Z. Zellforsch. 95, 37--57 (1969)

Histology and Ultrastructure of the Neural Lobe of the Lizard, Klauberina riversiana

E. M. RODRIGUEZ* and J. LA POFNTE**

Department of Pharmacology, University of Bristol, England

Received August 30, 1968

Summary. The pars nervosa of Klauberina riversiana belongs to a primitive tetrapod type which is characterized by the deep penetration of the infundibular recess, a thin-walled structure, and the virtual absence of pituicytes. The differential response of this gland to aldehyde fuchsin and periodic acid Schiff suggests the presence of two types of neurosecretory nerve endings. Ultrastructurally four kinds of nerve endings are distinguishable. Type l, probably a cholinergie nerve ending, contains only small clear vesicles ca. 400 A in diameter. The relative abundance of cholinergic nerve endings in this pars nervosa may be related to the necessity of transporting hormone through the ependymal cell. Type II, containing granulated vesicles about 1,000/~ in diameter and probably aminergic, is very rare. The two remaining types apparently secrete neurohypophysial hormones. They are Type III, containing dense granules ca. 1,500 A in diameter and Type IV containing pale granules ca. 1,500/~ in diameter. Evidence is reviewed which suggests that Type III nerve endings may secrete arginine vasotocin while Type IV endings may secrete (an)other hormone(s).

All these axons end only on the ependymal cells, the vascular processes of which form a continuous cuff over the basement membranes of the blood vessels. Hence the ependymal cells link the cerebrospinal fluid, the nerve endings and the blood vessels. Particles resolvable with the electron microscope are traced through a possible transport pathway from the granules, through the ependymal cells to the basement membrane. It is suggested that pituicytes replace ependymal cells and assume their transport functions in animals with massive neural lobes containing large numbers of nerve endings and blood vessels.

Introduction

Morphologically the pars nervosa of the Xantus i id lizard, Klauberina river- siana, belongs to a type tha t is commonly found in lower tetrapods (W~GSTRAND, 1966; SAINT G1-RONS, 1961). Deep pene t ra t ion of the in fund ibu la r recess into the gland results in a sac-like structure, t ha t is variously lobed in different species. The walls of the gland are th in and simple in structure. Pi tuicytes ma y be rare or absent as has been reported for the agamid lizard Calotes (NAYAR and PAHDALA~, 1964), the scincid lizard Tiliqua (GRIFFITItS, 1938) and the rhyncocephal ian, Sphenodon puctatum (WYETH and Row, 1923). Since little is known of the s t ructural details of this type of pars nervosa, its his tochemistry and u l t ras t ruc ture were studied. The simple s t ructure of the pars nervosa of Klauberina riversiana allowed comparisons of observat ions on the light and electron microscope levels to be made with relative ease result ing in subs tant ia l evidence for the existence of

* Fellow of the Consejo Nacional de Investigaciones Cientlficas y T6cnicas de la Repdblica Argentina.

** This investigation was supported in part by a Public Health Service fellowship 1 FZ HD 32,949-01 REP from the national Institute of Child Health and Human Development.

The authors wish to thank Professor H. HELLER for his constant interest and constructive criticism.

38 E.M. RODRIGUEZ and J. LA POINTE:

more than one kind of neurosecretory axon in this organ. Also the in t imate

relationship of ependymal cells with nerve endings t h a t are associated with the

sac-like s t ructure of this pars nervosa has resulted in a par t icular ly interest ing

picture of the t ranspor t of mater ia l f rom the neurosecretory nerve endings.

Finally, a s tudy of the fine s t ructure of a pars nervosa which v i r tua l ly lacks

pi tuicytes has allowed us to generalize about the function of this cell type in ver tebra tes t ha t do possess it.

