Tracing the neural connections between the subfornical organ and the supraoptic and paraventricular nuclei using neuroanatomical-tract tracing and immunoperoxidase methods in albino rats

Abstrak

Penelitian ini bertujuan untuk nwnastikan hubungan anatomik sarafi antata organutn subfornicale dengan nucleus 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.

136 Daryanto

Tracing

the Neural Connections

Supraoptic and Paraventricular

lJsing

Neuroanatomical-tract tracing

Daryanto

INTRODUCTION

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The subfornical organ is one of the circumventricular organs (Hofer, 1958, cit. Weindl, I973)r located in the roof of the third cerebral ventricle and attached to the ventral surface of the hippocampal commissure2'3 1""" Figure 1 and 2).

The subfornical organ has been a curiosity mostly

to

embryologists and comparative anatomists. The subfornical organ-has been identified

in

at least 131 vertebrate species.r Its topography is consistent among animals. It _is a midline tubercle at the dorsal aspect

of

the third ventricle near

the

interventricular foramina of Monro.

It

is only about 1 mm2 in total tissue area.

and appears

to

be uniform

in

size among ,p""i"r3'4 (Figure 2).

Med J Univ Indon

between the Subfornical Organ and

the

Nuclei

and

Immunoperoxidase Methods

in Albino Rats

The subfornical organ is embryologically derived from the telencephalic ependyma at the point where the tela choroidea and lamina terminalis

-"rg".2

The subfornical organ has attracted considerable attention because of its unusual characteristics and its location within the cerebral ventricle space. It is a small structure possessing distinct morphological features that set apart from other neuronal nuclear systems,3 al cells and a dense plexus

of

(Figure 2).

is highly vascular,3 and lacks

Miselis and colleagues has identified a substantial

system

of

efferent_f-ibers emanating from the SFO.6 Recent anatomical?'8 and physiological studiese

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Vol 3, No 3, July - Septenber

1994

Connections between Subfornical Organ and Supraoptic and Paraventricular Nuclei r37

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. 'f

[image:2.595.72.563.403.682.2]

138 Daryanto

cated that the SFO is in fact a nucleus with neurons that appears to innervate directly other neurons in the para-ventricular and supraoptic nuclei that release vasopres-sin from the posterior pituitary, as well as neurons in the meCian preoptic

nucleus

that

project

to

those nuclei.E

The SFO is known to participate in drinking in-duced by angiotensin-Illo'lf and the control of vaso-pressin release from the _lnsterior pituitary.12

The neurons

in

the subfornical organ have

ef-ferent

projections to both the paraventricular

and supraoptic nuclgi of the hypothalamus (electrophysio-logical studies)7 suggesting the importance

of these

pathways

for

controlling

body

fluid

balance and central cardiovascular regulation.

ence of afferents and efferents to and from the subfor-nical organ to both the supraoptic (SON) and para-ventricular (PVN) nuclei of the hypothalamus, a region known to contain

the cell

bodies

of oxytocin

and vasopressin-secreting n.uronrl4'16 and relulate the circulating levels of the hormones. Electrical stimula-tion to the region of the SFO influences the excitability of the magnocellular neurosecretory neurons and the

n both of SON and by electrical stimu-Ërponr"t8 and the ntrationl2 observed following intravenous administration of

angiotension-il.

The majority of neurons in the mammalian hypo-thalamic supraoptic

and

paraventricular nuclei are classic magnocellular neurosecretoary cells synthesiz-ing either vasopressin or oxytocin, and releasing these

p

their axon terminals in the

neurohypo-p

onse

to

appropriate stimule.lT'le

îïis

p

lled a

neurosecretion.lo'le

From

an anatomical viewpoint, these neurosecretory neurons in that receive a diversity of synaptic contracts, a third

of

which at least arise from extranuclear sources.

