Exp. Brain Res. 22, 525--540 (1975) @ by Springer-Verlag 1975
Quantitative Studies on the Supraoptic Nucleus in the Rat. II. Afferent Fiber Connections L. Z~borszky, Cs. L6r~nth, G.B. Makara and M. Palkovits 1st Department of Anatomy, Semmelweis University Medical School and Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary) Received January 17, 1975 Summary. A quantitative electron microscopic study of synaptic terminal degeneration was performed in the supraoptic nucleus (NSO) after a variety of major transeetions or ablations, destroying or interrupting in different combinations the afferent pathways known from earlier and own light microscopic degeneration studies. Solutions of a set of equations, expressing the percentage degenerations in synaptic profiles after different combinations in which the several pathways are interrupted by the various interferences, enabled the authors to give the following percentage numbers for afferent synapses from different sources. 32.7% of supraoptic afferents originate from the brain stem pxobably representing the monoaminergic innervation of this nucleus. The medial basal hypothalamus (21.0%), amygdala (13.5%), septum (13.5%), hippocampus (8.5%) and olfactory tubercle and further rostral cortical region (17.0%) are the other main sites of origin of supraoptic nucleus afferents. There are no supraoptic afferents from the optic nerve, superior cervical ganglion or fimbria hippocampi. Key words: Supraoptic nucleus - - Quantitative electron microscopy - - Afferent fiber connections - - R a t Introduction Two thirds of the axon terminals in the supraoptic nucleus (NSO) appear to be of intranuclear origin (L6rs et al., 1975), hence one third of the synaptic boutons ought to arise from the afferent pathways of the NSO; according to the preceding quantitative study (L6rs et al., 1975) this concerns 9.96X 106 synapses per m m 3 of nuclear tissue and 169 per cell. The fiber connections of the rat NSO have been dealt with at the light microscope level in several reports (Krieg, 1932; Nauta, 1956, 1958; Guillery, 1957; Morest, 1961 ; SzcntAgothai et al., 1968; Ungerstedt, 1971). This paper aims at a quantitative electron microscope level investigation into the participation of various afferent pathways in the total number and the several types of extrinsic synapses of the NSO. I n order to achieve this objective one needs (1) to know what territories contribute affereuts to the NSO, and (2) what percentage of afferent axon terminals belong to afferents of various origins. The first question can be solved in the traditional manner by making discrete focal lesions into the nuclear regions or the fiber bundles t h a t are either known or can be suspected to contribute afferents to the NSO. The solution of the second question rests entirely on EM level quantitative
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studies, which are the only ones t h a t m a y yield data on the n u m b e r of degenerated a n d n o r m a l terminals i n a n y finite v o l u m e of neural tissue. Since discrete focal lesions are rarely if ever suited to destroy or i n t e r r u p t all r e l e v a n t elements a n d since the n u m b e r of terminals given b y the i n d i v i d u a l afferent paths are m u c h too small to yield reliable q u a n t i t a t i v e data, one has to l u m p together several of the afferent p a t h w a y s b y m a j o r surgical i n t e r r u p t i o n s t h a t yield significant n u m b e r s of degenerated terminals. B y cross correlating the n u m e r i c a l d a t a received from such m a j o r i n t e r r u p t i o n s it is t h e n possible to arrive at a p p r o x i m a t e conclusions on the c o n t r i b u t i o n of other b r a i n regions to the afferents of the NSO.
Methods Albino rats of both sexes were anaesthetized with either 3.5 mg/10O g pentobarbital or ether inhalation. Different lesions (Table 1 and Figs. 1--3) were made stereotaxically by passing 2 mA of current for 10 sec through a small monopolar nichrome electrode insulated with tephlon except for 0.2 mm of the tip. The stereotaxic coordinates were determined using the atlas of KSnig and Klippel (1963). Surgical interventions are summarized on Table 2 and Fig. 4. Deaffercntations were performed as described by tIalgsz and Pupp (1965). Parasagittal cuts were made according to the description of Makara et al. (1972). A posterior mesodiencephalic transection (Fig. 4D) was made by lowering a 1 mm fragment of a razor blade on one side of the sagittal sinus to the floor of the skull at the level of the interpcduncular nucleus (distance from bregma: 5.1 ram). The hypothalamus was removed by suction through a glass funnel according to Makara et al. (197i). For silver stains of degeneration the animals were sacrified 3--4 days after the operation by perfusion with 0.9% saline followed by 10% formaline. After suitable fixation period 20 #m thick sections were cut in frontal or sagittal planes. Distribution of degenerated fibres and endings were examined on sections impregnated according to the Fink-Heimer method (1967). For EM degeneration studies the animals were killed 2 days after operation. The rats were perfused with Karnovsky's solution. After the usual procedure the small tissue blocks conraining the NSO were embedded into Durcupan. Sections were cut on t~eichert ultrotome, stained with uranyl acetate and lead citrate and examined with a TESLA II. B S143 electron microscope. The remaining parts of the brain were sectioned for identification of the lesions. The number of cross-sections of normal and degenerated synaptic boutons were counted directly under the electron microscope, in 50 • 50 pm areas corresponding to the meshes of the grids. A total number of 6317 synaptic boutons were counted on a surface area of 0.27 mm 2. The number of degenerated boutons was expressed in percent of the total of synaptic profiles encountered.
Results
Light Microscopic Observations Alter Discrete Focal Lesions (Table 1) Lesions destroying the ol]actory tubercle (Fig. 1A) a n d p a r t l y the nucleus a c c u m b e n s a n d oral parts of the medial forebrain b u n d l e (MFB) resulted in a h e a v y degeneration in the M F B which could be traced into the I~SO. A damage of the s t r i o h y p o t h a l a m i c a n d lateral cortieohypothalamic tracts (Gurdjian, 1927) could n o t be excluded i n this case. Massive degeneration i n the M F B a n d i n the NSO could be observed after septal lesions (Fig. 1B), which included the medial a n d p a r t l y the lateral septal nuclei or after a lesion destroying the fimbrial a n d dorsal septal nuclei with some i n v o l v e m e n t of the fornix (Fig. 1C). Degenerated fibres could be traced t h r o u g h the precommissural fornix a n d the M F B into the NSO following superior hippocampal (Fig. 1E) or fornix superior (Fig. 1D) lesions b u t n o t after d e s t r u c t i o n of the fimbria hippocampi (Fig. IF).
o
1A 1B
1C
Olfactory . . . . . . . . . . . . . Septal . . . . . . . . . . . . . .
~ornix
1F
Fimbria hippocampi
2B 2C
2D
2E
2F
3A 3B
--
--
Midline t h a l a m i c . . . . . . . . . Stria t e r m i n a l i s . . . . . . . . . .
. . . . . . . . . . . .
Medial-basal h y p o t h . . . . . . . . .
. . . . . . .
Amygdala
Supraoptic decussatio
Ventral tegmental . . . . . . . . . S u b s t a n t i a grisea c e n t . . . . . . . .
Enucleation . . . . . . . . . . . .
Ganglionectomy . . . . . . . . . .
5.8 4.3
F H , CC, C F V FS, 1%, R E , Ms
.
3.8
F H , CC
.
3.5
H I , GD, CC
.
6.0
FS, CC
1.6
SG .
.
2.0
A T V , SG, C P .
4.2 5.0
~NA, VM, D M , F S , CC M, H I , CC
.
5.3
A M , IC, ST, CC
.
5.8
ST, F]-I, CC
CC
6.8
0.4
0.6
0.5 1.4
2.5
2.1
0.6 0.0
3.6
1.0 a n d 2.0
0.2
0.5
0.5
8.0
SM, SL, D, CC F P , SF, SD, FS, CC
Coordinates lateral b
1.7
frontal a
8.5
T U , M, T S T H , CC
Regions destroyed (see list of abbreviations)
5.0
7.6
8.5 8.0
8.0
4.2
4.0 4.0 a n d 6.0
4.2
3.2
3.0
4.2
5.0
7.0
vertical e
a rostral to t h e i n t e r a u r i c u l a r line in m m , b lateral to t h e m i d l i n e in r a m , c d i s t a n c e b e l o w t h e t o p of t h e skull in r a m .