Material and Methods

Twenty female lizards of the species Klauberina riversiana, were collected from San Clemente Island, California, in January and were killed in the last week of February. While the animals were in captivity, ambient temperature ranged from 19 ~ C at night to 28 ~ C during the day. They were allowed to regulate their own body temperature at about 32 ~ C in a photo- thermal gradient provided by infrared lamps during daylight hours. They were fed once a day with larval meal worms, Tenebrio molitor, and water was provided ad libitum.

All the animals were killed by decapitation and the fixative was immediately injected into the cranial cavity through the perimedullar space opened by the decapitation. The brain was then exposed by raising the skull roof. By cutting the lateral regions of the cerebral hemispheres, both lateral ventricles were exposed, and more fixative was injected into the ventricular cavities. The whole brain, the pituitary, and the base of the skull were immersed in fresh fixative. The material for light microscopy was kept immersed in diluted Bouin's fixative (BA~ER, 1946) for 7 days, dehydrated and embedded in paraffin. The material for electron microscopy was kept in fixative for half an hour. Then the final pieces of tissue were dissected out, and re-immersed in fresh fixative to complete a period of 2 hr. of prefixation. As pre-fixative for electron microscopy a threefold mixture containing paraformaldehyde, glutaraldehyde and acrolein buffered to pH 7.5 with phosphate was used. The tissue was post fixed for two hours in phosphate-buffered 1% OsOa and embedded in araldite (GLAUERT and GLAUE~T, 1958).

For light microscopy saggital serial sections from the hypothalamo-hypophysial region were prepared. Alternate sections were stained with aldehyde fuchsin (AF) (GABE, 1953), periodic acid - - Schiff (PAS.) (P~,ARS~, 1960), PAS. --- Orange G (CoRnIER and HERLANT, 1957) and Mallory's trichrome stain.

For electron microscopy, ultrathin saggital sections of the neurointermediate lobe of the hypophysis were cut in a Porter-Blum ultramicrotome and were stained with a saturated solution of uranyl acetate in absolute methanol (2 rain) and with a lead citrate solution (VENABLE and COOGESHALL, 1965) for 1 or 2 rain. A Hitachi HU-11B electron microscope was used. Sections of approximately 1 ~z thickness were stained with toluidine blue-borax and used for orientation.

Results

Gross Anatomy. The pars nervosa of Klauberina riversiana is a simple, sl ightly assymetrical outpocket ing of the infundibular recess. The walls of the gland are

approx imate ly 40 ~ thick. I t is surrounded by the pars in termedia and a capil lary

bed is inserted between them (Fig. 1). Occasional capil lary loops ex tend into the substance of the pars nervosa. The wall of the pars nervosa is pene t ra ted by

openings. No communicat ions were observed between these openings and the

infundibular recess, nor are they lined with ependymal cells.

Histology. Three layers can be dist inguished in the neural lobe in mater ia l

s tained with toluidine blue, A F or P A S : The innermost layer consisting of the per ikarya of ependymal cells, a subependymal hilar region t h a t stains l ight ly wi th

Morphology of the Lizard Neural Lobe 39

Fig. 1. Median sagittal section through the hypophysial region of the lizard, Klauberina river- siana. I R infundibular recess; M E median eminence; N L neural lobe; HC hypophysial cleft; DL distal lobe. Anterior to the right. Ventricular perfusion with diluted Bouin's fluid in

Figs. 1 ~ . Masson's trichrome • 83

toluidine blue and PAS and strongly with AF and an outer palisade zone tha t stains darkly in all three stains (Figs. 2, 3 and 5).

The ependymal nuclei are ovoid and irregular. They stain darkly with basic stains (toluidine and Masson trichrome). No axons were observed to cross the ependymal layer. Basophilic processes of the ependymal cells traverse the hilar and palisade layers to end on the capillary walls (Fig. 4).

The hilar region is composed of nerve fibres which are AF positive and PAS negative. The palisade region is composed of axon endings most of which appear to be AF positive while only par t of them are PAS positive (Fig. 2 and 3). Pitui- cytes are rare and when they appear they are indistinguishable from the ependymal cells except for their position within the hilar or palisade regions (Figs. 1 and 4).