Oxytocin and vasopressin are typical neuronal hormones, i.e. hormones secreted into the circulation by nerve cells. The term neurosecretion was originally coined to,

describe

the

secretion

of

hormones by t"oaotr, l6

Like other peptide

hormones, both vasopressin and oxytocin granules have

a

characteristic neuro-physin associated

with

the granules

in

the neruons which secrete them, neurophysin I in the case of

oxy-Med J Univ Indon

tocin and neurophysin II in the case of vasopressin. The neurophysins were originally thought

to be binding

polypeptides, but it now appears that they are simply parts

of

the precursor molecules. The precursor for vasopressin is prepropresopressin and the precursor for oxytocin is prepro-oxyphysin.l6

The

lFO

is implicated in angiotensin Il-induced drinking,20 a centraliy mediated piessor reslnnse, and the release

of

vasopressin from the posterior pituita-rj1.t2'tt Recent ,"port. have described efferent

projec-li:ïi'o:fStupraoptic

nucleus arising from neurons in The aim of this investigation was to ascertain the anatomically nerve connections between the subforni-cal organ and the supraoptic and paraventricular nuclei

of

the hypothalamus,

by

utilizing

neuroanatomical-tract tracing and immunoperoxidase method.

MATERIALS

AND METIIODS

Neuroanatomical-tract tracing method

Seven adult albino rats

of

the Wistar strain weighing between 25O-35O g, were used

in

this experiment, Each animal was anesthesized intraperitoneally using 10% chloralhydrate and these rats were placed in the stereotaxic frame (Figure 3). One small hole

in the

skull was made into which a fine glass micropipette was placed, filled with a small amount of 4'-6-diami-dino-2-phenylindol dihydrochloride

(DAPI)

(Figure 3,1). The location of the hole were 1,40 mm caudal to the bregma and 1,0 mm lateral to the linea sagittalis, and the glass micropipette was lowered into the sub-fornical organ (Figure 4). The depth was 4,31 mm and at an angle of 12,5o to the sterotaxic vertical plane. The location of the hole and the depth of the penetration of the glass micropipette was decided empirically, based on the animal's weight and also from the rats brain atlas by Paxinos & Watson.23 The bregma was taken as the point ofreferrence to established a fixed relation between the measurement and geometry

of

the head where the hole is to be drilled.

Vol 3, No 3, July - September 1994 Connections bebreen Subfornical Organ and Supraoptic and Paraventricular Nuclei 139

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

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 Iantina ternrinalis; SO, hypothalanric supraoptic nucleus; OC, optic chiasn; PP, posterior pituitary; AP, anlerior pituitary (fron

140 Daryanto

The brain was further immersed in lO% formalin (pH 7,3)

for

a while and then cut serially

in

frontal slices at 20 um thickness in a cryostat. The specimen was air-dried

in

a dark box and covered

with

non-fluorescent glycerine. Two out

of

every five sections were mounted on clean glycerine coated glass slides and the exact site of injections as well as the extent

of

diffusion within each structure were examined. The

sections

were

microscopically examined under the fluorescent microscope (Nikon optiphot-EF) with a filter system of

U

+ 40K. Thereafter the exact location of the labeled cell was depicted in the enlarged map (18

X)

of the brain section (camera lucida draw-ings).

This investigation by utilizing neuroanatomical-tract tracing method was done at the Department

of

Anatomy, Kobe University School of Medicine, Kobe,

Japan.

Immunoperoxidase method

This investigation used one of the immunohistochemi-cal method, i.e. the immunoperoxidase method, and was based on the assumption

of

the production and secretion of neurosecretoric substances. The immuno-peroxidase method, was based on the reaction

of

an-tigen-antibody complexes. This investigation used 15 adult, male and female albino rats, primary antibody

( Rabbit anti-human N eur ophysitt), and Labe le d

Strept-Avidin-Biotin

(LSAB)

Kits,

and were done at the Department

of

Anatomical

Pathology,

Faculty of

Medicine, University

of

Indonesia, Jakarta and the Laboratory of Histology, Faculty of Medicine, Gadjah Mada University, Yogyakarta.

The staining procedure used in this investigation is one

of

the many immuno-enzymatic staining me-thods, i.e. the two-step indirect method

uing

labeled streptAvidin-Biotin

kit

on

parafine embedded

sec-tions.