2A
. . . . . . . . . .
Stria m e d u l l a r i s
1E
Hippoe~mpus . . . . . . . . . . .
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1D
. . . . . . . . . .
Fornix superior
. . . . . . . . . . . . . .
N u m b e r of figure a n d code in t h e t e x t
T y p e of t h e lesions
NO
No
Yes
Yes
Yes Yes
No
No
No Yes
No
Yes
Yes
Yes
Yes
Yes
Degeneration in t h e supraoptie nucleus
T a b l e 1. L e s i o n s for d e m o n s t r a t i n g n e r v e t e r m i n a l s in t h e s u p r a o p t i c n u c l e u s w i t h F i n k - H e i m e r silver d e g e n e r a t i o n m e t h o d
O1
~a o
O~
~0
t~
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Fig. ]. Drawings for localization of various electrical lesions. Frontal sections according to KSnig and Klippel (1963). Abbr. see in the text. (A) Lesion in the olfactory tubercle region, (B) Lesion in the septum, (C) Fornix lesion in the septum, (D) Lesion destroying fornix superior, (E) Twofold lesion in the hippocampus, (F) Fimbria hippocampi lesion
H e a v y ipsilateral and slight contralateral degenerations were caused by a hypothalamic lesion including the ventromedial and the arcuate nuclei as well as the supraoptic decussations (Fig. 2E and Fig. 6A, B). Interruption of the supraoptic decussation below the MFB (Fig. 2F) resulted in degeneration only in the ipsilateral supraoptic nucleus. Lesion in the midline thalamic region by penetration through the fornix superior, were followed by degeneration in the NSO (Fig. 2B). The same result was obtained after dorsomedial hypothalamic lesions. Lesions or damage in the stria medullaris (Fig. 2A) did not result in any degeneration in the supraoptic nucleus. No degeneration was demonstrable with the Fink-}Ieimer method following medial amygdaloid lesion (Fig. 21)) or after destruction of the stria terminalis (Fig. 2C). Degenerated fragments could be observed after lesions of either the ventral tegmental area (Fig. 3A) or the central meseneephalic gray matter (Fig. 3B), or after a transection at the diencephalon-mesencephaIon border (Figs. 4E and 6C).
Afferent Fibers of the Supraoptic Nucleus
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l t
s ~"
Fig. 2. Drawings for localization of various electrical lesions. Frontal sections according to K6nig and Klippel (1963). Abbr. see in the text. (A) Lesion destroying the stria medullaris, (B) Midline thalamic lesion, (C) Lesion destroying the stria terminalis, (D) Lesion in the nucleus amygdaloideus medialis, (E) Lesion in the medial basal hypothalamus, (F) Lesion destroying the supraoptic decussation
c-
Fig. 3. Diagrams of various electrolytic lesions. Frontal sections. Abbr. see in the text. (A) Lesion in the ventral tegmental area, (B) Lesion in the substantia grisea centralis
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A
B
C
Fig. 4. Drawings for localization of various major surgical interferences in rat dicncephalon. (A--D) parasagittal, (E--F) frontal sections. Abbr. see in the text. (A) Complete isolation of the medial basal hypothalamus, (B) Anterior hypothalamic deafferentation, (C) Sucking away the entire hypothalamus, (E) Parasagittal section through in the lateral hypothalamus, (F) Frontal section at the diencephalon-mesencephalonborder
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D
i
1 Fig. 4D--F
However, it is important to note that the lesion of the central gray matter resulted in degeneration in the NSO, only if the lesion encroached upon the neighbouring tegmental region, which contains the ascending noradrenergie fibres (Ungerstedt, 1971).
Fig. 5. Electron micrographs of the NSO. Bar scale: 0.5/~ .(A) After complete isolation of the medial basal hypothalamus, (B) septal lesion. Arrow points to synaptie region. Og oligodendroglial cell, D dendrite, db degenerated bouton
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Fig. 6. Terminal degeneration in the NSO following a lesion of the medial hypothalamus: (A and B) frontal section and diencephMic-meseneephMie transection, (C) sagittal section. Fink-Heimer silver degeneration method The Fink-Heimer degeneration findings were substantiated under the electron microscope, for virtually all types of focal lesions, although no a t t e m p t was made to quantitate degeneration findings as discrete focal lesions were not considered as suitable for real quantitative conclusions.
Electron Microscopic Quantitative Studies o] Terminal Degeneration A/ter Major Surgical Interruptions o[ the AGerent Systems o] the NSO Some degenerating terminals were found even after lesions that did not yield degeneration in the silver stain, such as medial amygdaloid (2D) loci. Lesions in the olfactory region (1A) resulted in moderate numbers of degenerating boutons in the NSO. Neither a complete medial basal hypothalamie isolation (4A) yielding 14% degenerated boutons of the total, nor paramedian sagittal sections (4E) separating the NSO from the medial hypothalamus and from the other side of the brain (resulting in 12.2~o degeneration) produced a degeneration in the NSO t h a t would approach 20% of the total population of axon terminals. Less than I 0 % of the total NSO terminals was found to be degenerated after a coronal plane transection at the dieneephalic-mesencephalic border (4F) destroying all possible ascending fibers from the brainstem. No changes were noticed in the NSO 2 days after eye enucleation or after cervical sympathectomy. The numerical degeneration findings are summarized in Table 2. I n order to understand this table it is important to keep in mind t h a t major surgical inter37 Exp.Brain Res. Vol. 22
4C 4D 4E 4F 1B 2D 1A
. . . . . .
4. S u c k i n g a w a y t h e entire h y p o t h a l a m u s
5. P o s t e r i o r h y p o t h a l a m i c d e a f f e r e n t a t i o n . . . . . .
6. P a r a s a g i t t a l t r a n s e c t i o n in t h e d i e n c e p h a l o n . . 7. D i e n c e p h a l i c - m e s e n c e p h a l i c t r a n s e c t i o n . . . . . .
8. SepVal lesion . . . . . . . . . . . . . . . . . 9. A m y g d a l a lesion . . . . . . . . . . . . . . . .
10. Olfactory lesion
(3)
85 44 22 12 21
453 397 560
3 101 12
156 609 573 606
-88
458 627
697
b) Degenerated
a) Normal
Synaptic boutons c o u n t e d in representative area
(4)
3.8
3.0
4.9
7.3
12.2
1.9 16.6 2.1
0.0 14.0
Normal and degenerated boutons ratio in ~
(5)
0
A
S, H I
B
H. B
HI S, H I , B, H :B (partly)
-H I , I-I, :B
l~egion d e s t r o y e d or s e p a r a t e d from the NSO (see list of abbreviations)
(6)
17.0
13.5
22.0
32.7
54.7
8.5 74.4 9.4
0.0 62.8
Degenerated b o u t o n s in percentage of degene r a t i o n in total NSOisolated r a t s ~
S = s e p t u m (3.0%), H I = hippocampus (1.9%), 0 = o l f a c t o r y t u b e r c l e (3.8%), H ~ h y p o t h a l a m u s (4.7%), B = b r a i n s t e m (7.3%), A = a m y g dala (3.0%). 22.30/0 of s y n a p t i c a l b o u t o n s were d e g e n e r a t e d following t o t a l N S O isolation ( L 6 r s et al., 1975) ; t h i s v a l u e c o r r e s p o n d s to 100~ .
. . . . . . . . . . . . . . . .
-4A 4B
1. I n t a c t control . . . . . . . . . . . . . . . . . 2. C o m p l e t e isolation of t h e m e d i a l b a s a l h y p o t h a l a m u s 3. A n t e r i o r h y p o t h a l a m i c d e a f t ~ r e n t a t i o n . . . . . .