Ultrastructure. The ependymal cells contain large dense nuclei (Fig. 6) with prominent nucleoli (Fig. 5). The mitochondria, rough and smooth endoplasmic reticulum, lysosomes, filaments, and Golgi membranes vary in amount from cell to cell. Extensive interdigitation of the cell membranes occurs where ependymal cells contact each other. Microvilli occur with variable density (Figs. 7 and 8); in suitable sections they are often very long. They contain filaments, and spines about 120 A in length appear on their surfaces. Similar spines appear on the cell membrane adiacent to microvilli and lining the pinocytotic vesicles. The pino- cytotic vesicles are abundant at the bases of microvilli. Dense granular material occurs in a layer under the apical cell membrane which is in contact with the cerebro-spinal fluid (CSF) (Fig. 8).

40 E.M. I~ODRIGUEZ and J. LA POINTE:

Fig. 2. Sagittal section through the neurointermediate lobe showing the location of Gomori positive material. I R infundibular recess; E ependyma; H R hilar region; P R pallisade region;

I L intermediate lobe; Aldehyde fuchsin. • 740 Fig. 3. Sagittal section adjacent to the section shown in Fig. 2 showing the location of PAS

positive material. Periodic acid Shift. • 740 Fig. 4. Sagittal section through the neurointermediate lobe showing the vascular processes (arrows) of the ependymal cells. I R infundibular recess; P "pituicyte" ; C capillary; I L inter-

mediate lobe; Masson's trichrome. • 1,000

Morphology of the Lizard Neural Lobe 41

Fig. 5. One ~z section through the neurointermediate lobe showing the locations of electron micrographs. The numbers of the squares refer to the respective Figs. 6, 10, 9 and 11. Note the holes (arrows) in the neural lobe. I R infundibular recess; H R hilar region; P R pallisade region;

C capillaries; I L intermediate lobe; HC hypophysial cleft. Toluidine blue. x 550

42 E.M. RODRIGUEZ and J. LA POI~TE :

Fig. 6. Electron micrograph of ependymal perikaryon and adjacent hilar region. See Fig. 5 for orientation. I I , I I I and I V refer to three kinds of nerve endings described in the text. Arrows indicate aggregations of small vesicles in the region of the nerve endings in contact with

the ependymal ce]l. I R infundibular recess; L Lysosome; NF nerve fibres. • 15,000

The vascular processes of the ependyma l cells t raverse the ent ire th ickness of the wall of the pars nervosa to end on the enveloping basement membrane . These processes produce small angular branches in bo th the hi lar and pal isade regions and large d ichotomous branches in the pal isade region only. The large d ichotomous branches reuni te on the basement membrane to form vascula r feet (Fig. 11). They cover the ent ire basement m e m b r a n e as far as has been observed.

Where the branches of ad j acen t cells meet on the basement m e m b r a n e thei r cell membranes in te rd ig i t a t e ex tens ive ly (Fig. 17). The vascu la r processes and feet conta in a b u n d a n t smooth endoplasmic re t iculum, microtubules , f i l aments and

Morphology of the Lizard Neural Lobe 43

Fig. 7. Electron micrograph of the apical region of an ependymal cell. Arrows indicate pino- cytotic vesicles at the bases of the microvitli, x 27,000

Fig. 8. A section through the apical region of an ependymal cell where the microvilti are scarce, compared to tile region shown in Fig. 7. Arrows indicate pinocytotic vesicle formation. Tile bracket marks a region of denser cytoplasm Iaking organeIles. M mit~chondrion, x 50,000

small particles 50 ,k in diameter can be observed in the smooth endoplasmic reticulum. The mitochondria are large and dense (Figs. 12, 13 and 17). Occasional rough endoplasmic ret iculum and lysosomes occur in the processes. There is occasional evidence of phagoeytosis in the form of large bodies containing inclusions resembling granules from neurosecretory axons, multilamellate bodies, and large lipid droplets. Extensive processes of the basement membrane penetrate the vascular feet (Figs. 11 and 17). Smooth endoplasmic ret iculum and vesicles can be observed opening onto these processes (Fig. 17). Pinocytot ic vesicles with a dense particle content appear on the ependymal cell membrane (Figs. 17 and 18). They are more common in the region of the vascular feet and some are coated (Fig. 19).

