Okajjmas Folia Anat. Jpn., 58(4-6) : 419-442, March 1982 Distribution of Neurons Innervating the Supraoptic Nucleus of the Rat with Special Reference to Neighbouring Relay Neurons Using HRP and Golgi Methods By KOICHI IIJIMA Department of Anatomy, Akita University School of Medicine, Akita, Hondo, 010, Japan -Received for Publication, September 17, 1981- Key words : Supraoptic nucleus , Relay neurons, Rat, HRP, Rapid Golgi. Summary : Detailed histochemical studies have been performed on the distribution of the nuclei innervating the supraoptic nucleus (SO) in the whole brain after horseradish peroxidase (HRP) injections through fine glass micropipettes (2-8μm tip diameter) into the SO of 39 Wistar rats. The chromatogenic reaction was carried out according to Mesulam's No. 8 procedure (1976). In the 3 cases, where injected HRP was most exactly restricted to the SO, HRP-labeling was limited to some small neurons located just dorsal to the SO. The other successful HRP injec- tions into the SO resulted in labeling rather few neurons which are sparsely distri- buted in various regions of the brain. The following conclusions were drawn from the analysis of these successful cases. The rapid Golgi method was used to check the morphology and possible functions of these small neurons revealed in the 3 cases . Every nuclei of the subthalamus and posthypothalamus innervate the SO whereas probably very few neurons of the hypothalamic arcuate and ventromedial nuclei send axons to it. The afferent monoaminergic fibers arise largely from the B7 and B8, and to a lessor extent from the A6 and A10 cell groups. There is a high possibility that the mesencephalic central gray directly innervates the SO. The septum, diagonal band nucleus and amygdala send very few projections, whereas the olfactory tubercle and pyriform cortex may not. It remains unanswered whether the hippocampus projects to it or not. Small non-secretory neurons in and around the SO may be important relay neurons to large neurosecretory ones. A recent electrophysiological study in studies (Leranth at al., 1975 ; ZAborszky vitro (Haller and Wakerley, 1980) has de- et al., 1975) have estimated two things monstrated that the osmoreceptor is loca- that only one third of axon terminals in ted separately from the supraoptic nucleus the SO probably originate outside the (SO) of the rat, indicating that function- nucleus, and the propotion of the number ing of the SO is controlled by other nu- of afferent fibers originating in various clei. subcortical structures to that of total aff- Quantitative electron microscopic (EM) erents to the SO. These results were 419 420 K. lijima based on EM obervations on degenerat- by means of the HRP method, and to ing axon terminals in the SO resulting study the merphology and possible role from transection of afferent pathways of of small non-secretory neurons in and various sources in various combinations. around the SO by the rapid Golgi method. However, neither of the studies describe the distribution of neurons innervating Material and Methods the SO at the light microscopic (LM) level. Although Swaab et al. (1975) have 39 female Wistar rats (200 g body we- found that about 10% of the neurons in ight) were used in an HRP study. They the hypothalamic neurosecretory nuclei were anesthetized with sodium pentobar- of the rat without oxytocin nor vasop- bital (60 mg/kg i.p.) and subjected to ressin according to their immuno-histo- surgical operation in a stereotaxic appa- fluorescence study, the roles of these ratus so that KOnig and Klippel's stereo- cells as well as small neurons just dorsal taxic atlas (1963) could be used for locali- to the SO have not attracted special atten- zation of the SO. A glass micropipette tion hitherto. (2-8 pm tip diameter) filled with HRP It has not been determined by the HRP (Sigma type VI) which was dissolved into and Golgi studies (Millhouse, 1973 ;Bodoky 2% dimethylsulfoxide (DMSO) aqueous and Rethelyi, 1977) whether the hypotha- solution to a final concentration of 40% lamic arcuate (AR) and ventromedial (VM) (W /V) was vertically inserted to a depth nuclei project directly to the SO or not. of 8.5 mm below the cortical surface at Previous studies by the Fink-Heimer me- a point 1.7 mm lateral to the midline and thod and EM did not reveal the direct 6.4 mm anterior to the interaural line. projections to the SO from the olfactory DC currents (2-4 /IA) were passed for 5 tubercle, hippocampus and amygdaloid to 10 min between the micropipette and complex (Leonard and Scott, 1971; Za.bo- the indifferent electrode on the skull rszky et al., 1972). The relationship with the micropipette as anode. Some- between the raphe nuclei and SO is not times a