An in situ Study of the Hypothalamic Neurosecretory System ofthe Freshwater Catfish Ailia chila' (Ham.) Shamim Haider and A . G. Sathyanesan Surgical Research Laboratory, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India (Received May 18, 197 1 )

Abstract The hypothalamo-neurohypophysial complex of Ailia coila is well demonstrated with the help of in situ staining procedure. Both pars magnocellularis and pars parvocelIularis components of the nucleus preopticus contribute to the formation of the right and the left main neurosecretory tracts. Anterior one third of these tracts are loosely set and posteriorly they became more compact. From the posterior two thirds of the main tracts several pairs of lateral tracts were given off which join at the midline to form the paired median tracts. The median and the main tracts jointly enter the pituitary as the common tract. The common tract on entering the pituitary often divides into two or more branches and enter the pars intermedia independently.

The rostra1 pars distalis is least innervated by the neurosecretory axons. Since the proximal pars distalis has varying amount of AF-positive cells, and the pars intermedia has the bulk of the neurosecretory axons both these regions are stained dark in the in situ preparations. Bulk prepara- tions provide a clear topographic picture of the entire neurosecretory system, which is very difficult 'to visualise in tissue sections and in their reconstructions.

After the introduction of Gomori's chrome-alum-hematoxy lin-phloxine ( Barg- mann 1949) and Gomori's aldehyde fuch- sin (Gabe 1953; Dawson 1953) to dif- ferentially stain the neurosecretory system, the hypothalamo-neurohypophysial ( HN) complex of several species of teleosts has been studied. However, in 1963 Dodd and Kerr observed that fewer than 30 tele- ostean species of the 23,000, believed to exist have been studied using the special techniques demonstrating the neuro- secretory material (NSM) . During the subsequent years several more species were investigated and this subject also has been reviewed from time to time (Arvy et al. 1959; Dodd and Kerr 1963; Jnrrgensen

and Larsen 1967; Perks 1969 and others). Several newer staining procedures ( Adams and Sloper 1956; Olsson 1958, 1959) including fluorescence technique (Sterba 1961) have been employed to study the neurosecretory system. However, all these techniques were employed to stain tissue sections and the interpretation of the structure of the HN complex was made largely through their reconstructions.

Subsequent to the introduction of bulk staining technique by Braak (1962) to demonstrate the HN complex in situ, the neurosecretory system of several species of fishes was studied in toto (Leatherland et al. 1966; Leatherland and Dodd 1969; Knowles and Vollrath 1966; Sathyanesan

Actu Zoologica 53: 23-30 (1972)

24 Shamim Haider and A . G. Sathyanesan

1969 a, b, c ) . Since the in situ prepara- tions provide a three dimensional picture of the HN complex it is easier to follow the circuitous courses of the neurosecre- tory tract. The details obtained through such studies are difficult or impossible to observe in tissue sections or in their re- constructions.

In this communication the structure and the distributional pattern of the neurosecretory axonal system of A . coila are studied using both in situ staining techniques and microtome-sections.

Material and Methods Over 30 fishes used in this investigation were collected from the river Ganges at Varanasi. All were killed by decapitation and the entire brain was fixed in Bouin’s fluid. Fifteen of them were stained in bulk adopting the modified Gabe’s AF technique as used earlier (Sathya- nesan 1969 c) to demonstrate the hypothalamo- neurohypophysial complex in bulk. 100 to 700 ,uq thick freehand section of the whole preparations were made through sagittal, frontal and transverse planes to study the distributional pattern of the neurosecretory tracts. The other 15 brains were processed and paraffin sections were cut a t 5, 8 and 10 ,urn thick and stained in AF, using Halmi‘s Orange- G-fastgreen-chromotrope 2R combination as counter stain. In some cases Ehrlich’s hema- toxylin is used as a nuclear stain after AF.