These methods utilize the high affinity of avidin or streptavidin for biotin. Avidin has four binding sites for biotin. This method require a biotinylated antibody as a

link

antibody.2a

In

tÀis investigaiion the Rabbit anti-Human-Neurophysin was used as a primary an-tibody.

RESULTS

Neuroanatomical-tract tracing study

Seven animals received injection of DAPI in the sub-fornical organ.

MedJ Univ Indon

Figure 5 is a frontal section of the brain, showing the site of DAPI injection, and the SFO filled with the injected DAPI (anow).

Figure 6 is a frontal section of the rat brain at the stereotaxic coordinates of IA (interaural) 7,60 mm and bregma

-

1,40 mm and

IA

7,20 mmand bregma

-

1,80 mm showing the sites of the DAPI labeled parent cell bodies

in

the supraoptic nucleus (a) and paraventri-cular nucleus (b). The color

ofthe

labeled parent cell body is a clear white upon a blue colored background. The results revealed that the parent nerve cell bodies

i4

both paraventricular and supraoptic nuclei

of

the hypothalamus are clearly labeled. This figure also depicts the topography of the neurons in the supraoptic and paraventricular nuclei of hypothalamus.

The results of this investigation demonstrated that the single fluorescent tracer (DAPI) has labeled the parent cell bodies of the neurons in the paraventricular and supraoptic nuclei.

Figure '7.

A

_{camera lucida drawing the frontal} section of the rat brain, at the stereotaxic coordinate of: (a) IA (interaural) 7,60 mm and bregma - 1,40 mm and

lA

7,2O mm and bregma

- 1,80

mm

to

depicts the topography and the sites

of

the

DAPI

labeled parent cell bodies in the supraoptic nucleus, paraventricular nucleus, the caudate-putamen nuclues and in the dorsal hippocampal nucleus.

Immunoperoxidase study

The result

of

this investigation by using one

of

the immunoperoxidase methods using the LSAB kits re-vealed that the subfornical organ gave a positive reac-lion (brown reaction) and the location

of

the neuro-secretoric substances which gave the positive reaction

is extracellular (see Figure 8 and 9). On the other hand, figure I

I

and 12 ab (frontal sections of the rat brain at the stereotaxic coordiates between

IA

7,60 mm and bregma

-

1,40 mm and IA 7,2O mm and bregma

-

1,80

mm) showed a positive reaction (brown reaction) in the cell bodies of the magnocellular neurosecretory system (intracellular)

in

the paraventricular and supraoptic nuclei of the hypothalamus. The location of the brown reaction suggested that the neurosecretoric materials were synthetized and secreted by the nerve cell bodies

of

the magnocellular neurosecretory system

in

those nuclei.

DISCUSSION

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Vol 3, No 3, July - September

1994

Connections berween Subfornical Organ and Supraoptic and Paravenlricular

Nuclei l4l

Figure 5. A _frontal section of the rat brain at the stereotclric coordinates berween IA 7,60

nn

and bregna - 1,40, and IA 7,2O nnr 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

ofthe sites ofDAPI labeled parent cell bodies in the paraventricular nucleus (a) and IÀ

exanple of the sites of DAPI labeled parent cell bodies in the supraoptic nucleus (b).

142 Daryanto _{Med J Univ Indon}

<ll"r".

-1:î3 m.M.

-

à1".,.

-1:33:":: [image:7.595.102.536.97.393.2] [image:7.595.112.547.449.696.2]

-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).

[image:8.595.110.518.84.332.2]

Vol 3, No 3, July - September

1994

_{Connections between Subfornical Organ andSupraoptic and} paraventricular

Nuclei

₁₄₃

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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.

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

1994

connections betvveen Subfornical organ _{andSupraoptic and paraventricular Nuclei 145'}

of the _{neuroanatomical- tract tracing methods using a}

single _{fluorescent tracer 4',6-}

diamidino-2-phenylin-dol dihydrochloride (DAPI) (which was devàloped by

Heimer and Robards)Æ

and

one

of

the

immuno-peroxidase methods,

i.e.

the Labeled

StreptAvidin-Biotin techniques (LSAB), which was developed by Naish et a/.2a

Injection of a single fluorescent tracer (DApI) in

seven adult albino rats of the Wistar strain, de monstrat-ed that DAPI had labeled the parent cell bodies of the

neuron

in

the fourteen nuclei and areas

in

the brain,

including the supraoptic and paraventricular nuclei

of

the hypothalamus.