train of square wave pulses (0.5 well known (Zaborszky et al., 1975 ; Pal- sec in diurection, 2-4 1211 in intensity) at kovits and Za.borszky, 1979). Histofluo- a rate of 1/sec was delivered for 10 to rescence, autoradiographic and HRP stu- 20 min instead of DC currents. After a dies demonstrate the presence of a pro- survival time of 48 h, animals were anest- jection from the A6 to the SO (Loizou, hetized with sodium pentobarbital (60 mg 1969 ; Jones and Moore, 1977 ; Iijima and /kg i.p.). Following an intracardial injec- Ogawa, 1980), but it is not clear whether tion of 1 % sodium nitrate solution puls other noradrenergic nuclei such as the 0.1 ml of heparin (1000 units/ml), perfusion Al, A2, A5, A7 and A8 cell groups project was carried out through the heart with to the SO or not, although their efferent a warm Ringer solution followed by a fibers are involved in the medial forebrain sequence of 2 phosphate buffered (0.1 M) bunble (MFB) (Lindvall and BjOrklund, aldehyde mixtures ; the 1st containing 1% 1974). paraformaldehyde and 1.25 % glutaral- The HRP method permits us to study dehyde, the 2nd 2 % paraformaldehyde the distribution of all the nuclei innervat- and 2.5 % glutaraldehyde. Thereafter, ing the SO at the LM level. The present the brain was removed from the skull investigation was undertaken to study it and stored overnight in 0.1 M phosphate throughout the whole brain and throw buffer (pH=7.4) containing 5 % sucrose light on these questions mentioned above at 4°C. The whole brain was cut into Neurons Innervating the Rat SO 421 45 ,um serially on a freezing microtome. according to the d!stribution patterns of All the sections were reacted using be injected HRP, and summarized in table 1. nzidine dihydrochloride (BDHC) after a Labeled neurons except for those in raphe slight modification of Mesulam's No 8 nuclei were observed ipsilateral to the procedure (1976). They were counters- injection site in all cases (Diagrams 1-5 ; tamed with a 1 % neutral red aqueous Figs. 1-18, 20-24). solution. Group I. 4 cases (22, 24, 29, 32) were 18 female Wistar rats (200g body included in this group. This group (except weight) were used for the rapid Golgi for case 32) was characterized by the method (Romeis, 1968). The optimal in- finding that injected HRP was completely cubation period was 24 h for small neurons restricted to the SO with some small just dorsal to the SO. Celloidin sections HRP-labeled neurons in nearby tissue were cut into 50 rim. In order to diffe- (Diagram 1G-1; Figs. 1, 2). Photomicro- rentiate them from SO neurosecetory graphs of the injection sites are illustrated neurons, karyometry was carried out by in Figs. 1-7. using a phasecontrast microscope (Nikon, In cases 22 and 24, injected HRP co- DLL x 100) and a screw-micrometer eye- vered mostly the ventral part of the SO piece (Nihonkogaku Co.) on both groups leaving small rostral and dorsal parts of of neurons. The eyepiece allowed a ma- the nucleus free of HRP. In both cases rgin of error less than 0.1 pm. Both the no labeling could be detected throughout largest (A) and shortest (B) axes of each the brain except for the perinclear tissue nucleus were measured. The nuclear of the SO, where small labeled neurons volume was calculated according to the were recognizable (table 1, Diagram 1G-1). formula In case 29, where injected HRP covered the SO almost completely and labeled some V,----47/3(A/2)(B/2)2. small neurons just dorsal to it (Figs. 1, 2), The mean nuclear volume and standard we could not find convincing evidence of deviation were obtained from these data. labeling in any parts of the brain, alt- The t-test was carried out to check the hough a cluster of granule cell-like struc- presence or absence of significant diffe- tures in the hippocampus and a large rence between the 2 groups of neurons. round cell-like one in the linear nucleus (A10 cell group) appeared to be labeled Results with HRP. In these 3 cases, where HRP in- jection into the SO seemed most successful, I. The HRP Method we could detect signs of HRP-labeling The follwing descriptions were limited only in some small neurons located just to the 10 cases where the injection sites dorsal to the SO. were well localized to the principal part In connection with these findings, the of the SO (Leranth et al., 1975) with no results obtained from case 32 were de- (cases 22, 24, 29)(Diagram 1G-1) or negli- sdribed in table 1, where HRP was in- gible (cases 19, 21, 25, 26, 32, 35, 36) jected slightly lateral to the center of Diagrams 1G-2, 1G-3, 1G-4) diffusion of the SO, but contamination of the perinu- HRP in the surrounding tissue and 3 cases clear tissue with HRP was negligible (cases 2, 3, 34) (Diagram 1C) where the (Fig.