0 bservations The nucleus preopticus (NPO) of A . coilu is situated on either side of the infundib- ular cavity dorsal to the base of the optic nerve (Fig. 1). In a cross sectional view the intact NPO is in the form of an elongated, broad, compact band of tissue, having few loosely scattered neurons around it (Fig. 4). The dorsal half of the NPO is formed of larger cells constituting

the pars magnocellularis (PMC) (Figs. 2, 5, 7 ) and the ventral half is made up of smaller cells which go to form the pars parvocellularis (PPC) (Fig. 7 ) . Some of the PMC cells are bi- or even multipolar (Fig. 5) and their axons may be in con- tact with the axons or neuronal bodies of other neurosecretory cells. The cells of both the PMC and PPC contribute to the formation of the right and the left main tracts. In thick frontal sections of the in situ preparation the formation of these tracts can be well demonstrated (Figs. 1, 3 ) . In the anterior one third of the left and right main tracts the neuro- secretory axons are loosely set. Hence, this region looks like a hazy curtain (Figs. 1, 9). However, in a higher magnification, individual beaded axons are clearly dis- cernible in this region (Figs. 2, 3 ) . Pos- terior to this hazy curtain-like area the neurosecretory tracts approximate and become more compact. Subsequently, from this compact region six or more pairs of lateral tracts separate off and join at the midline to form a closely set pair of median tracts. They were so closely approximated that its double na- ture is very faintly discernible (Figs. 9, 10). The median and the main tracts jointly enter the pituitary as the common neurosecretory tract. In some cases, this common tract separates off into two or more tracts at the region of the rostral pars distalis and penetrate the pars inter- media independently (Fig. 11). Although, smaller neurosecretory tracts were seen ramifying among the cells of the proximal pars distalis, they were almost absent in the rostral pars distalis (Fig. 8). Due to the presence of large number of AF- positive cells in the proximal pars distalis in bulk preparations larger area of this component is also stained dark as the pars intermedia where the neurosecretory

F i g . 1 . Thick freehand frontal sections of the in situ stained hypothalamic neurosecretory system showing the formation of the right and left main tracts. The diffuse curtain-like region is seen adjacent to the NPO. X 75. Fig. 2 . Two neurons belonging to the region of the pars magnocellularis. X 270. Fig. 3. Thick freehand frontal section of the

Neurosecretion in Ailia coda 25

in situ stained nucleus preopticus showing long beaded axons (arrows). X 95. F i g . 4 . Thick freehand transverse section of in situ stained nucleus preopticus showing the formation of left and right main tracts (ar- rows). ~ 9 5 . Fig. 5. A group of neurons from pars magno- cellularis. Arrow shows a multipolar neuron. x 270.

26 Shamim Haider and A . G. Sathyanesan

Fig. 6. Thin frontal section showing the loca- tion of the nucleus preopticus on either side of the third ventricle. Arrow shows the colloid-like material in the ventricular space. x 120. Fig. 7. Thin parasagittal section of the nucleus preopticus showing the distribution of neurons of the pars magnocellularis and pars parvocel- lularis. x 180. Fig. 8. Sagittal section of the pituitary showing its component parts and the distribution of the neurosecretory axons. x 80. Fig. 9. Ventral view of the in situ stained hypothalamo-neurohypophysial complex show- ing its component parts. x 45.

a x p s ramify extensively and have peri- vascular endings.

Parasagittal view of the bulk stained HN complex as well as microtome pre- parations show that the cells of the PMC are scattered to a larger area to form a T- shaped configuration (Fig. 7) . However, the horizontal limb of the -T- has only sparse distribution of neurons (Fig. 7 ) . The cells of the NPO are situated very close to the ependymal lining of the third ventricle (Fig. 6) and occasionaly few

Neurosecretion in Ailia coda 27

neuronal bodies and axons were noticed among the ependymal cells. The third ventricle contains AF-positive homogene- ous material which is largely contributed by its ependymal lining. Often the apical processes of the ependymal cells were seen in contact with the secretory material. The position of the neuronal bodies and axons of the NPO does suggest the pos- sibility of some NSM getting access into the cavity.

Fig. 10. Ventral view of the bulk stained middle segment of the hypothalamo-neurohypophysial complex showing the formation of the lateral and median tracts and the union of the median and main tracts to form the common tract. x 50. Fig. 11. Bulk stained pituitary showing the bifurcation (arrow) of the common tract in the pars distalis prior entering the pars inter- media. ~ 7 5 . Fig. 12. Side view of the in situ hypothalamo- neurohypophysial complex showing its various components. Arrows show the common tract. x 40.