(2) Code number in figures and Text
(1)
T y p e of t h e surgical interferences
Table 2. D e g e n e r a t e d n e r v e t e r m i n a l s following v a r i o u s t r a n s e c t i o n s a n d lesions of fibers t e r m i n a t i n g in t h e s u p r a o p t i c n u c l e u s
2-
9
b~
Afferent Fibers of the Supraoptic Nucleus
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ferences are labeled in column (2) by their code numbers refering mainly to the several drawings of Fig. 4 and to lesser extent to some drawings of Figs. 1 and 2. The same code numbers are used also in the text. The regions destroyed or separated from the NSO are listed in column (5) by using the abbreviations given in an appendix. One has to realize that such large cuts disconnect from the NSO often more than one of the brain regions contributing afferents. The percentage number of degenerated synapses is indicated in two different forms: in column (4) in percent of the total synapse population and in column (6) in percent of the poi~ulation of extrinsic terminals. Since only 22.3% of the synaptic boutons were found previously (L6rs et al., 1975) to be of extrinsic source, this w l ~ e would correspond to 100% in column (6). The percentage values of individual brain regions indicated below the lower horizontal line in Table 2 have been calculated by cross correlation of the data in column (4) and correspond therefore to percentages of the total synapse population. Discussion
By appl3dng the double strategy of smaller discrete and/or local lesions for qualitative, and major surgical interferences for quantitative evaluation, the proportions of afferents to the NSO from different parts of the limbic system (olfactory tubercle, septum, hippocampus, and amygdala) from the medial basal hypothalamus, and from the lower brainstem could be estimated with reasonable accuracy. The procedure by which one could arrive at the percentage values given in Table 2 is indirect in the ease of the medial basal hypothalamus and of the septum, because these regions could not be isolated or their connections with the NSO interrupted without major damage to other structures that contribute additional afferents. However, the percentage numbers of afferents given by these regions could still be established by cross correlation o f the quantitative data received from different lesions destroying the afferents in various combinations. Although the whole procedure might appear as rather unusual, it is easy to convince oneself that percentage data obtained by the interruption in different combinations of the major afferent systems yielded values that are self consistent. Considerable confidence for the procedure can be gained from the simple fact that if one adds the final percentage values received for contributions by the six separate regions mentioned above and indicated in Table 2 below the lower horizontal line, the sum is 23.7% which is almost exactly the value received for the actually observed degenerating synapse in the preceding study (L6rs et al., 1975) for the extrinsic afferents of the NSO. The strategy applied by the surgical transections can be understood better from Fig. 7, in which the several interferences are diagrammatieally indicated by dashed lines labelled with the appropriate code numbers corresponding to the drawings of Figs. 1, 2 and mainly 4. The coarse lines terminating in arrows represent the major known (labelled) and unknown (unlabelled) afferent paths of the NSO. The box symbolizing the hippocampus (CA1) has been superimposed on the larger box corresponding to the medial basal hypothalamus partly because the diagram tries to give a schematic view from above, but mainly because a quite extensive damage to the hippocampus is caused by the isolation of the hypothala37*
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/,A
Fig. 7. Composite diagram of neural pathways that terminate in the NSO. Broken lines represent various transections and lesions as defined in Figs. 1, 2 and 4 as well as in Table 2 m u s (4A). B y considering w h a t coarse lines, r e p r e s e n t i n g t h e p a t h w a y s , i n t e r s e c t with which d a s h e d lines s t a n d i n g for t h e surgical interferences i t is e a s y to d e d u c e from Fig. 7 t h e various t y p e s of afferents t h a t can be s u p p o s e d to undergo degeneration after t h e several cuts. On t h e basis of this figure also certain formalizations can be m a d e for t h e relations b e t w e e n t h e percentage occurrence of d e g e n e r a t e d t e r m i n a l s after various interferences. These relations are as follows: la) 4C-~4B(HI)-+4F(B)+S+H lb) 1B Z+4B(HI) S = 1B~4B S -~ 1 B ( S + H I ) - t - 4 B ( H I ) 4C ~ 4 B ~ - 4 F ~ - ( 1 B I B ) - [ - H S = 4 . 9 - - 1 . 9 ~ 3.0 H ~ 4C--4F--1B U -----1 6 . 6 - - 7 . 3 - - 4 . 9 ---- 4.4 2) 4A : 4 B ( H I ) + 4 F ( B ) ~ - H 3) 4E -~ 4 F ( B ) - ] - H H ~ 4A 4 B - - 4 F H ~ 4E---4F(B) H ~ 1 4 . 0 - - 1 . 9 - - 7 . 3 ---- 4.8 H ~ 12.2--7.3 ~ 4.9 As can be seen t h e observed ratios of d e g e n e r a t e d t e r m i n a l s are in v e r y good a g r e e m e n t w i t h t h e logical e x p e c t a t i o n s arising from t h e formalization. The M F B carries t h e m a j o r i t y of t h e N S O afferents (L~rs et al., 1972). Ascending M F B fibers originating from various regions of t h e b r a i n s t e m (Nauta, 1956; Guillery, 1957; N a u t a a n d K u y p e r s , 1958; Cowan et al., 1964; Z s et al., 1973) give rise to a considerable fraction of t h e t o t a l afferent i n p u t (7.3 [32.7]0//0, column 6, T a b l e 2) 1. Most of these afferents correspond p r o b a b l y t o m o n o a m i n e r g i c fibers (Ungerstedt, 1971 ; Sachs et al., 1973) a n d should r e p r e s e n t
Afferent Fibers of the Supraoptic Nucleus
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the morphological substrate of brain stem influence on the vasopressin release from the NSO (Mills and Wang, 1964; Woods et al., 1969). The descending MFB fibers reaching the NSO originate in the septum (3.0 [13.51%) and the olfactory tubercle (3.8 [17.01%). The former observation confirms the results of Johnson (1965) and Powell and l~orie (1967); and Tangapregassom et al. (1974). The high percentage of olfactory afferents is probably due to the fact that the lesions interrupt also fibers of more rostral (cortical: piriform cortex Gurd]ian, 1925, 1926; Nauta, 1956; Powell et al., 1965) origin, collected by the MFB. This study has revealed the existence of hippocampal supraoptic connexions. These fibers can be assumed to run through the fornix superior, because both hippocampal lesions (1E) or damage of fornix superior (1D) cause degeneration in the NSO, while large fimbrial lesions (IF) do not. Consequently only the sector CA1 may contribute to this pathway (l~aisman et al., 1966). In contrast with the present results no termination of hippocampal fibers in the NSO was mentioned by Nauta (1958) and Raisman et al. (1966). The contribution of this pathway to the NSO afferent terminals can be inferred directly from the results of a 4B type transection, this being the only pathway to NSO interrupted by such a lesion of the fornix superior (Zs et al., 1972). The degeneration in the NSO following the lesion of the medial amygdaloid area (2D) is moderate (3.0 [13.51%). Since the lesion and the electrode track cause additional damage to the ventral amygdalofugal pathway and the stria terminalis, this has also to be taken into consideration. The interruption of the stria terminalis (2C) does not yield any degeneration demonstrable either with the Fink-Heimer method or under the EM, which is in agreement with other studies (Helmet and Nauta, 1969; Leonard, 1971; De Olmos and Ingram, 1972). I t is reasonable, therefore, to assume that the amygdalo-supraoptic fibers are running in the ventral amygdalofugal path, as already suggested by Nauta (1961) for the monkey. However there is some doubt (Leonard and Scott, 1971) about the amygdaloid contribution to this pathway in the rat. Some fibers from the periamygdaloid cortex traversing the lesioned area should be taken also into consideration. Sucking away the entire hypothalamus from dorsal approach (4C) yields an abundant degeneration of synaptic terminals in the NSO (16.6 [74.4]%), corresponding well to the lesion of the septum and the hippocampal fibers by intraduction of the funnel and by removal of the medial basal hypothalamus and the MFB. Isolation of the medial basal hypothalamus (4A) destroys both the hypothalamie sources of NSO afferents and the MFB, and causes additional damage by introduction of the Hals knife to the fornix superior. Hence the quantitative result in degeneration (14.0 [62.8]%) should correspond to the result of 4C minus the septal fibers (i. e. 16.6--3 ~ 14). The paramedian sagittal cut (4E) along the lateral edge of the MFB interrups the same fiber systems as 4A with the only exception of the hippoeampal fibers (see Fig. 7). Thus the numerical result in terminal degeneration of the parasagittal section (12.2 [54.7]%) and the value (1.9 [8.5]%) found for hippocampal fibers (4B) corresponds well to the yield of 4A (12.2~1.9 ~ 14). 1 From the two numbers the first refers always to percentage of total synapse population and the second [in square brackets] to the percentage of extrinsic afferent synapses.