In this study, the projections of nerve fibers to the

subfornical organ

from

those nuclei, especially the

supraoptic

and

the

paraventricular

nuclei,

_were verified by the retrogradely transported DAPI. These results that all labeled nerve fibers and cell bodies that were traced from the SFO were afferents of the

supra-optic and paraventricular nuclei, according to the ob-servations that labeled axons can be followed to retro-gradely labeled neurons that may given rise to them.

Similar findings have been reported

by

inves-tigators using other techniques,

for

examplès, using electrophysical methods, which stated thatlh"re

*".é

some _{connections between the SFO and the suoraootic} and paraventricular nuclei

of

the hypothalamus,14,15 and using the autoradiographic

and

tracer's

antero-grade transportation method which demonstrated the

connections between the SFO and the median preoptic, supraoptic and paraventricular nucleus, Based upon an

electrophysiological method and.r_lsing acetylcholine,

saralasin and atropin Saro et al. l

/

demonstrated that there were some connections between the subfornical

organ and the paraventrilar nucleus.

The results of this investigation

by

utilizing the

immunoperoxidase method

on

15 rats

of

the LMR

strain, showed a positive repons by giving brown reac_

tioninthe subfornical organ. The location of the brown

reaction, however, is extracellular. So this result

re-vealed that

the

SFO contains neurosecretoric ma_

terials, which was positive to the immunoperoxidase

method. This method used Rabbit anti-human neuro_

physin as a primary antibody, and LSAB kit; the biotin linked

to

the primary antibody, produces a

biotiny-lated conjugate which, when added to the tissue sec-tion, localizes the sites of antigen within the section.2T

The neurophysin,

is

a precursor

of

vasopresin and

oxytocin

hormones.

Moreover,

the existences

of

neurosecretoric

material

extracellularly proves that

this neurosecretoric

material

was

synthetised and

produced by another nuclei _{in the brain. This results}

ensured that, in fact, no secretory

cell

were found in

the SFO.

The origin of the neurosecretoric material which gave

a

positive result

by

giving

a

brown reaction,

turned out to be the _{neurosecretoric material found in}

the cytoplasma of the cell bodies

of

the magnocellular

neurosecretory system

of,the

supraoptic and para_

ventricular

nuclei

of

the hypothalamus. The

neuro-secrete's existence

in

the cell bodies

of

those nuclei

proved that

this

neurosecrete was synthetised and

produced by those nuclei.

Similar findings have been reported by other

in-vestigators, i.e. Stutinsky le and Gantng l6

*iri"h

"tut"d

that the neurosecretoric material was synthetised and

produced

by

the

cell

bodies

of

the magnocellular

neurosecretory system

in

the supraoptic

and

para_

ventricular nuclei

of

the hypothalamus. This

neuro-secretoric material was then transported to the

neuro-hypophysis via the hypothalamo-hypophysealis-tracr through the neurosecretion process (see Figure l3).

There was some neurosecrete material in the SFO

which gave positive reaction to the immunoperoxidase

staining methods, eventhough in small amounts. This

proved that the SFO sends efferent projections in small

amounts to those nuclei (see Figure 13).

This investigation using the LSAB kits,

in

fact,

can be used as a tracer to trace the nerve fiber

connec-tions between the

subfornical

_tic

andparaventricularnucleioft

_he

results were

in

concordance

with

the other result by

using the neuroanatomical-tract tracing method.

This

_{investigation, concluded that}

by

_utilizing

neuroanatomical-tract _{tracing method and}

immuno-peroxidase method (LSAB kits) the subfornical organ

was proved to be anatomically connected to the supra_ optic and paraventricular nuclei of the hypothalamus.

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t46 Daryanto MedJ Univ Indon

Figure 13. A schenatic sagittal drawing of the rat diencephalon 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}

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