a, interneurons in the perinuclear region, relaying input to the SON from a variety of afferent source.\. Indeed. electrophysiological and immunocytochemical studies have suggested that SON neurons receive an input from adjacent perinuclear GABA and cholinergic neurons ( 17. II. 77,). Septal stimulation is thought to exert an inhibitory effect on the SON by wa] of these GABA producing intemeurons (51,661. It therefore seems likely that the various areas shown. in the present study. to project dorsal to the SON may also directly influence SON activity by way of intcrneurons found in the perinuclear zone. Our study additionally reveals that the perinuclear zone on both side\ projecth to each SON. It seems reasonable to postulate that by virtue ot’ these connections that the two SON nuclei could function in a coordinated fashion, and the cells found dorsal to the nuclei may provide the pathway for this communication between the two SON regiona. ACKNOWLEDGEMENTS

This study was supported by a grant from the MRC of Canada. We would like to thank Mr. Perumal for his excellent technical assistance.

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