136 Daryanto Med J Univ Indon

Tracing the Neural Connections between the Subfornical Organ and the Supraoptic and Paraventricular Nuclei lJsing Neuroanatomical-tract tracing and Immunoperoxidase Methods in Albino Rats

Daryanto

Abstrak

Penelitian ini bertujuan untuk nwnastikan hubungan anatomik sarafi antata organutn subfornicale dengan supraopticus dan nucleus paraventricularis hypothalani, dengan nenggwtakan metode pelacakan neuroanatonti yang nrenggunakan 4',6-diamidino- 2- phenylittdol dihydrochloride (DAPI) dan salah satu metode inunoperoksidase yang n,enggunaknn kit "l-abeled StreptAvidin- Biotin" (LSAB) dan antibodi kelinci terhadap neurofsin manusia sebagai antibodi priner. Pada penelitian ini digunakan 7 ekor tikus putih dewasa galur Wistar untuk pelacakan neuroanato,ni dan 15 eknr tikus putih dewasa galur Lenbaga Maknnan Rakyat, Jakarta (LMR) untuk pengkajian inunoperoksidase. Hasil penelitian menunjukkan bahwa secara anatonûk organum subfornicale dihubungknn dengan nucleus supraopticus dan nucleus parav entricularis hypothalani.

Abstract

The obiective of this investigation is to ascertain the anatomical connections between the subfornical organ and the supraoptic and paraventricular nuclei of the hypothalanus by utilizing a neuroanatomical-tract tracing nrcthod using a single tracer 4', 6-diatttidino-2-phenylindol dihydrochloride (DAPI) and one of the inmrunoperoxidase nrcthods, using l-abeled StreptAvidin- Biorin (LSAB) kit and Rabbit anti Hunnn neurophysin as a prinnry antibody. The neuroanatonical-tract tracing study used 7 adult albino rat of the Letnbaga Makanan Ralcyat, Jakarta (LMR) strain. The results revealed that the subfornical otgan was anatonûcally connected v,ith the supraoptic and paraventricular nuclei of the hypothalanus.

Keywords : Subfornical organ, supraoptic nucleus, paraventricular nucleus, neural connections, neuroanatonical, tract tracirlg nrcthod, itnnunoperoxidase method.

INTRODUCTION The subfornical organ is embryologically derived from the telencephalic ependyma point The subfornical organ is one of the circumventricular at the where the tela choroidea and lamina organs (Hofer, 1958, cit. Weindl, I973)r located in the terminalis -"rg".2 The subfornical organ roof of the third cerebral ventricle and attached to the has attracted considerable attention because of its unusual characteristics and its ventral surface of the hippocampal commissure2'3 1""" Figure 1 and 2). location within the cerebral ventricle space. It is a small possessing The subfornical organ has been a curiosity mostly structure distinct morphological features to embryologists and comparative anatomists. The that set apart from other neuronal nuclear systems,3 subfornical organ-has been identified in at least 131 al cells and a dense plexus of vertebrate species.r Its topography is consistent among (Figure 2). animals. It is a midline tubercle at the dorsal aspect of is highly vascular,3 and lacks the third ventricle near the interventricular foramina of Monro. It is only about 1 mm2 in total tissue area. Miselis and colleagues has identified a substantial and appears to be uniform in size among ,p""i"r3'4 system of efferent_f-ibers emanating from the SFO.6 (Figure 2). Recent anatomical?'8 and physiological studiese indi-

Departnent of Histology, Faculry of Medicine Gadjah Mada University, Yogyakarta, Indonasia Vol 3, No 3, July - Septenber 1994 Connections between Subfornical Organ and Supraoptic and Paraventricular Nuclei r37

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I

Figure 1. Median sagittal view of the rat brain, showing the location of the subfornical organ a,,n,'tg 'the circunventricular or- gans, AP, area postretna; ME, nedian enûnence; NL, neural lobe; OVLT, organu,n vasculosuttr of the lamina terninalis; PIN, pineal organ; Rec. recessus collicularis ofthe aqueduct; SFO,_subfornical organ; SCO, subcontnissural organ; I, cerebral third ventricle; II, choroid plexuses (from Weindl & Sofroniew, 197q.2Ô

