Fetal hypothalamic transplants: Neuronal and neurovascular interrelationships

Neuroscience Letters, 51 (1984)93-98

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Elsevier Scientific Publishers Ireland Ltd. NSL 02965

FETAL HYPOTHALAMIC TRANSPLANTS: NEURONAL AND NEUROVASCULAR INTERRELATIONSHIPS

DAVID E. SCOTT Department o f Anatomy, University o f Missouri - Columbia, School o f Medicine, Columbia, MO 65212 (U.S.A.)

(Received June 15th, 1984; Accepted July 17th, 1984)

Key words: brain transplantation - neuropeptides - neurosecretion - plasticity - hypothalamus

This investigation is one of a series aimed at developing an understanding of the neuroanatomical correlates of neuronal and vascular interactions that occur between heterografts of normal fetal hypothalamic tissue transplanted into the third cerebral ventricle of adult Brattleboro rats with homozygous autosomal diabetes insipidus (DI). Rapid vascular invasion of fetal neurografts occurred within 3 days and arose from the mantle plexus of portal capillaries in the underlying host median eminence. Host vessels also invaded from the periventricular stratum and preoptic area. In the ventral regions of heterografts neurites were observed to terminate upon perivascular spaces that surrounded local capillaries. Despite the development of apparently normal neurovascular zones, none of the DI host recipients demonstrated an amelioration of polydipsia or polyuria characteristic of diabeties insipidus.

T h e o r g a n i z a t i o n o f the e n d o c r i n e laypothalamus a n d a d j a c e n t m e d i a n e m i n e n c e has been the subject o f intense s c r u t i n y over the last q u a r t e r century. With the advent of sophisticated i m m u n o c y t o c h e m i c a l techniques, a r e m a r k a b l e range o f n e u r o p e p t i d e h o r m o n e s have been synthesized, characterized a n d u l t i m a t e l y localized within the m e d i a n e m i n e n c e a n d other c i r c u m v e n t r i c u l a r organs as well [11]. Despite intensive p r o g r a m s o f research designed to characterize the biochemical n e u r o a n a t o m y o f these n e u r o e n d o c r i n e transducers, relatively little is k n o w n a b o u t the n e u r o n a l a n d n e u r o v a s c u l a r i n t e r a c t i o n s that transpire b e t w e e n host h y p o t h a l a m i c tissues a n d f r a g m e n t s o f fetal h y p o t h a l a m u s a n d cortex which are stereotaxically i n t r o d u c e d into the third cerebral ventricles o f host rats. The present i n v e s t i g a t i o n is o n e o f a series f r o m this l a b o r a t o r y directed t o w a r d a n assessment o f these i n t e r r e l a t i o n s h i p s a n d the u n d e r l y i n g m e c h a n i s m s o f n e o v a s c u l a r i z a t i o n , a x o n a l targeting a n d synaptogenesis that m a y transpire between cellular elements o f the host a n d those o f fetal h y p o t h a l a m i c n e u r o g r a f t s . M a t u r e male B r a t t l e b o r o rats with chronic a u t o s o m a l h o m o z y g o u s diabetes insipidus were e m p l o y e d as recipient hosts. T i m e d , p r e g n a n t , L o n g - E v a n s females were killed b y cervical dislocation a n d 17-day post-coitus fetuses were r e m o v e d a n d 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

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stored in sterile, ice-cold Eagles medium. Following the techniques of Gash and Sladek [4], the rostral portion of the anterior hypothalamus of each fetus was dissected away from the surrounding brain. Fragments of anterior hypothalamus and cortex were minced with ophthalamic blades and re-emersed in sterile Eagles medium. The fetal hypothalamic tissue and media was stereotaxically introduced via an 18-gauge spinal needle with an indwelling stylet into the third cerebral ventricle of host Brattleboro rats whose brains were ultimately prepared for correlative microangiography, immunocytochemistry and transmission electron microscopy following the techniques of Scott and Sherman [7] and Sherman and Paull [9]. Fetal cortical fragments were stereotaxically introduced as controls. Microangiography. By three days post-surgery, vascular varicosities were observ-

Fig. 1. Basomedial hypothalamus of Brattleboro rat killed 9 days following stereotaxic surgery and placement of normal hypothalamic fragment. Portal vessels (arrows) from the host median eminence (ME) can be observed to penetrate into the parenchyma of heterograft (G) which obliterates most of the ventricular lumen of the host (V). × 162.5.