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A n isolated r e m o v a l of t h e h y p o t h a l a m u s w i t h o u t a d d i t i o n a l d a m a g e to o t h e r structures n o t being feasible, t h e c o n t r i b u t i o n of t h e medial basal h y p o t h a l a m u s to t h e afferents of t h e N S O can only be c a l c u l a t e d indirectly. B y using t h r e e different c o m b i n a t i o n s of d a m a g e , formalized in equations (la), (2) a n d (3) t h e calculated value was found to be 4.4%, 4.8%, a n d 4 . 9 % respectively. This fraction of afferents is quite considerable as i t c o n t r i b u t e s to 21~ i.e. a fifth of t h e t o t a l afferent s y n a p s e p o p u l a t i o n of t h e NSO. A l t h o u g h t h e findings give no evidence of t h e possible origin of these afferents w i t h i n t h e h y p o t h a l a m u s , it is reasonable to assume t h a t a t least p a r t of t h e m corresponds to t h e a r e u a t e nucleus - - periv e n t r i c u l a r d o p a m i n e r g i c cell groups (DahlstrSm a n d F u x e , 1964; B j 6 r k l u n d a n d Nobin, 1973). A p r o b a b l y m i n o r c o n t r i b u t i o n b y d a m a g e of t h e s u p r a o p t i e deeussiation (Minderhoud, 1967 ; Leontowich, I970) can n o t be e n t i r e l y excluded. On t h e whole a b o u t 5 0 % of t h e N S O afferents originate from t h e limbic s t r u c t u r e s (olfactory tubercle, s e p t u m , a m y g d a l a , h i p p o c a m p u s ) , which seems to agree well w i t h physiological findings on t h e role of t h e same s t r u c t u r e s in vasopressin release from NSO (I-Iayward a n d Smith, 1963; Powell a n d I~orie, 1967; W o o d s et al, 1969). The r e m a i n i n g half of t h e afferents consists of 3 0 % b r a i n s t e m afferents p r o b a b l y m a i n l y adrenergic a n d serotoninergic fibers a n d of 20% hypot h a l a m i c fibers p r o b a b l y largely of d o p a m i n e r g i c character. The m e n t a l s t r a t e g y u n d e r l y i n g this s t u d y as well as t h e m e t h o d i c a l a p p r o a c h are u n d o u b t e d l y unusual, a n d a d d i t i o n a l l y t h e extensive convergence u p o n t h e NSO m a y raise some d o u b t s against t h e results. I n d e e d t h e t r a d i t i o n a l views of t h e N S O as a n e u r o s e c r c t o r y nucleus h a v i n g its own osmoreceptors is n o t easily reconciled whit such a convergence of afferents from r a d i c a l l y different sources. However, several recent physiological studies m e n t i o n e d above, would p o i n t in t h e same direction. A AB AC AL AM ATV C CA CC CFV CO CP D DM DS F FtI FLM FP FS G GD HI IC
= = = = = = = = = = = = = = -= = -= = = = = =
List of Abbreviations nucleus accumbens nucleus amygdaloideus basalis nucleus amygdaloidcus centralis nucleus amygdaloideus lateralis nucleus amygdMoideus mcdialis area tegmenti ventralis (TsM) c~udate-putamen eommissura ant~erior corpus eallosum commissura fornicvis ventralis chiasma opticum commissura posterior nucleus traetus diagnolis nucleus dorsomedialis deeussationes supraoptica columna fornicis fimbri~ hippocampi fasciculus longitudinalis medialis fornix praeeommissuralis fornix superior globus pallidus gyrus dentatus hippocampus capsula interna
Afferent Fibers of the Supraoptic Nucleus
539
IP LM
= nucleus interpeduncularis = lemniscus medialis M = medial forebrain bundle (MFB) MM nucleus medialis thalami, pars medialis NA nucleus arcuatus R nucleus rhomboideus RE = nucleus reuniens I~V = nucleus ruber S = stria medullaris thalami SD = nucleus dorsalis septi SF = nucleus fimbrialis septi SG = substantia grisea centrslis SL = nucleus lateralis septi SM = nucleus medislis septi SN = substantia nigra ST = nucleus interstitialis striae terminalis T = traetus olfaetorius lateralis TD = tractus diagonalis (Broea) TO traetus opticus TSTH = tractus striohypothalamicus TU tuberculum olfaetorium nucleus ventromedislis V~
Acl~nowledgements. The suthors are indebted to Prof. J. Szentdgothai for valuable advice, critical comments a n d suggestions. References
Bj5rklund, A., Nobin, A. : Fluorescence histochemicM and microsp3ctrofluorometrie mapping of dopamine and noradrenaline cell groups in the rat diencephalon. Brain P~es. 51,193--205 (1973) Cowan, W. ~ . , Guillery, P~. W., Powell, T. P. S. : The origin of the mamillary peduncle s n d other hypothMamic connexions from the midbrain. J. Anat. (Lond.) 98, 345--363 (1964) DahlstrSm, A., Fuxe, K. : Existence of monoamine containing neurons in the cell bodies of brain stem neurons. Acts physiol, stand. 232, Suppl. 62, 1--53 (1964) De OImos, J.S., Ingram, W.I~.: The projection field of the stria terminMis in the rat brain. An experimental study. J. comp. Neurol. 146, 303--334 (1972) Fink, 1~. P., Heimer, L. : Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4, 369--375 (1967) Guillery, R.W. : Degeneration in the hypothalamic connexions of tile albino rat. J. Anat. (Lond.) 91, 91--115 (1957) Gurdjian, E.S. : Olfactory connections in the albino rat, with special reference to the stris medullaris and the anterior commissure. J. comp. Neurol. 38, 127--163 (1925) Gnrdjian, E.S.: The diencephalon of the albino rat. J. comp. Neurol. 43, 1--114 (1927) ttalgsz, B., Pupp, L. : Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotropic are. Endocrinology 77, 553--563 (1965) Hayward, J.N., Smith, W . K . : Influence of limbic system on neurohypophysis. Arch. Neurol. (Chic.) 9, 171--177 (1963) I~Ieimer, L., Nauta, W . J . I-I. : The hypothalamic distribution of the stria terminalis in the rat. Brain Res. 18, 284--297 (1969) Johnson, T.N. : An experimental study of the fornix and hypothalamo-tegmental tracls in the cst. J. comp. Neurol. 125, 29--40 (1965) KSnig, J. F. 1~., Klippel, R. A. : The R a t Brain : A Stereotsxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams and Wilkins 1963 Krieg, J . : The hypothalamus of the albino rst. J. eomp. Neurol. 55, 19--89 (1932) Leonard, C.M., Scott, J. W. : Origin and distribution of the amygdalofngal pathways in the rat : a n experimental-neuroanatomieal study. J. comp. Neurol. 141, 313--330 (1971)
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Leontovieh, T.A.: The neurons of the magnocellular neurosecretory nuclei of the dog's hypothalamus. J. Hirnforsch. 11,499--517 (1970) L6rs Cs., Z~borszky, L., Makara, G.B., Palkovits, M. : Degeneration yon Synapsen im Nucleus supraopticus nach Unterbrechung der afferenten Systeme des Hypothalamus. Ergebn. Anat. Anz. 180, 587--593 (1972) L6rs Cs., Zs L., Marton, J., Palkovits, M.: Studies on the supraoptie nuclei in the rat. [. Synapses. Exp. Brain Res. 22, 509--523 (1975) Makara, G.B., Stark, E., M6sz~ros, T. : Corticotrophin release induced by E. coli endotoxin after removal of the medial hypothalamus. Endocrinology 88, 412--414 (1971) Makara, G.B., Stark, E., Marton, J., M6szs T. : Corticotrophin release induced by surgical trauma after transection of various afferent nervous pathways to the hypothalamus. J. Endocr. 53, 389--395 (1972) Mills, E., Wang, S.C. : Liberation of antidiuretic hormone: Location of ascending pathways. Amer. J. Physiol. 207, 1399--1404 (1964) Minderhoud, J.M. : Observations on the supra-optic decussations in the albino rat. J. comp. NeuroI. 129, 297--312 (1967) Morest, D.K. : Connexions of the dorsal tegmental nucleus in rat and rabbit. J. Anat. (Loud.) 95, 229--246 (1961) Nauta, W. g. H. : An experimental study of the fornix in the rat. J. comp. Neurol. 