Figure 2. Median sagittal view of the rat brain, showing the structure of the subfornical organ, stained by henrutoxylin-eosin (HE) staining technique. I, dorsal coluntn; II, the bod1, ofthe SFo; III, ventral column; IV, cerebral third ventricle; V. choroid plexuses oT the cerebral third ventricle. 138 Daryanto Med J Univ Indon

cated that the SFO is in fact a nucleus with that tocin and neurophysin II in the case of . The appears to innervate directly other neurons in the para- were originally thought to be binding ventricular and supraoptic nuclei that release vasopres- polypeptides, but it now appears that they are simply sin from the , as well as neurons in parts of the precursor molecules. The precursor for the meCian preoptic nucleus that project to those vasopressin is prepropresopressin and the precursor nuclei.E for is prepro-oxyphysin.l6 The SFO is known to participate in drinking in- The lFO is implicated in angiotensin Il-induced duced by angiotensin-Illo'lf and the control of vaso- drinking,20 a centraliy mediated piessor reslnnse, and pressin release from the lnsterior pituitary.12 the release of vasopressin from the posterior pituita- The neurons in the subfornical organ have ef- rj1.t2'tt Recent ,"port. have described efferent projec- ferent projections to both the paraventricular and nucleus arising from neurons in supraoptic nuclgi of the (electrophysio- li:ïi'o:fStupraoptic logical studies)7 suggesting the importance of these The aim of this investigation was to ascertain the pathways for controlling body fluid balance and anatomically nerve connections between the subforni- central cardiovascular regulation. cal organ and the supraoptic and paraventricular nuclei of the hypothalamus, by utilizing neuroanatomical- tract tracing and immunoperoxidase method.

MATERIALS AND METIIODS ence of afferents and efferents to and from the subfor- Neuroanatomical-tract tracing method nical organ to both the supraoptic (SON) and para- Seven adult albino rats of the Wistar strain weighing ventricular (PVN) nuclei of the hypothalamus, a region between 25O-35O g, were used in this experiment, known to contain the cell bodies of oxytocin and Each animal was anesthesized intraperitoneally using vasopressin-secreting n.uronrl4'16 and relulate the 10% chloralhydrate and these rats were placed in the circulating levels of the . Electrical stimula- stereotaxic frame (Figure 3). One small hole in the tion to the region of the SFO influences the excitability skull was made into which a glass of the magnocellular neurosecretory neurons and the fine micropipette was placed, filled with a small amount of 4'-6-diami- n both of SON and dino-2-phenylindol dihydrochloride (DAPI) (Figure by electrical stimu- 3,1). The location of the hole were 1,40 mm caudal to Ërponr"t8 and the the bregma and 1,0 mm lateral to the linea sagittalis, ntrationl2 observed and the glass micropipette was lowered the sub- following intravenous administration of angiotension- into il. fornical organ (Figure 4). The depth was 4,31 mm and at an angle of 12,5o to the sterotaxic vertical plane. The majority of neurons in the mammalian hypo- The location of the hole and the depth of the penetration thalamic supraoptic and paraventricular nuclei are of the glass micropipette was classic magnocellular neurosecretoary cells synthesiz- decided empirically, based on the animal's ing either vasopressin or oxytocin, and releasing these weight and also from the rats brain atlas p their terminals in the neurohypo- by Paxinos & Watson.23 The bregma was taken p onse to appropriate stimule.lT'le îïis as the point ofreferrence to established a fixed relation p lled a neurosecretion.lo'le From an between the measurement and geometry of the head anatomical viewpoint, these neurosecretory neurons in where the hole is to be drilled. that receive a diversity of synaptic contracts, a third of The injection volume ranges from 0,03 - 0,04 ml which at least arise from extranuclear sources. of 2,5% DAPI. The solution was delivered into the Oxytocin and vasopressin are typical neuronal SFO via a glass micropipette (Figure 3,1) connected to hormones, i.e. hormones secreted into the circulation a Hamilton's microsyringe (Figure 3,3) over a period by nerve cells. The term neurosecretion was originally of 1 - 5 minutes. Each animal was allowed to survive coined to, describe the secretion of hormones by for 48 - 72 hours and individually housed in a cage l6 t"oaotr, covered by a paper box and given food andwaler ad Like other hormones, both vasopressin lib inthe dark room, and sacrified by intraventricular and oxytocin granules have a characteristic neuro- perfusion of lOTo formalin in phosphate buffer (Mil- physin associated with the granules in the neruons lonig), under 10% chloralhydrate deep anaesthesia; which secrete them, neurophysin I in the case of oxy- afterwards the whole brain was removed. Nuclei 139 Vol 3, No 3, July - September 1994 Connections bebreen Subfornical Organ and Supraoptic and Paraventricular