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ed within the parenchyma of fetal hetereografts. The organization of vessels was incomplete and often marked by the accumulation of immunoreactive red blood cells and focal zones completely devoid of vascular invasion. Six days post-surgery one could note a significant increase in the capillary density of fetal explants. It was also evident that a remarkable degree of vascular input was derived from branches of the mantel plexus of hypophyseal portal vessels that grew dorsally from the underlying median eminence of the host. These were observed to vertically penetrate hypothalamic explants from the underlying median eminence and commonly arborized into smaller secondary and tertiary vessels (Fig. 1). Other sources of vascular input into the fetal hypothalamic explants were derived from the lateral wall of the host periventricular stratum and paraventricular (magnocellular) bed nuclei (Fig. 2). Like the primary vessels of the median eminence, these also arboriz-

Fig. 2. Photomicrograft of Brattleboro host that demonstrates multiple sources of blood vessels (arrows) arising from host median eminence (ME) and the periventricular stratum (PS) which are seen to invade the heterograft (G). V, lumen of third cerebral ventricle. × 227.5.

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Fig. 3. Transmission electron microgram of neurovascular zone in the ventral one-third of a hypothalamic heterograft in a Brattleboro host rat killed 60 days following stereotaxic surgery. A dominant feature is the presence of a fenestrated capillary (CAP) in the upper left quadrant of the field. Notable here are axon profiles (*) filled with dense core vesicles. These profiles are observed in close proximity to perivascular spaces (PS) that typically surround fenestrated vessels of these hypothalamic neurografts. C, collagen fibrils, x 10,080. ed

extensively

micrographs

into

secondary

of the ventral

and

one-third

tertiary

branches.

of the explant

Transmission

revealed

electron

the presence

of

f e n e s t r a t e d vessels w i t h d i s t i n c t p e r i v a s c u l a r s p a c e s ( F i g . 3). H o w e v e r , t h e m i d c e n -

97 tral and dorsal regions of fetal hetereografts did not exhibit fenestrated vessels and instead harbored capillaries that lacked fenestra and appeared more classical in their anatomical organization. Control rats having received fragments of fetal cortex at no time exhibited neurovascular (neurohemal) zones. In host rats killed 30 days post-surgically, fenestrated capillaries in the ventral one-third of hypothalamic explants demonstrated numerous axon profiles (neurites) that appeared to terminate upon the abluminal vasal lamina of perivascular spaces that surrounded these fenestrated vessels (Fig. 3). These neurites were loosely organized and appeared to harbor neurosecretory (dense core) vesicles of various diameters (Fig. 3). The population of neurites observed to terminate upon or near perivascular spaces of heterograft capillaries appeared relatively pure with few if any intervening glial or ependymal processes. No changes were observed in the physiological parameters of the water balance in recipient host rats which continued to demonstrate profound polyuria and polydypsia despite stereotaxic placement of normal fetal hypothalamic fragments. This is consistent with earlier published studies from this laboratory [1, 8]. The development of neurovascular (neurohemal) zones [5] in viable hypothalamic heterografts that flourish in the third cerebral ventricle of the host rats is predictable. Two reasons for their presence can be offered. The first is that they may arise from the fetal anlage of portal vessels that may have been introduced into the host ventricle during stereotaxic surgery [1]. The second and more plausible reason is, as can be seen from the data of the present investigation, that they simply arise from portal vessels of the host. This latter system invades the heterograft from the median eminence below and arborizes extensively within it. A question that may be difficult to answer is whether there is a fusion of intrinsic vascular anlage of the fetal explant with extrinsic vessels derived from the host. Despite the presence of bonafide neurovascular (neurohemal) zones coupled with neurophysin positive magocelluler neurons and arginine vasopressin positive afferent fields seen in the fetal explants in earlier studies from this and other laboratories [2, 4, 8], no evidence of a functional return of water balance in recipient Brattleboro host rats was noted in this investigation. The molecular basis for the autosomal homozygous diabetes insipidus characteristic of the Brattleboro strain of rat has recently been described by Schmale and Reichter [6]. Their elegant biochemical observations support the notion that the C-terminus of the neurophysin molecule is significantly altered and that a number o f cysteine residues, originally present in normal neurophysin, are in effect replaced by other amino acid residues in the mutant form. Their thesis supports the view that this replacement has a significant effect upon the stereochemistry of the mutant neurophysin. They have further convincingly suggested that the C-terminus of the mutant neurophysin is altered to such a degree that it no longer can protect the hormone (arginine vasopressin (AVP)) from enzymatic degradation. In theory, the surgical