104,247--270 (1956) Nauta, W. J. H. : Hippocampal projections and related neural pathways to the midbrain in the rat. Brain 81,319--341 (1958) Nauta, W.J.H.: Fibre degeneration following lesions of the amygdaloid complex in the monkey. J. Anat. (Loud.) 95, 515--531 (1961) Nauta, W.J.H., Kuypers, H.G.J.M. : Some ascending pathways in the brain stem reticular formation. In: Reticular Formation of the Brain, pp. 3--30. Ed. by Jasper, H.H. et al., Boston: Little, Brown and Company 1958 Powell, T.P.S., Cowan, W.M., Raisman, G.: The central olfactory connections. J. Anat. (Lond.) 99, 791--813 (1965) Powell, E.W., Rorie, D.K. : Septal projections to nuclei functioning in oxytocin release. Amer. J. Anat. 120, 605--610 (1967) Raisman, G. : The connections of the septum. Brain 89, 317--348 (1966) Raisman, G., Cowan, W.M., Powell, T.P.S. : An experimental analysis of the efferent projection of the hippocampus. Brain 89, 83--108 (1966) Sachs, C., Jonsson, G., Fuxe, K. : Mapping of central noradrenaline pathways with 6-hydroxyDOPA. Brain Res. 63, 249--261 (1973) Szent~gothai, J., FlerkS, B., Mess, B., Hals B.: Hypothalamic Control of the Anterior Pituitary, p. 399. Budapest: Akad6miai Kind5 1968 Tangapregassom, A.M., Tangapregassom, M.I., Soulairac, A., Soulairac, M.L.: Effects des 16sions septales sur l'ultrastructure du noyau supra-optique rat. Ann. Endocr. (Paris) 35, ]49--152 (1974) Ungerstedt, U. : Stereotaxic mapping of monoamine pathways in the rat brain. Acta physiol. scand. 867, Suppl. 1--48 (1971) Woods, W.H., Holland, R. C., Powell, E.W. : Connections of cerebral structures functioning in neurohypophysial hormone release. Brain Res. 12, 26--46 (1969) Zs L., L4rs Cs., Palkovits, M.: Faserdegeneration im Hypothalamus und im limbischen System nach konventionellen (dorsomedialen) Penetrationen. Ergebn. Anat. Ariz. la9, 595--600 (1972) Zs L., L6rs Cs., Marton, J., Palkovits, M. : Afferent brainstem pathways to hypothalamus and to limbic system in the rat. In: Hormones and Brain Function, pp. 449~457. Ed. by K. Liss~k. Budapest: Akad6miai Kind5 1973 Dr. L. Z~borszky 1st :Department of Anatomy Semmelweis University Medical School H - 1450 Budapest IX. Tiizolt5 u. 58 Hungary
Quantitative Studies on the Supraoptic Nucleus in the Rat. II. Afferent Fiber Connections L. Z~borszky, Cs. L6r~nth, G.B. Makara and M. Palkovits 1st Department of Anatomy, Semmelweis University Medical School and Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary) Received January 17, 1975 Summary. A quantitative electron microscopic study of synaptic terminal degeneration was performed in the supraoptic nucleus (NSO) after a variety of major transeetions or ablations, destroying or interrupting in different combinations the afferent pathways known from earlier and own light microscopic degeneration studies. Solutions of a set of equations, expressing the percentage degenerations in synaptic profiles after different combinations in which the several pathways are interrupted by the various interferences, enabled the authors to give the following percentage numbers for afferent synapses from different sources. 32.7% of supraoptic afferents originate from the brain stem pxobably representing the monoaminergic innervation of this nucleus. The medial basal hypothalamus (21.0%), amygdala (13.5%), septum (13.5%), hippocampus (8.5%) and olfactory tubercle and further rostral cortical region (17.0%) are the other main sites of origin of supraoptic nucleus afferents. There are no supraoptic afferents from the optic nerve, superior cervical ganglion or fimbria hippocampi. Key words: Supraoptic nucleus - - Quantitative electron microscopy - - Afferent fiber connections - - R a t Introduction Two thirds of the axon terminals in the supraoptic nucleus (NSO) appear to be of intranuclear origin (L6rs et al., 1975), hence one third of the synaptic boutons ought to arise from the afferent pathways of the NSO; according to the preceding quantitative study (L6rs et al., 1975) this concerns 9.96X 106 synapses per m m 3 of nuclear tissue and 169 per cell. The fiber connections of the rat NSO have been dealt with at the light microscope level in several reports (Krieg, 1932; Nauta, 1956, 1958; Guillery, 1957; Morest, 1961 ; SzcntAgothai et al., 1968; Ungerstedt, 1971). This paper aims at a quantitative electron microscope level investigation into the participation of various afferent pathways in the total number and the several types of extrinsic synapses of the NSO. I n order to achieve this objective one needs (1) to know what territories contribute affereuts to the NSO, and (2) what percentage of afferent axon terminals belong to afferents of various origins. The first question can be solved in the traditional manner by making discrete focal lesions into the nuclear regions or the fiber bundles t h a t are either known or can be suspected to contribute afferents to the NSO. The solution of the second question rests entirely on EM level quantitative
526
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studies, which are the only ones t h a t m a y yield data on the n u m b e r of degenerated a n d n o r m a l terminals i n a n y finite v o l u m e of neural tissue. Since discrete focal lesions are rarely if ever suited to destroy or i n t e r r u p t all r e l e v a n t elements a n d since the n u m b e r of terminals given b y the i n d i v i d u a l afferent paths are m u c h too small to yield reliable q u a n t i t a t i v e data, one has to l u m p together several of the afferent p a t h w a y s b y m a j o r surgical i n t e r r u p t i o n s t h a t yield significant n u m b e r s of degenerated terminals. B y cross correlating the n u m e r i c a l d a t a received from such m a j o r i n t e r r u p t i o n s it is t h e n possible to arrive at a p p r o x i m a t e conclusions on the c o n t r i b u t i o n of other b r a i n regions to the afferents of the NSO.
Methods Albino rats of both sexes were anaesthetized with either 3.5 mg/10O g pentobarbital or ether inhalation. Different lesions (Table 1 and Figs. 1--3) were made stereotaxically by passing 2 mA of current for 10 sec through a small monopolar nichrome electrode insulated with tephlon except for 0.2 mm of the tip. The stereotaxic coordinates were determined using the atlas of KSnig and Klippel (1963). Surgical interventions are summarized on Table 2 and Fig. 4. Deaffercntations were performed as described by tIalgsz and Pupp (1965). Parasagittal cuts were made according to the description of Makara et al. (1972). A posterior mesodiencephalic transection (Fig. 4D) was made by lowering a 1 mm fragment of a razor blade on one side of the sagittal sinus to the floor of the skull at the level of the interpcduncular nucleus (distance from bregma: 5.1 ram). The hypothalamus was removed by suction through a glass funnel according to Makara et al. (197i). For silver stains of degeneration the animals were sacrified 3--4 days after the operation by perfusion with 0.9% saline followed by 10% formaline. After suitable fixation period 20 #m thick sections were cut in frontal or sagittal planes. Distribution of degenerated fibres and endings were examined on sections impregnated according to the Fink-Heimer method (1967). For EM degeneration studies the animals were killed 2 days after operation. The rats were perfused with Karnovsky's solution. After the usual procedure the small tissue blocks conraining the NSO were embedded into Durcupan. Sections were cut on t~eichert ultrotome, stained with uranyl acetate and lead citrate and examined with a TESLA II. B S143 electron microscope. The remaining parts of the brain were sectioned for identification of the lesions. The number of cross-sections of normal and degenerated synaptic boutons were counted directly under the electron microscope, in 50 • 50 pm areas corresponding to the meshes of the grids. A total number of 6317 synaptic boutons were counted on a surface area of 0.27 mm 2. The number of degenerated boutons was expressed in percent of the total of synaptic profiles encountered.