Figurej.Afigureshowingaposirionoftheratinthestereotaxicapparatus.I,aglassnticropipettefilledbyasingleJluorescent nicrosyrmge' tràcer DAP1;-2, polyethylene tube (PE-20); 3, Hamilton's

cî the location of the subfornical organ ailnnç the other nuclei a Hatnilton's ntycrosyrine (Figure 3, 3) at the stereotaxic behind the bregma, and 1,0 nm lateral to the sulcus sagittalis the stereotaric vertical plane. CC, corpus callosuttr; HC' hyp- angular septal nucleus; MS, nredial septal nucleus; AC' anterio-r cular nucleus: OT, optic tract; OVLT, organu"t vasculosun ofthe posterior pituitary; AP, anlerior pituitary (fron Iantina ternrinalis; SO, hypothalanric supraoptic nucleus; OC, optic chiasn; PP, Sgro et aI.) Arrow indicates the exact location of the organ' 140 Daryanto MedJ Univ Indon

The brain was further immersed in lO% formalin Figure 5 is a frontal section of the brain, showing (pH 7,3) for a while and then cut serially in frontal the site of DAPI injection, and the SFO filled with the slices at 20 um thickness in a cryostat. The specimen injected DAPI (anow). was air-dried in a dark box and covered with non- Figure 6 is a frontal section of the rat brain at the fluorescent glycerine. Two out of every five sections stereotaxic coordinates of IA (interaural) 7,60 mm and were mounted on clean glycerine coated glass slides bregma - 1,40 mm and IA 7,20 mmand bregma - 1,80 and the exact site of injections as well as the extent of mm showing the sites of the DAPI labeled parent cell diffusion within each structure were examined. bodies in the supraoptic nucleus (a) and paraventri- The sections were microscopically examined cular nucleus (b). The color ofthe labeled parent cell under the fluorescent microscope (Nikon optiphot-EF) body is a clear white upon a blue colored background. The results revealed that the parent nerve bodies with a filter system of U + 40K. Thereafter the exact cell i4 both paraventricular and supraoptic nuclei of the location of the labeled cell was depicted in the enlarged hypothalamus are clearly labeled. This figure also map (18 X) of the brain section (camera lucida draw- depicts the topography of the neurons in the supraoptic ings). and paraventricular nuclei of hypothalamus. This investigation by utilizing neuroanatomical- The results of this investigation demonstrated that tract tracing method was done at the Department of the single fluorescent tracer (DAPI) has labeled the Anatomy, Kobe University School of Medicine, Kobe, parent cell bodies of the neurons in the paraventricular Japan. and supraoptic nuclei. Figure '7. A camera lucida drawing the frontal Immunoperoxidase method section of the rat brain, at the stereotaxic coordinate of: (a) IA (interaural) 7,60 mm and bregma - 1,40 mm and This investigation used one of the immunohistochemi- lA 7,2O mm and bregma - 1,80 mm to depicts the cal method, i.e. the immunoperoxidase method, and topography and the sites of the DAPI labeled parent was based on the assumption of the production and cell bodies in the supraoptic nucleus, paraventricular secretion of neurosecretoric substances. The immuno- nucleus, the caudate-putamen nuclues and in the dorsal peroxidase method, was based on the reaction of an- hippocampal nucleus. tigen-antibody complexes. This investigation used 15 adult, male and female albino primary rats, antibody Immunoperoxidase study ( Rabbit anti-human N eur ophysitt), and Labe le d Strept- Avidin-Biotin (LSAB) Kits, and were done at the The result of this investigation by using one of the Department of Anatomical Pathology, Faculty of immunoperoxidase methods using the LSAB kits re- Medicine, University of Indonesia, Jakarta and the vealed that the subfornical organ gave a positive reac- Laboratory of Histology, Faculty of Medicine, Gadjah lion (brown reaction) and the location of the neuro- Mada University, Yogyakarta. secretoric substances which gave the positive reaction The staining procedure used in this investigation is extracellular (see Figure 8 and 9). On the other hand, is one of the many immuno-enzymatic staining me- figure I I and 12 ab (frontal sections of the rat brain at thods, i.e. the two-step indirect method uing labeled the stereotaxic coordiates between IA 7,60 mm and streptAvidin-Biotin kit on parafine embedded sec- bregma - 1,40 mm and IA 7,2O mm and bregma - 1,80 tions. mm) showed a positive reaction (brown reaction) in the These methods utilize the high affinity of avidin cell bodies of the magnocellular neurosecretory system or streptavidin for biotin. Avidin has four binding sites (intracellular) in the paraventricular and supraoptic for biotin. This method require a biotinylated antibody nuclei of the hypothalamus. The location of the brown as a link antibody.2a In tÀis investigaiion the Rabbit reaction suggested that the neurosecretoric materials were synthetized anti-Human-Neurophysin was used as a primary an- and secreted by the nerve cell bodies tibody. of the magnocellular neurosecretory system in those nuclei.