44 E .M. I~ODRIO:UEZ and J. LA 1)OII~TE : Morphology of the Lizard Neural Lobe

Fig. 9

Fig. 10. Section through the pallisade region similar to that shown in rectangle 10 in Fig. 5. I l I and I V mark two types of nerve swellings. Note that the small vesicles indicated by the

arrows occur only in the vicinity of ependymal processes (E). • 18,000

Fig. 9. A section through a port.ion of the hitar region similar to that shown in the rectangle in Fig. 5. 1, 2 and 3 mark three types of nerve fibres described in the text. I , l I I and I F mark different types of nerve endings also described in the text. Note the aggregations of small vesicles (8F) in the regions of the nerve endings in ~x)ntact with the ependymal celt. Note the presence of vacuoles (F) in the nerve endings of both TbTes I I I and tV. The broken arrow indicates a lateral branch of the ependymal vascular process. M mitochondrion; N nucleus of

the ependymal cell; • 20,000

46 E.M. RODRIGUEZ and J. LA POI•TE:

Fig. 11. Vascular processes of ependymal cells (E). The region corresponds to rectangle 11 of Fig. 5. Lateral processes and dichotomous branches are indicated by solid arrows. Note tha t the dichotomous branches reunite to form a vascular cuff. Broken arrows indicate processes of the basement membrane (BM). I, 1 i i and IV indicate three types of nerve endings. The broken line marks the limits of an aggregation of small vesicles against the ependymal process.

N nucleus, x 10,500

]~Iorphology of the Lizard Neural Lobe 47

Fig. 12. Nerve endings (Types I, H I and IV) on the vascular process of the ependymal cell in the hflar region. M mitochondria; M T microtnbules; F filaments; SR smooth endoplasmic reticulum; LP lateral process of t, he ependymal vascular process; S V small vesicles, x 24,000

48 E.M. RODRIGUEZ and J. LA POINTE:

Fig. 13. Cross section of the ependymal vascular process showing smooth endoplasmic reticulum (SR) in a radiating pattern and filaments (F). I I I and IV denote two types of nerve swellings. Where the swelling of the nerve fibre in the lower right-hand corner of the photograph contacts the ependymal vascular process there is an aggregation of small

vesicles (marked by the broken line). G neurosecretory granules; M mitochondrion; M T microtubules. • 47,000

Fig. 14. Contact of type I nerve endings with an ependymal process. The broken line delimits a peripheral aggregation of small vesicles (SV) in the contact region. Solid arrows indicate region of contact of small vesicles with the axon membrane. Broken arrows indicate the pres- ence of particles ca. 50 A in diameter inside the smooth endoplasmic reticulum (SR) of the ependymal process. V vacuoles; G neurosecretory granules; The area in the rectangle is

enlarged in Fig. 16. • 40,000

Fig. 15. Type I nerve ending. A connection of the neurosecretory granule (G) membrane with the smooth endoplasmic reticulum (arrow). V vacuoles; E ependymal process. • 120,000

Fig. 16. Enlargement of the area in the rectangle in Fig. 14. Solid arrows indicate particles ca. 50 A in diameter. The broken line delimits an aggregation of these particles within and between cell membranes in a region of contact between a Type I nerve ending (C) and an ependymal process (E). Note the absence of 50 A particles in the intercellular space between

nerve endings (broken arrow). • 172,000

Three k inds of n e r v e f ibres cou ld be d i s t i ngu i shed in t h e pars n e r v o s a of