Results
Light Microscopic Observations Alter Discrete Focal Lesions (Table 1) Lesions destroying the ol]actory tubercle (Fig. 1A) a n d p a r t l y the nucleus a c c u m b e n s a n d oral parts of the medial forebrain b u n d l e (MFB) resulted in a h e a v y degeneration in the M F B which could be traced into the I~SO. A damage of the s t r i o h y p o t h a l a m i c a n d lateral cortieohypothalamic tracts (Gurdjian, 1927) could n o t be excluded i n this case. Massive degeneration i n the M F B a n d i n the NSO could be observed after septal lesions (Fig. 1B), which included the medial a n d p a r t l y the lateral septal nuclei or after a lesion destroying the fimbrial a n d dorsal septal nuclei with some i n v o l v e m e n t of the fornix (Fig. 1C). Degenerated fibres could be traced t h r o u g h the precommissural fornix a n d the M F B into the NSO following superior hippocampal (Fig. 1E) or fornix superior (Fig. 1D) lesions b u t n o t after d e s t r u c t i o n of the fimbria hippocampi (Fig. IF).
o
1A 1B
1C
Olfactory . . . . . . . . . . . . . Septal . . . . . . . . . . . . . .
~ornix
1F
Fimbria hippocampi
2B 2C
2D
2E
2F
3A 3B
--
--
Midline t h a l a m i c . . . . . . . . . Stria t e r m i n a l i s . . . . . . . . . .
. . . . . . . . . . . .
Medial-basal h y p o t h . . . . . . . . .
. . . . . . .
Amygdala
Supraoptic decussatio
Ventral tegmental . . . . . . . . . S u b s t a n t i a grisea c e n t . . . . . . . .
Enucleation . . . . . . . . . . . .
Ganglionectomy . . . . . . . . . .
5.8 4.3
F H , CC, C F V FS, 1%, R E , Ms
.
3.8
F H , CC
.
3.5
H I , GD, CC
.
6.0
FS, CC
1.6
SG .
.
2.0
A T V , SG, C P .
4.2 5.0
~NA, VM, D M , F S , CC M, H I , CC
.
5.3
A M , IC, ST, CC
.
5.8
ST, F]-I, CC
CC
6.8
0.4
0.6
0.5 1.4
2.5
2.1
0.6 0.0
3.6
1.0 a n d 2.0
0.2
0.5
0.5
8.0
SM, SL, D, CC F P , SF, SD, FS, CC
Coordinates lateral b
1.7
frontal a
8.5
T U , M, T S T H , CC
Regions destroyed (see list of abbreviations)
5.0
7.6
8.5 8.0
8.0
4.2
4.0 4.0 a n d 6.0
4.2
3.2
3.0
4.2
5.0
7.0
vertical e
a rostral to t h e i n t e r a u r i c u l a r line in m m , b lateral to t h e m i d l i n e in r a m , c d i s t a n c e b e l o w t h e t o p of t h e skull in r a m .
2A
. . . . . . . . . .
Stria m e d u l l a r i s
1E
Hippoe~mpus . . . . . . . . . . .
. . . . . . . .
1D
. . . . . . . . . .
Fornix superior
. . . . . . . . . . . . . .
N u m b e r of figure a n d code in t h e t e x t
T y p e of t h e lesions
NO
No
Yes
Yes
Yes Yes
No
No
No Yes
No
Yes
Yes
Yes
Yes
Yes
Degeneration in t h e supraoptie nucleus
T a b l e 1. L e s i o n s for d e m o n s t r a t i n g n e r v e t e r m i n a l s in t h e s u p r a o p t i c n u c l e u s w i t h F i n k - H e i m e r silver d e g e n e r a t i o n m e t h o d
O1
~a o
O~
~0
t~
528
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Fig. ]. Drawings for localization of various electrical lesions. Frontal sections according to KSnig and Klippel (1963). Abbr. see in the text. (A) Lesion in the olfactory tubercle region, (B) Lesion in the septum, (C) Fornix lesion in the septum, (D) Lesion destroying fornix superior, (E) Twofold lesion in the hippocampus, (F) Fimbria hippocampi lesion
H e a v y ipsilateral and slight contralateral degenerations were caused by a hypothalamic lesion including the ventromedial and the arcuate nuclei as well as the supraoptic decussations (Fig. 2E and Fig. 6A, B). Interruption of the supraoptic decussation below the MFB (Fig. 2F) resulted in degeneration only in the ipsilateral supraoptic nucleus. Lesion in the midline thalamic region by penetration through the fornix superior, were followed by degeneration in the NSO (Fig. 2B). The same result was obtained after dorsomedial hypothalamic lesions. Lesions or damage in the stria medullaris (Fig. 2A) did not result in any degeneration in the supraoptic nucleus. No degeneration was demonstrable with the Fink-}Ieimer method following medial amygdaloid lesion (Fig. 21)) or after destruction of the stria terminalis (Fig. 2C). Degenerated fragments could be observed after lesions of either the ventral tegmental area (Fig. 3A) or the central meseneephalic gray matter (Fig. 3B), or after a transection at the diencephalon-mesencephaIon border (Figs. 4E and 6C).
Afferent Fibers of the Supraoptic Nucleus
529
l t
s ~"
Fig. 2. Drawings for localization of various electrical lesions. Frontal sections according to K6nig and Klippel (1963). Abbr. see in the text. (A) Lesion destroying the stria medullaris, (B) Midline thalamic lesion, (C) Lesion destroying the stria terminalis, (D) Lesion in the nucleus amygdaloideus medialis, (E) Lesion in the medial basal hypothalamus, (F) Lesion destroying the supraoptic decussation
c-
Fig. 3. Diagrams of various electrolytic lesions. Frontal sections. Abbr. see in the text. (A) Lesion in the ventral tegmental area, (B) Lesion in the substantia grisea centralis
36*
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L. Z~borszky et al.
A
B
C
Fig. 4. Drawings for localization of various major surgical interferences in rat dicncephalon. (A--D) parasagittal, (E--F) frontal sections. Abbr. see in the text. (A) Complete isolation of the medial basal hypothalamus, (B) Anterior hypothalamic deafferentation, (C) Sucking away the entire hypothalamus, (E) Parasagittal section through in the lateral hypothalamus, (F) Frontal section at the diencephalon-mesencephalonborder
Afferent Fibers of the Supraoptie Nucleus
531
D
i
1 Fig. 4D--F
However, it is important to note that the lesion of the central gray matter resulted in degeneration in the NSO, only if the lesion encroached upon the neighbouring tegmental region, which contains the ascending noradrenergie fibres (Ungerstedt, 1971).
Fig. 5. Electron micrographs of the NSO. Bar scale: 0.5/~ .(A) After complete isolation of the medial basal hypothalamus, (B) septal lesion. Arrow points to synaptie region. Og oligodendroglial cell, D dendrite, db degenerated bouton
Afferent Fibers of the Supraoptie Nucleus
533
Fig. 6. Terminal degeneration in the NSO following a lesion of the medial hypothalamus: (A and B) frontal section and diencephMic-meseneephMie transection, (C) sagittal section. Fink-Heimer silver degeneration method The Fink-Heimer degeneration findings were substantiated under the electron microscope, for virtually all types of focal lesions, although no a t t e m p t was made to quantitate degeneration findings as discrete focal lesions were not considered as suitable for real quantitative conclusions.