98 i n t r o d u c t i o n o f h ea lt h y fetal h y p o t h a l a m i c f r a g m e n t s h a r b o r i n g the anlage o f n o r ma l vasopressinergic s u p r a o p t i c p a r a v e n t r i c u l a r and s u p r a c h i a s m a t i c n e u r o n s ( a m o n g o t h er elements) should be able to c o m p e n s a t e for this c o n g e n i t a l deficit. T h e failure to re-establish n o r m a l w a t e r c o n s u m p t i o n values an d urine o s m o l a r i t y in the present i n v es t i g at i o n m a y bear u p o n o th e r f a c t o r s which m a y d e p e n d u p o n the critical

mass

of

vasopressin-producing

neurons

that

survive

within

fetal

h e t e r o g r a f t s . Clearly the anlage o f o t h e r cellular elements such as glia, e p e n d y m a a n d n o n - p e p t i d e r g i c parivicellular n e u r o n s , are i n c o r p o r a t e d within these explants as well. These latter anlage m a y c o m p e t e with v a s o p r e s s i n - p r o d u c i n g n e u r o n s f o r the local d e v e l o p i n g b l o o d supply. F u r t h e r , p o p u l a t i o n densities o f actively g r o w i n g axons, dendrites and gliat processes m a y serve to inhibit the g r o w t h and d e v e l o p m e n t o f necessary n u m b e r s o f m a g n o c e l l u l a r axons and neurites in c o n j u n c t i o n with adequate numbers

o f f e n e s t r a t e d vessels to achieve the o p t i m u m

number

of

n e u r o v a s c u l a r zones for the t r a n s f e r ( t r a n s d u c t i o n ) o f A V P into the systemic circ ul at i o n . Serial analysis and cell counts are n o w u n d e r w a y to q u a n t i f y the relative p o p u l a t i o n s o f m a g n o c e l l u l a r ( A V P - p r o d u c i n g ) n e u r o n s to help resolve an d conf r o n t these intriguing and e n i g m a t i c questions. This study was s u p p o r t e d by U S P H S G r a n t 19197. 1 Fink, G. and Smith, G.C., Ultrastructural features of the prenatal development of the hypothalamicpituitary axis in the rat, J. Anat., 108 (1977) 207. 2 Gash, D.M. and Scott, D.E., Fetal hypothalamic transplants in the third ventricle of the adult rat brain, Cell Tiss. Res., 211 (1980) 191+206. 3 Gash, D.M., Sladek, C.D. and Sladek, Jr., J.R., A model system for analyzing functional development of transplanted peptidergic neurons, Peptides, Suppl. 1 (1980) 125-134. 4 Gash, D.M. and Sladek, Jr., J.R., Vasopressin neurons grafted into Brattleboro rats: viability and activity, Peptides, Suppl. 1 (1980) 11-14. 5 H6fer, H., Zur Morphologie der circumventricularem Ongane des Zwischenhirms der Saugetiere, Vorh. Dtch. Zool. Ges. Frankfort/M8 (1958) 202-251. 6 Schmale, H. and Richter, D., Single base deletion in the vasopressive gene is the cause of diabetes insipidus in Brattleboro rats, Nature (Lond.), 308 (1984) 705-709. 7 Scott, D.E. and Sherman, D.M., Neuronal and neurovascular integration following transplantations of the fetal hypothalamus into the third cerebral ventricle of adult Brattleboro rats. Neurological transplants. 1., Brain Res. Bull., in press. 8 Scott, D.E., Sherman, D.M., Gibbs, F.P., Paull, W.K. and Gash, D.M., The neuroanatomical and neurovascular organization of nomial fetal hypothalamic explants in the third cerebral ventricle of Brattleboro rats with homozygous diabetes insipidus. In W.K. Paull, D.E. Scott and T.A. Mitchell (Eds.), Brain-Endocrine Interaction, V, W.K. Peptides l, Ankho, Fayetleville, NY, U.S.A., 1984, pp. 169-183. 9 Sherman, D.M. and Paull, W.K., A new method for the visualization of vascular networks in nonperfused fixed specimen material, Stain. Tech., in press. 10 Sladek, Jr., J.R., Scholer, J., Notter, M.D. and Gash, D.M., lmmunohistochemical analysis of vasopressive neurons transplanted into the Brattleboro rat. In H+ Sokol and H+ Valtin (Eds.), Brattleboro Rat, N.Y. Ann. Acad. Sci., 1983, pp. 102-114+ 11 Weindl, A., Neuroendocrine aspects of circumventricular organs. In W+F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Oxford University Press, 1973, pp. 3-22.