Klauber ina b y the i r c o n t e n t s : 1) con ta ins dense granules , 1500 A in d i a m e t e r , in a d d i t i o n to mic ro tubu le s , f i l a m e n t s a n d m i t o c h o n d r i a . 2) con ta ins pa le g ranu les

Morphology of the Lizard Neural Lobe 49

4 z. Zellforsch., Bd. 95

Figs. 14--16

50 E . M . RODRIGVEZ and J . LA POn~TE :

Figs. 17- -19

Morphology of the Lizard Neural Lobe 51

1500 A in diameter in addit ion to mierotubules, filaments and mitochondria. 3) contains the same organelles but no granules (Fig. 9).

Nerve endings are distinguishable from nerve fibres by their larger diameter, by the absence of microtubules and filaments and by the presence of small vesicles 400 A in diameter. Four kinds can be distinguished : Nerve ending Type I contains only small vesicles and Type I I , which is very rare, contains granulated vesicles about 1000 A in diameter as well as the small vesicles. The two remaining nerve endings contain granules about 1500 A in diameter. Type I I I contains dense granules and Type IV contains pale granules (Figs. 9 and 12). Nerve endings of Type I I I were at least twice as abundan t as Type IV endings (Fig. 10).

Nerve endings occur only in contact with the cpendymal cells. There are few nerve endings and m a n y fibres in the hilar region where the vascular processes of the ependymal cells traverse the wall of the gland without extensive branching. However, the nerve endings become much more abundan t and the fibres rare in the palisade region in conjunction with the extensive dichotomous branching of the vascular processes. Hence the hilar region can be distinguished from the palisade region by the relative content of nerve fibres and nerve endings (compare Figs. 9 and 10).

Within the nerve endings the limiting membranes of pale or dense granules which are continuous with tubular structures resembling smooth endoplasmic ret iculum can occasionally be observed (Fig. 15). These tubules contain fine particles about 50 A in diameter. Large irregular vesicles of variable content occasionally appear in the nerve ending (Fig. 14 and 15). Small vesicles are abun- dan t in the region of the nerve ending in contact with the ependymal cell (Figs. 9 and 14). Fine particles, about 50 A in diameter frequently appear near thecell membranes, both within and between cells, where contact occurs between nerve endings and ependymal cells (Figs. 16 and 17). Similar particles are also observed within pinocytot ic vesicles of the ependymal cells. These vesicles are seen free in the cytoplasm and contact ing the cell membrane facing the nerve endings and the basement membrane (Figs. 17, 18 and 19).

Fig. 17. Contact of the vascular cuff of the ependymal cell (E) with the basement membrane (BM). Solid arrows indicate branches of the ependymal vascular process. Note that the dichotomous branches reunite to form a vascular cuff. Broken arrows indicate dense regions with aggregations of intercellular particles. The broken line delimits an aggregation of small vesicles in a Type I nerve ending (I). 1V, Type IV nerve endings; PV pinocytotic vesicles in the epcndymal cuff; BM basement membrane; B M P and arrows, processes of the basement membrane into the ependymal cuff; smooth endoplasmic reticulum (SR) can be seen opening onto the processes of the basement membrane. M mitochondria ; Y vacuole; L1 lateral infold- ing at junction between ependymal cells, x 36,000. Insert: Pinocytotic vesicles (PV) in the ependymal cuff (E) opening toward the nerve ending (NE) and toward a process of the base-

ment membrane (BMP). x 40,000

Fig. 18. Type I and I I I nerve endings in contact with the ependymal cuff. Arrows indicate aggregations of intercellular material. PV pinocytotie vesicles; BM basement membrane.

• 70,000

Fig. 19. A coated pinoeytotic vesicle (PV) in the ependymal cuff where it is contacted by a nerve ending (NE). SR, smooth endoplasmie reticulum; M mitochondrion; BM basement

membrane. • 82,000

4*

52 E .M. RODRIGUEZ and J. LA POINTE :

D i s c u s s i o n

A. Type o / A xons and their Possible Functional Signi/ieance

Histochemically, there are at least two kinds of neurosecretory endings in the pars nervosa, since more endings stain with AF than stain with PAS.