Electron Microscopic Quantitative Studies o] Terminal Degeneration A/ter Major Surgical Interruptions o[ the AGerent Systems o] the NSO Some degenerating terminals were found even after lesions that did not yield degeneration in the silver stain, such as medial amygdaloid (2D) loci. Lesions in the olfactory region (1A) resulted in moderate numbers of degenerating boutons in the NSO. Neither a complete medial basal hypothalamie isolation (4A) yielding 14% degenerated boutons of the total, nor paramedian sagittal sections (4E) separating the NSO from the medial hypothalamus and from the other side of the brain (resulting in 12.2~o degeneration) produced a degeneration in the NSO t h a t would approach 20% of the total population of axon terminals. Less than I 0 % of the total NSO terminals was found to be degenerated after a coronal plane transection at the dieneephalic-mesencephalic border (4F) destroying all possible ascending fibers from the brainstem. No changes were noticed in the NSO 2 days after eye enucleation or after cervical sympathectomy. The numerical degeneration findings are summarized in Table 2. I n order to understand this table it is important to keep in mind t h a t major surgical inter37 Exp.Brain Res. Vol. 22
4C 4D 4E 4F 1B 2D 1A
. . . . . .
4. S u c k i n g a w a y t h e entire h y p o t h a l a m u s
5. P o s t e r i o r h y p o t h a l a m i c d e a f f e r e n t a t i o n . . . . . .
6. P a r a s a g i t t a l t r a n s e c t i o n in t h e d i e n c e p h a l o n . . 7. D i e n c e p h a l i c - m e s e n c e p h a l i c t r a n s e c t i o n . . . . . .
8. SepVal lesion . . . . . . . . . . . . . . . . . 9. A m y g d a l a lesion . . . . . . . . . . . . . . . .
10. Olfactory lesion
(3)
85 44 22 12 21
453 397 560
3 101 12
156 609 573 606
-88
458 627
697
b) Degenerated
a) Normal
Synaptic boutons c o u n t e d in representative area
(4)
3.8
3.0
4.9
7.3
12.2
1.9 16.6 2.1
0.0 14.0
Normal and degenerated boutons ratio in ~
(5)
0
A
S, H I
B
H. B
HI S, H I , B, H :B (partly)
-H I , I-I, :B
l~egion d e s t r o y e d or s e p a r a t e d from the NSO (see list of abbreviations)
(6)
17.0
13.5
22.0
32.7
54.7
8.5 74.4 9.4
0.0 62.8
Degenerated b o u t o n s in percentage of degene r a t i o n in total NSOisolated r a t s ~
S = s e p t u m (3.0%), H I = hippocampus (1.9%), 0 = o l f a c t o r y t u b e r c l e (3.8%), H ~ h y p o t h a l a m u s (4.7%), B = b r a i n s t e m (7.3%), A = a m y g dala (3.0%). 22.30/0 of s y n a p t i c a l b o u t o n s were d e g e n e r a t e d following t o t a l N S O isolation ( L 6 r s et al., 1975) ; t h i s v a l u e c o r r e s p o n d s to 100~ .
. . . . . . . . . . . . . . . .
-4A 4B
1. I n t a c t control . . . . . . . . . . . . . . . . . 2. C o m p l e t e isolation of t h e m e d i a l b a s a l h y p o t h a l a m u s 3. A n t e r i o r h y p o t h a l a m i c d e a f t ~ r e n t a t i o n . . . . . .
(2) Code number in figures and Text
(1)
T y p e of t h e surgical interferences
Table 2. D e g e n e r a t e d n e r v e t e r m i n a l s following v a r i o u s t r a n s e c t i o n s a n d lesions of fibers t e r m i n a t i n g in t h e s u p r a o p t i c n u c l e u s
2-
9
b~
Afferent Fibers of the Supraoptic Nucleus
535
ferences are labeled in column (2) by their code numbers refering mainly to the several drawings of Fig. 4 and to lesser extent to some drawings of Figs. 1 and 2. The same code numbers are used also in the text. The regions destroyed or separated from the NSO are listed in column (5) by using the abbreviations given in an appendix. One has to realize that such large cuts disconnect from the NSO often more than one of the brain regions contributing afferents. The percentage number of degenerated synapses is indicated in two different forms: in column (4) in percent of the total synapse population and in column (6) in percent of the poi~ulation of extrinsic terminals. Since only 22.3% of the synaptic boutons were found previously (L6rs et al., 1975) to be of extrinsic source, this w l ~ e would correspond to 100% in column (6). The percentage values of individual brain regions indicated below the lower horizontal line in Table 2 have been calculated by cross correlation of the data in column (4) and correspond therefore to percentages of the total synapse population. Discussion
By appl3dng the double strategy of smaller discrete and/or local lesions for qualitative, and major surgical interferences for quantitative evaluation, the proportions of afferents to the NSO from different parts of the limbic system (olfactory tubercle, septum, hippocampus, and amygdala) from the medial basal hypothalamus, and from the lower brainstem could be estimated with reasonable accuracy. The procedure by which one could arrive at the percentage values given in Table 2 is indirect in the ease of the medial basal hypothalamus and of the septum, because these regions could not be isolated or their connections with the NSO interrupted without major damage to other structures that contribute additional afferents. However, the percentage numbers of afferents given by these regions could still be established by cross correlation o f the quantitative data received from different lesions destroying the afferents in various combinations. Although the whole procedure might appear as rather unusual, it is easy to convince oneself that percentage data obtained by the interruption in different combinations of the major afferent systems yielded values that are self consistent. Considerable confidence for the procedure can be gained from the simple fact that if one adds the final percentage values received for contributions by the six separate regions mentioned above and indicated in Table 2 below the lower horizontal line, the sum is 23.7% which is almost exactly the value received for the actually observed degenerating synapse in the preceding study (L6rs et al., 1975) for the extrinsic afferents of the NSO. The strategy applied by the surgical transections can be understood better from Fig. 7, in which the several interferences are diagrammatieally indicated by dashed lines labelled with the appropriate code numbers corresponding to the drawings of Figs. 1, 2 and mainly 4. The coarse lines terminating in arrows represent the major known (labelled) and unknown (unlabelled) afferent paths of the NSO. The box symbolizing the hippocampus (CA1) has been superimposed on the larger box corresponding to the medial basal hypothalamus partly because the diagram tries to give a schematic view from above, but mainly because a quite extensive damage to the hippocampus is caused by the isolation of the hypothala37*
L. Zs
536
et al.