28 Shamim Haider and A . G. Sathyanesan

Discussion In situ staining has been succ'essfully adopted to demonstrate the neurosecretory system of the European eel AnguilEa an- guilla (Leatherland et al. 1966; Leather- land and Dodd 1969; Knowles and Voll- rath 1966) and in the Indian freshwater teleosts Clarias batrachus (Sathyanesan 1969 a, b, c) Heteropneustes fossilis (Sathyanesan 1969 b; Sathyanesan and Haider 1970 a ) Rita rita (Sathyanesan 1969 b; Sathyanesan and Haider 1970 b). The distributional pattern of the neuro- secretory tract exhibits a wide variation in some species. In Channa punctatus the fine neurosecretory axons of the NPO unite at the region of the nucleus itself to form 4 to 6 or more pairs of thicker tracts which run along the lateral aspect of the hypothalamus and enter the neuro- hypophysis independently at the level of the proximal pars distalis (Haider and Sathyanesan 1971 a ) . The same authors ( 1971 b) described a modification of what is found in C. punctatus, in Macrognathus aculeatus where the fibres of the NPO unite to form a pair of loosely set tracts which run laterally and enter the pituitary independently at the level of proximal pars distalis. In A . coila, the left and the right main tracts instead of entering the pituiary directly, give rise to a series of lateral tracts which in turn form sthe median tracts. Thus the base of the in- fundibulum adjacent to the pituitary stalk of this species has a network of neuro- secretory ramifications. The comparable area of the teleosts Clarias batrachus (Sathyanesan 197 1) and Heteropneustes fossilis (Sathyanesan and Haider 1971 a, b) has been described as an incipient median eminence where the primary capillary plexus ramify and come in con- tact with the neurosecretory network and

form the portal vessels which enter the pituitary. In the European eel, Leather- land et al. (1965) observed the ending of some axons at the ventral floor of the hypothalamus above the anterior margin of the rostra1 pars distalis, which they designated as subterminal region. In the birds, Scharrer and Scharrer (1963) sug- gested the formation of tortuous loops in the neurosecretory tracts as a device to increase the storage capacity for the NSM. In A . coila also formation of additional neurosecretory tracts like the lateral and median ones from the main tracts might serve the purpose of increasing the storage and releasing area for the NSM.

Explanation of Abbreviations CT - Common tract DCN -Diffuse curtain-like region of the left

HN - Hypothalamo-hypophysial complex IST - Infundibular stalk LAT -Lateral neurosecretory tract L T MD - Median neurosecretory tract NSM - Neurosecretory material NPO - Nucleus preopticus PPD - Proximal pars distalis PI - Pars intermedia PMC - Pars magnocellularis PPC - Pars parvocellularis RPD - Rostra1 pars distalis RT -Right main neurosecretory tract Tb - Third ventricle

and right main tracts

- Left main neurosecretory tract

Acknowledgements We are thankful to Dr. K. N. Udupa, Director, Institute of Medical Sciences and Dr. L. M. Singh, Officer Incharge of the Surgical Research Laboratory, Banaras Hindu University, Varanasi, for providing all facilities and encouragement. One of

Neurosecretion in Ailia coda 29

us (SH) is indebted to the University Grants Commission of India for the award of a Senior Research Fellowship during the tenure of which this work was carried out.

References Adams, C . W. M., Sloper, J . C . 1956. The

hypothalmic elaboration of posterior pitui- tary principle in man, rat and the dog; histochemical evidence derived from a per- formic acid alcian blue reaction for cystine. J . Endocrinol. 13: 221-228.

Arvy, L . , Fontaine, M. , Gabe, M . 1959. La voie neuroskcrbtnce hypothalamo-hypo- physaire des tklkostbens. J . Physiol. 51:

Bargmann, W. 1949. Ober die neurosekretori- sche Verkniipfung von Hypothalamus und Neurohypophyse. Z. Zelljorsch. 34: 610- 634.

Braak, H. 1962. Uber die Gestalt des neuro- sekretorischen Zwischenhirn-Hypophysen- system von Fischen. Z . Zellforsch. 58: 265- 276.

Dawson, A . B. 1953. Evidence for the termina- tion of neurosecretory fibres within the pars intermedia of the hypophysis of the frog, Rana pipiens. Anat . Rec . 115: 63-70.

Dodd, J . M., Kerr, T . 1963. Comparative morphology and histology of the hypo- thalarno-neurohypophysial system. Symp. Zool. SOC. (London) 9: 5-27.

Gabe, M . 1953. Sur quelques applications de la coloration par le fuchsine-paraldkhyde. Bull. Micr. Appl. 3: 153-162.