Four kinds of nerve endings could be distinguished with the electron microscope. I t is probable that the endings containing only small vesicles (Type I) and the endings containing small granulated vesicles (Type I I ) are the terminations of the fibres which do not contain granules. Type I axons are probably cholinergic and Type I I axons are probably aminergie (LEDERIS, 1967). LEDERIS further proposed that cholinergic and aminergic nerve endings in the pars nervosa may play a role in controling blood flow through tha t organ. I t is interesting to note that both the cholinergie and aminergic endings are rare in the mammalian (LEDERIS, 1967) and the toad pars nervosa (RODRIGUEZ, in preparation). The abundance of possibly cholinergie endings in Klauberina and in Zonotrichia (BERN et al., 1966) where neurosecretory material must cross the ependymal cell suggests tha t acetylcholine may have an important function in the transport of neurosecretory material across this barrier.

One of us (E. M. R.) has applied the same electron microscope technique to the toad neural lobe as used in the present paper and he has found tha t the toad neural lobe also has two kinds of granulated fibres, one of them corresponds to Type I I I of the lizard, but the endings containing pale granules (Type IV) are missing in the toad neural lobe. These facts seem to indicate that the pale granules of the lizard neural lobe are not artifacts, since the same technique does not reveal them in the toad neural lobe.

The nerve endings of Types I I I and IV probably represent different kinds of axons. I t seems unlikely that the pale and dense granules represent different physiological states of one kind of granule for the following reasons: 1) the two kinds of granules are always found in separate nerve endings or fibres and no intermediate stages between the two kinds of granules have been observed. 2) Both kinds of granules occur in the nerve fibres far from the nerve endings where physiological changes are more likely to occur. 3) The pale granules are not to be confused with "empty granules". E m p t y granules lacking electron dense material appear in both kinds of axons in the regions of contact with epcndymal cells. Another possibility cannot be ruled out : The pale granules could be ones that have simultaneously lost part of their electron dense material in each axon where they occur in response to a strong stimulus as seems to be the case in lactating rats after suckling (MonROE and SCOTT, 1966). However, we think this is unlikely in the present case in light of the arguments above and considering the fact tha t we have studied the animals under normal conditions. Morever, some authors have shown that after certain stimuli it is possible to observe transitional forms from dense to empty granules but within the same nerve ending (GERSHENFELD et al., 1960; LED]~RIS, 1964).

Since aminergic and cholinergic endings elsewhere in the central nervous system do not stain with AF or PAS, it is unlikely that they stained with these reagents in the pars nervosa. The axons that did stain were probably the ones containing the large granules. The two kinds of axons which were distinguished histoehemi-

Morphology of the Lizard Neural Lobe 53

cally may correspond to the two types of granulated axons distinguished with the electron microscope. However, we are unable to say at present which type of axon is PAS negative or which is PAS positive. The presence of more than one type of neurosecretory fibre in the neural lobe has been postulated in other species.

HOWE (1959) stained serial sections of the rat neural lobe in AF and a specific stain for arginine. Only some of the Gomori positive nerve endings stained with the arginine stain, leading him to speculate that oxytocin and arginine vasopressin occur in separate nerve endings. HELLER (1961) has postulated that different neurohypophysial hormones are produced and secreted by different kinds of neurons. SOKOL and VALTIN'S (1967) observation of the absence of stainable material from definite areas of the neurohypophysis of rats with congenital diabetes insipidus supports Heller's hypothesis. The concept of the association of different neurohypophysial hormones with different nerve endings or different granules receives further support from the studies of LED]~RIS (1962; 1964), KAUZ and LI~DERIS (1965), CAMPBELL and HOLMES (1966) and ZAMBRANO and DE Ro- BERTm (1968).