RESULTS DISCUSSION Neuroanatomical-tract tracing study In this investigation the neural connection between the Seven animals received injection of DAPI in the sub- subfornical organ and supraoptic and paraventricular fornical organ. nuclei of the hypothalamus were verified using one Vol 3, No 3, July - September 1994 Connections berween Subfornical Organ and Supraoptic and Paravenlricular Nuclei l4l

- 1,40, and IA 7,2O nnr Figure 5. A frontal section of the rat brain at the stereotclric coordinates berween IA 7,60 nn and bregna and bregma - 1,EO mnt, showing the exantples of the sites of DAPI injection in the subfornical org,an.

Figure 6. A frontal section of the rat broin at îhe stereotLxic coordinates of IA 7,60 mnt and bregtna - 1,40 nutt showing an exanrple ofthe sites ofDAPI labeled parent cell bodies in the paraventricular nucleus (a) and IÀ 7,20 mn and bregnn - l,8O mm showing an exanple of the sites of DAPI labeled parent cell bodies in the supraoptic nucleus (b). 142 Daryanto Med J Univ Indon

m.

- à1".,. -1:33:":: -b

Figure 7. A canera Lucida drawing of the frontal section of the rat brain, at the stereotaûc coordinates of: (a) IA (interaural) 7,60 nunandbregna- l,40nunshowingthesitesof the DAP(injectioninthesubfornical organ (1) and labeled parent cell bodies in the paraventricular nucleus (2); (b) IA 7,20 mm and bregna - l,8O mm showing the sires of the DAPI labeled parent cell bodies in the dorsal hippocatnpal nucleus (3), the supraoptic nucleus (4), the caudate-putanen (5).

Figure 8. Photonùcrograph of a nredian sagittal view of the rat brain, showing an example of the sites of positive reaction (brown reaction) to the lnbeled StreptAvidin-Biotin staining iltethod (LSAB) in the subfornical organ. The location ofthe brown reaction is extracellular. X 40. Vol 3, No 3, July - September 1994 Connections between Subfornical Organ andSupraoptic and paraventricular Nuclei 143

Figure 10. Photon icrograph of a ntedian sagittal view of the rat brain, showing a negative control of the subfornical organ, stained by the LSÀB staining method, but without adding a primary antibody. X 400. 144 Daryanto MedJ Univ Indon

Figure 11. Photonicrograph ofafrontal section ofthe rat brain at the stereotaxic coordinates berween IA 7,60 ntnr and bregnrc - 1,40 mm, and IA 7,20 mm andbregma - 1,80 nm showing an example of sites of positive reaction (brown reaction) to the lnbeled StreptAvidin-Biotin staining methods. The location of the brown reaction is in the neme cell bodies of the nngnocellulcr neurosecretory system (intracellular) in the supraoptic and paraventricular nuclei of the hypothalanws. The locaîion of the brown reaction suggested that the neurosecrerc were synthetized and prod.uced by these nerve cell bodies of the nngnocellular neurosecretory systen in those nuclei. X 40.