Haider, S.,, Sathyanesan, A . G . 1971 a. Com- parative study of the hypothalamo-neuro- hypophysial complex in some Indian fresh- water teleosts. Acta Anat. ( In press.)

- 197 1 b. Hypothalamo-neurohypophysial com- plex of the teleost Macrognuthus aculeatus (BL). Acta Zool. (Stockh.) ( In press.)

Jsrgensen, B., Larsen, L . 0 . 1967. Neuro- endocrine mechanisms in lower vertebrates. In: Neuroendocrinology. (Martini, L., Ga- nong, W. F. eds) pp. 485-521. Academic Press, New York.

Knowles, F., Vollrath, L . 1966. Neurosecretory innervation of the pituitary of the eels

1031-1085.

Anguilla and Conger. Phil. Trans. Roy. SOC. (London) 250: 31 1-342.

Leatherland, J . F., Budtz, P . E. , Dodd, J . M . 1966. Zn situ studies on the hypothalamo- neurohypophysial complex of the European eel Anguilla anguilla (L.) Gen. Comp. Endocrinol. 7: 234-244.

Leatherland, J . F., Dodd, J . M . 1969. Histol- ogy and the fine structure of the preoptic nucleus and hypothalmic tracts of the European eel Anguifla anguilla (L.) Phil. Trans. Roy . SOC. (London) 256: 135-145.

Olsson, R . 1958. Studies on the subcommissural organ. Acta zool. (Stockh.) 39: 71-102.

- 1959. The neurosecretory hypothalamus system and the adenohypophysis of myxine. Z . Zellforsch. 51: 97-107.

Perks, A . M . 1969. The neurohypophysis. In: Fish Physiology. (Hoar, W. S. , Randall, D. J. eds.) Vol. 2, pp. 1-93, Academic Press, New York.

Sathyanesan, A . G. 1969 a. Neurosecretory axonal pattern in the hypothalamus of some fishes. In: Zirkumventrikulare Organe und Liquor. (Sterba, G. ed.), pp. 255-259, Gustav Fischer, Jena. 1969 b. Stereometric structure of the hypo- thalamo-neurohypophysial complex in some Indian catfishes. Neuroendocrinology. 5: 10-23. 1969c. An in situ study of the preoptico- neurohypophysial complex of the freshwater teleost Clarias batrachus (L) . Z . Zellforsch.

1970. The architectural pattern of the neurosecretory system in the freshwater teleost Rita rita (Ham.) with a note on their post-hypophysectomy changes. Zeit. Mikr.-anat. Forsch. 82: 493-507. 197 1. Hypothalamo-hypophysial vasculariza- tion and its relationship with the neuro- secretory system in the catfish Clarias batrachus (L). Zool. Beitrage 17: 1-1 1. 197 1. Hypothalamo-hypophysial vasculariza- tion in a teleost fish with special reference to its tetrapodan features. Acta Anatom. ( In press).

98: 202-216.

Sathyanesan, A . G. , Haider, S . 1970a. Hypo- thalamo-neurohypophysial complex of the teleost Heteropneustes fossilis (BL) with some experimental evidence on the regener- ation of the neurosecretory tracts. Indian J . Exptl. Biol. 8: 174-178.

- 1970 b. The architectural pattern of the

30 Shamim Haider and A . G. Sathyanesan

neurosecretory system in the freshwater teleost Rita rita (Ham.) with a note on their post-hypophysectomy changes. Zeit . Mikr.-anat. Forsch. 82: 493-507.

- 1971 a. Presence of hypothalamo-hypophys- ial portal system in the teleost Heterop- neustes fossilis (BL). Experientia 27: 462- 463.

- 1971 b. Tetrapod-like features of the hypo- thalamo-hypophysial vascularization in the air breathing teleost Heteropneustes fossilis (BL). Gen. Comp. Endocrinol. 17 : 360- 370.

Scharrer, E., Scharrer, B . 1963. Neuroendo- crinology. Columbia University Press, New York.

Sterba, G . 1961. Fluoreszenzmikroskopische Untersuchungen iiber die Neurosekretion beim Bachneunauge (Larnpetra planeri Bloch). Z . Zellforsch. 65: 763-789.

Dr. A. G. Sathyanesan Surgical Research Laboratory College of Medical Sciences Banaras Hindu Universic- Varanasi-5, India