Snakes are thought to secrete both arginine vasotocin and oxytocin or meso- tocin (MuNSICK, 1966 ; PICKERING, 1967). Since lizards are closely related to snakes it seems reasonable to suppose that they also secrete at least two neurohypo- physial hormones. These observations lead us to consider the possibility tha t the two kinds of neurosecretory axons in the neurohypophysis of Klauberina may secrete different hormones. The fact tha t Type I I I nerve endings are about twice as abundant as Type IV endings provides the basis for a tentative identification of this type of ending. HELLER and PICKERING (1961) and FOLLETT (1967) have shown that the pressor/oxytocic value of neural lobes of the grass-snake, tortoise and green turtle is about 0.7. This corresponds to a molar ratio of about 2 for arginine vasotocin (AVT)/oxytocin or mesotocin. The dense granules might therefore contain AVT. The pale granules might contain oxytocin or mesotocin or both hormones. However, in view of the possibilities of species differences and poly- morphism within a species (FERcUSON and HELLER, 1965) it would be best to reserve judgment with respect to these latter principles. One is probably on safer ground with respect to the ubiquitous AVT.

B. Release Pathway

The relationships between the nerve endings, ependymal cells, and the basement membrane in the neurohypophysis of Klauberina provides interesting evidence on the hormone release mechanism in this species. All the axons of the neuro- hypophysis appear to end on all parts of the ependymal cells within the hilar and palisade regions. Because of the relationship between the nerve endings and the surface of the ependymal cells, the nerve endings are much more abundant in the palisade region where the vascular processes of the ependymal cells undergo extensive dichotomous branching. This basic and probably functional relationship resulting in increased density of nerve endings in the palisade region explains the stronger response of this region to staining with AF and PAS. The formation of a "cuff" by the vascular processes of the ependymal cells which entirely covers the basement membrane, and the fact tha t axons terminate on the ependymal cells,

54 E.M. RODRIGUEZ and J. LA POINTE:

indicate tha t neurosecretory material must be transported through the ependymal cells before it can reach the basement membrane and thence the capillaries. Apparently a similar "cuff" is formed by ependymal cells and pituicytes in the white-crowned sparrow (BERN et al., 1966).

In many other species, ncurosecretory endings of the pars nervosa end directly on the basement membrane. The interposition of the ependymal cells between the nerve endings and the basement membrane of Klauberina offers an interesting situation in which it appears to be possible to trace the fate of neurosecretory material beyond the nerve ending. Our observations suggest two possibilities for the release of neurosecretory material from the granules: l) by diffusion through the granule membrane, or 2) by release of material into tubular structures. The appearance of granules which seem to be in all stages of depletion in the vicinity of the synapse suggests that the neurosecretory material is diffusing into the axo- plasm. The appearance of free particles in the axoplasm similar to those observed within the granules tends to support this interpretation. On the other hand, the connections between neurosecretory granules and the tubular structures, and the appearance of particles in the lumen of these tubular formations suggest that these particles may be neurosccretory material being transported from the granules by the tubules. Observations of the connection of granules with the tubular structures are rare. Nevertheless, such a mechanism could account for the release of all or most of the hormonal material from the neurosecretory granules on the following grounds: l) the association of the granules with tubules may be much more frequent than is observable since the probabili ty tha t a section through a given granule of 1500 A will show a connection of about 400 Jk in dia- meter is low. 2) the ratio of granules acting as storage sites for hormone to those involved in active release in the normal animal is very high. Since we have observed no connections of the tubular formations with the axon membrane, it is possible that the particles are released into the axoplasm before crossing the axon mem- brane. The presence of neurosecrctory material within the tubular structures may account for GINSBURO and IRELAND'S (1966) and KAUZ and LEDERIS' (1965) evidence for a second hormone-containing compartment in the nerve endings other than the neurosecretory granules.