/,A
Fig. 7. Composite diagram of neural pathways that terminate in the NSO. Broken lines represent various transections and lesions as defined in Figs. 1, 2 and 4 as well as in Table 2 m u s (4A). B y considering w h a t coarse lines, r e p r e s e n t i n g t h e p a t h w a y s , i n t e r s e c t with which d a s h e d lines s t a n d i n g for t h e surgical interferences i t is e a s y to d e d u c e from Fig. 7 t h e various t y p e s of afferents t h a t can be s u p p o s e d to undergo degeneration after t h e several cuts. On t h e basis of this figure also certain formalizations can be m a d e for t h e relations b e t w e e n t h e percentage occurrence of d e g e n e r a t e d t e r m i n a l s after various interferences. These relations are as follows: la) 4C-~4B(HI)-+4F(B)+S+H lb) 1B Z+4B(HI) S = 1B~4B S -~ 1 B ( S + H I ) - t - 4 B ( H I ) 4C ~ 4 B ~ - 4 F ~ - ( 1 B I B ) - [ - H S = 4 . 9 - - 1 . 9 ~ 3.0 H ~ 4C--4F--1B U -----1 6 . 6 - - 7 . 3 - - 4 . 9 ---- 4.4 2) 4A : 4 B ( H I ) + 4 F ( B ) ~ - H 3) 4E -~ 4 F ( B ) - ] - H H ~ 4A 4 B - - 4 F H ~ 4E---4F(B) H ~ 1 4 . 0 - - 1 . 9 - - 7 . 3 ---- 4.8 H ~ 12.2--7.3 ~ 4.9 As can be seen t h e observed ratios of d e g e n e r a t e d t e r m i n a l s are in v e r y good a g r e e m e n t w i t h t h e logical e x p e c t a t i o n s arising from t h e formalization. The M F B carries t h e m a j o r i t y of t h e N S O afferents (L~rs et al., 1972). Ascending M F B fibers originating from various regions of t h e b r a i n s t e m (Nauta, 1956; Guillery, 1957; N a u t a a n d K u y p e r s , 1958; Cowan et al., 1964; Z s et al., 1973) give rise to a considerable fraction of t h e t o t a l afferent i n p u t (7.3 [32.7]0//0, column 6, T a b l e 2) 1. Most of these afferents correspond p r o b a b l y t o m o n o a m i n e r g i c fibers (Ungerstedt, 1971 ; Sachs et al., 1973) a n d should r e p r e s e n t
Afferent Fibers of the Supraoptic Nucleus
537
the morphological substrate of brain stem influence on the vasopressin release from the NSO (Mills and Wang, 1964; Woods et al., 1969). The descending MFB fibers reaching the NSO originate in the septum (3.0 [13.51%) and the olfactory tubercle (3.8 [17.01%). The former observation confirms the results of Johnson (1965) and Powell and l~orie (1967); and Tangapregassom et al. (1974). The high percentage of olfactory afferents is probably due to the fact that the lesions interrupt also fibers of more rostral (cortical: piriform cortex Gurd]ian, 1925, 1926; Nauta, 1956; Powell et al., 1965) origin, collected by the MFB. This study has revealed the existence of hippocampal supraoptic connexions. These fibers can be assumed to run through the fornix superior, because both hippocampal lesions (1E) or damage of fornix superior (1D) cause degeneration in the NSO, while large fimbrial lesions (IF) do not. Consequently only the sector CA1 may contribute to this pathway (l~aisman et al., 1966). In contrast with the present results no termination of hippocampal fibers in the NSO was mentioned by Nauta (1958) and Raisman et al. (1966). The contribution of this pathway to the NSO afferent terminals can be inferred directly from the results of a 4B type transection, this being the only pathway to NSO interrupted by such a lesion of the fornix superior (Zs et al., 1972). The degeneration in the NSO following the lesion of the medial amygdaloid area (2D) is moderate (3.0 [13.51%). Since the lesion and the electrode track cause additional damage to the ventral amygdalofugal pathway and the stria terminalis, this has also to be taken into consideration. The interruption of the stria terminalis (2C) does not yield any degeneration demonstrable either with the Fink-Heimer method or under the EM, which is in agreement with other studies (Helmet and Nauta, 1969; Leonard, 1971; De Olmos and Ingram, 1972). I t is reasonable, therefore, to assume that the amygdalo-supraoptic fibers are running in the ventral amygdalofugal path, as already suggested by Nauta (1961) for the monkey. However there is some doubt (Leonard and Scott, 1971) about the amygdaloid contribution to this pathway in the rat. Some fibers from the periamygdaloid cortex traversing the lesioned area should be taken also into consideration. Sucking away the entire hypothalamus from dorsal approach (4C) yields an abundant degeneration of synaptic terminals in the NSO (16.6 [74.4]%), corresponding well to the lesion of the septum and the hippocampal fibers by intraduction of the funnel and by removal of the medial basal hypothalamus and the MFB. Isolation of the medial basal hypothalamus (4A) destroys both the hypothalamie sources of NSO afferents and the MFB, and causes additional damage by introduction of the Hals knife to the fornix superior. Hence the quantitative result in degeneration (14.0 [62.8]%) should correspond to the result of 4C minus the septal fibers (i. e. 16.6--3 ~ 14). The paramedian sagittal cut (4E) along the lateral edge of the MFB interrups the same fiber systems as 4A with the only exception of the hippoeampal fibers (see Fig. 7). Thus the numerical result in terminal degeneration of the parasagittal section (12.2 [54.7]%) and the value (1.9 [8.5]%) found for hippocampal fibers (4B) corresponds well to the yield of 4A (12.2~1.9 ~ 14). 1 From the two numbers the first refers always to percentage of total synapse population and the second [in square brackets] to the percentage of extrinsic afferent synapses.
538
L. Zs
et al.
A n isolated r e m o v a l of t h e h y p o t h a l a m u s w i t h o u t a d d i t i o n a l d a m a g e to o t h e r structures n o t being feasible, t h e c o n t r i b u t i o n of t h e medial basal h y p o t h a l a m u s to t h e afferents of t h e N S O can only be c a l c u l a t e d indirectly. B y using t h r e e different c o m b i n a t i o n s of d a m a g e , formalized in equations (la), (2) a n d (3) t h e calculated value was found to be 4.4%, 4.8%, a n d 4 . 9 % respectively. This fraction of afferents is quite considerable as i t c o n t r i b u t e s to 21~ i.e. a fifth of t h e t o t a l afferent s y n a p s e p o p u l a t i o n of t h e NSO. A l t h o u g h t h e findings give no evidence of t h e possible origin of these afferents w i t h i n t h e h y p o t h a l a m u s , it is reasonable to assume t h a t a t least p a r t of t h e m corresponds to t h e a r e u a t e nucleus - - periv e n t r i c u l a r d o p a m i n e r g i c cell groups (DahlstrSm a n d F u x e , 1964; B j 6 r k l u n d a n d Nobin, 1973). A p r o b a b l y m i n o r c o n t r i b u t i o n b y d a m a g e of t h e s u p r a o p t i e deeussiation (Minderhoud, 1967 ; Leontowich, I970) can n o t be e n t i r e l y excluded. On t h e whole a b o u t 5 0 % of t h e N S O afferents originate from t h e limbic s t r u c t u r e s (olfactory tubercle, s e p t u m , a m y g d a l a , h i p p o c a m p u s ) , which seems to agree well w i t h physiological findings on t h e role of t h e same s t r u c t u r e s in vasopressin release from NSO (I-Iayward a n d Smith, 1963; Powell a n d I~orie, 1967; W o o d s et al, 1969). The r e m a i n i n g half of t h e afferents consists of 3 0 % b r a i n s t e m afferents p r o b a b l y m a i n l y adrenergic a n d serotoninergic fibers a n d of 20% hypot h a l a m i c fibers p r o b a b l y largely of d o p a m i n e r g i c character. The m e n t a l s t r a t e g y u n d e r l y i n g this s t u d y as well as t h e m e t h o d i c a l a p p r o a c h are u n d o u b t e d l y unusual, a n d a d d i t i o n a l l y t h e extensive convergence u p o n t h e NSO m a y raise some d o u b t s against t h e results. I n d e e d t h e t r a d i t i o n a l views of t h e N S O as a n e u r o s e c r c t o r y nucleus h a v i n g its own osmoreceptors is n o t easily reconciled whit such a convergence of afferents from r a d i c a l l y different sources. However, several recent physiological studies m e n t i o n e d above, would p o i n t in t h e same direction. A AB AC AL AM ATV C CA CC CFV CO CP D DM DS F FtI FLM FP FS G GD HI IC
= = = = = = = = = = = = = = -= = -= = = = = =
List of Abbreviations nucleus accumbens nucleus amygdaloideus basalis nucleus amygdaloidcus centralis nucleus amygdaloideus lateralis nucleus amygdMoideus mcdialis area tegmenti ventralis (TsM) c~udate-putamen eommissura ant~erior corpus eallosum commissura fornicvis ventralis chiasma opticum commissura posterior nucleus traetus diagnolis nucleus dorsomedialis deeussationes supraoptica columna fornicis fimbri~ hippocampi fasciculus longitudinalis medialis fornix praeeommissuralis fornix superior globus pallidus gyrus dentatus hippocampus capsula interna
Afferent Fibers of the Supraoptic Nucleus
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IP LM
= nucleus interpeduncularis = lemniscus medialis M = medial forebrain bundle (MFB) MM nucleus medialis thalami, pars medialis NA nucleus arcuatus R nucleus rhomboideus RE = nucleus reuniens I~V = nucleus ruber S = stria medullaris thalami SD = nucleus dorsalis septi SF = nucleus fimbrialis septi SG = substantia grisea centrslis SL = nucleus lateralis septi SM = nucleus medislis septi SN = substantia nigra ST = nucleus interstitialis striae terminalis T = traetus olfaetorius lateralis TD = tractus diagonalis (Broea) TO traetus opticus TSTH = tractus striohypothalamicus TU tuberculum olfaetorium nucleus ventromedislis V~
Acl~nowledgements. The suthors are indebted to Prof. J. Szentdgothai for valuable advice, critical comments a n d suggestions. References
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