a b

Figure 12 ab. Photomicrograph of afrontal section of the rat brain at the stereotaxic coordinates between IA 7,60 ntnr and bregma 1,40 mm, and IA 7,20 mrn and bregma - I,8O mm, showing an example of sites of positive reaction (brown reaction) in the neme cell bodies of the magnocellular neurosecretory system (intracellularly) in the paraventricular (a) and supraoptic nuclei (b) of the hypothalamus. X 400. Vol 3, No 3, JuIy - septenber connections 1994 betvveen Subfornical organ andSupraoptic and paraventricular Nuclei 145'

of the neuroanatomical- tract tracing methods using a The origin of the neurosecretoric material which single fluorescent tracer 4',6- diamidino-2-phenylin- gave a positive result by giving a brown reaction, dol dihydrochloride (DAPI) (which was devàloped by turned out to be the neurosecretoric material found in Heimer and Robards)Æ and one of the immuno- the cytoplasma of the cell bodies of the magnocellular peroxidase methods, i.e. the Labeled StreptAvidin- neurosecretory system of,the supraoptic and para_ Biotin techniques (LSAB), which was developed by ventricular nuclei of the hypothalamus. The neuro- Naish et a/.2a secrete's existence in the cell bodies of those nuclei Injection of a single fluorescent tracer (DApI) in proved that this neurosecrete was synthetised and seven adult albino rats of the Wistar strain, de monstrat- produced by those nuclei. ed that DAPI had labeled the parent cell bodies of the Similar findings have been reported by other in- the le l6 in fourteen nuclei and areas in the brain, vestigators, i.e. Stutinsky and Gantng *iri"h including the supraoptic and paraventricular "tut"d nuclei of that the neurosecretoric material was synthetised and the hypothalamus. produced by the cell bodies of the magnocellular In this study, the projections of nerve fibers to the neurosecretory system in the supraoptic and para_ subfornical organ from those nuclei, especially the ventricular nuclei of the hypothalamus. This neuro- supraoptic and the paraventricular nuclei, were secretoric material was then transported to the neuro- verified by the retrogradely transported DAPI. These hypophysis via the hypothalamo-hypophysealis-tracr results that all labeled nerve fibers and cell bodies that through the neurosecretion process (see Figure l3). were traced from the SFO were afferents of the supra- There was some neurosecrete material in the SFO optic and paraventricular nuclei, according to the ob- which gave positive reaction to the immunoperoxidase servations that labeled can be followed to retro- staining methods, eventhough in small amounts. This gradely labeled neurons that may given rise to them. proved that the SFO sends efferent projections in small Similar findings have been reported by inves- amounts to those nuclei (see Figure 13). tigators using other techniques, for examplès, using This investigation using the LSAB kits, in fact, electrophysical methods, which stated thatlh"re *".é can be used as a tracer to trace the nerve fiber connec- some connections between the SFO and the suoraootic tions between the subfornical tic and paraventricular nuclei of the hypothalamus,14,15 andparaventricularnucleioft he and using the autoradiographic and tracer's antero- results were in concordance with the other result by grade transportation method which demonstrated the using the neuroanatomical-tract tracing method. connections between the SFO and the median preoptic, This investigation, concluded that supraoptic and paraventricular nucleus, Based upon an by utilizing neuroanatomical-tract tracing electrophysiological method and.r_lsing acetylcholine, method and immuno- l / peroxidase method (LSAB kits) the saralasin and atropin Saro et al. demonstrated that subfornical organ was proved to be anatomically there were some connections between the subfornical connected to the supra_ organ and the paraventrilar nucleus. optic and paraventricular nuclei of the hypothalamus. 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sFo

Figure 13. A schenatic sagittal drawing of the rat depict the locatiott of the subfornical organ (SFO) lying in the of,thg^third ventricle underthe hippocanpal conunissure (HC) and fornft (Ft); inaccordance with recent ta,t/'te neurosecretoric ,ilaterial is to be transported through a neurosecrelion process in the hypothalanro- hypophysealis- tact (HHT) (solid heary lines) from the supraoptic and paraventricular nuclei of the hl,pothala,nus to the poste- rior pituitary (PP), some nerve fibers also projected to the SFO from those nuclei (intennittent lines). This neme fibers transport asnallamountofneurosecretoricnnterialto the SFO and release it to the blood capillariesvia rcnùnal nerve ending (frotn Sgro et at.)t7

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