The distribution of particles near the cell membranes and in the intercellular space where the nerve endings contact the ependymal cells suggests t ransport of the particles between the two kinds of cells. This interpretation is further strength- ened by the appearance in this contact region of pinocytotic vesicles and smooth endoplasmic reticulum containing similar particles in the ependymal cells. Both structures can be seen opening onto processes of the basement membrane, complet- ing a possible transport pathway from the neurosecretory granules (Fig. 21). Transport in the cuff region appears to be mainly be means of pinocytotic vesicles. On the other hand, the smooth endoplasmic reticulum extends throughout the ependymal cell and probably accounts for transport from regions more distant from the cuff.

C. Linking Function o/the Ependyma Structurally the ependymal cell forms a common link between the cerebro-

spinal fluid (CSF), the nerve endings of the pars nervosa and the blood vascular system. Specialization of the ependymal cell for absorption of material from the

Morphology of the Lizard Neural Lobe 55

Fig. 20. Diagramatic section through the wall of the pars nervosa showing an ependymal cell and the spatial relationships of primary components to it. The break in the ependyma] process indicates that it has been foreshortened in the diagram for purposes of clarity. I R infundibular recess; M V microvilli; P V pinocytotic vesicles; D desmosome; G Golgi apparatus; SR smooth endoplasmic reticulum; L lysosome; M T microtubules; F filaments; BM basement membrane; 1, 2 and 3, three kinds of nerve fibres; I, I I , I I I and IV, different kinds of nerve endings

(see text for description) Fig. 21. Diagram of proposed pathway for release of neurosecretory material from the nerve ending (NE) through the ependymal cuff (E) to the basement membrane (BM). Fifty A particles (represented by solid dotts) are shown being released from neurosecretory granules (NG) into either the smooth endoplasmic reticulum (SR) or into the axoplasm. The 50 A particles congregate in the synaptic region of the nerve ending and in the adjacent inter- cellular space suggesting that they are released from the nerve ending. They appear to be transported across the ependymal cuff by means of pinocytotic vesicles (PV) and the smooth endoplasmic reticulum which in turn opens onto the basement membrane. M mitochondria;

S V small vesicles; F filaments; B M P process of the basement membrane

CSF opens up the possibility of a funct ional relationship between the CSF and the nerve endings. The occurrence of neurohyohphysia l hormone in the CSF of other ver tebrate species is of interest in this regard (HELLER et al., 1968).

56 E.M. RODRIGUEZ and J. LA POINTE:

The absence of definitive pi tuicytes in the neural lobe of Klauberina riversiana is suggestive ~4th regard to the funct ion of these cells in animals which possess them. The ependymal cells in Ktauberina evidently p lay a role in the t ranspor t and release of neurosecretory material. I n animals with more massive neural lobes, e.g. toads and frogs, some birds and reptiles, and mammals , the ependymal cells differentiate into pituicytes (WI~GSTRAND, 1966). One reason for this could be the replacement of ependymal cells with pituicytes where the surface area exposed to eerebrospinal fluid limits the number of ependymal cells, and the number of nerve fibres and blood vessels is increased enormously in proportion. The pituieytes in massive neural lobes m a y be involved in t ranspor t and release of neurosecretory hormones as are the ependymal cells in thin neural lobes. The int imate association of pi tuicytes with nerve endings and blood vessels ( R n ~ s and DgAGsl~, 1955; W~TT~OWS~d, 1967 ; 1968), and the proliferation of pituicytes in s t imulated glands (S~Lu and HAnL, 1943; CHAmBeRS, 1945; RS~v~LS, 1958; D~CH~N, 1962) are facts which are both consistent with this hypothesis.

Relerenees

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Dr. E. M. I:~ODRIGUEZ Dept. of Pharmacology University of Bristol Medical School Bristol, BS 8, 1TD, England

Dr. J. LAPOINTE Department of Biology New Mexico State University Box 3 AT, La~ Cruces, New Mexico 88001 USA