Cell Biology International Reports, Vol. 14, No. 2, February 1990 143

FACTORS AFFECTING NEWRITE OUTGROWTEI OF OCCIPITAL

CORTICAL EXPLANTS -

Chau , R.H.W. ,’ Zhao, L-P.,’ Jen L-S.’ and Chan, S.O,‘*

Department of Anatomy, University of Hong Kong, 5, Sassoon Road, Hong Kong and The Chinese University of

Hong Kong, Hong Kong.

Running title: Neurite qutgrowth of cortical explants.

ABSTRACT

The effects of various substrata including laminin, collagen gel, collagen I, and human amniotic basement membrane on neurite outgrowth of occipital cortical and diencephalic explants were studied. The results showed that the extent and pattern of growing neurites of cortical explants varied considerably depending on the substrata used. While an elaborated network of growing neurites was observed when cortical explants were plated on laminin, the most extensive neurite outgrowth was observed when collagen gel was used as the substratum. In contrast, diencephalic explants did not grow on most of the substrata. The significance of the findings are discussed.

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"To whom correspondence should be addressed in Hong Kong.

S.O. Ghan's present address is Department of Human Anatomy, University of Oxford, Oxford, OX1 3QX, U.K.

0309-1651/90/020143-1 l/$03.00/0 @ 1990 Academic Press Ltd.

Cell Biology International Reports, Vol. 74, No. 2, February 1990

INTRODUCTION

Much attention has been given to the structural and functional organization of the visual cortical neurons under normal and abnormal conditions in the past fifteen years (Hubel and Wiesel, 1974. Jen et al, 1978. Jaeger and Lund, 1980. Rothblat and Hayes, 1982. Diao et al, 1983. Jen and Zeki, 1983. Jen et al, 1984. Stanfield and O’Leary, 1985. Chang et al, 1986. Florence and Casagrande, 1987. Jeffery and Parnavelas, 1987. Thong and Dreher, 1987. Craner et al, 1989). However, little is known as yet about factors which are involved in determining the pattern of neurite outgrowth of cortical neurons in developing visual cortex. The purpose of this study is to provide some information concerning this issue by comparing the extent and pattern of neurite outgrowth of occipital cortical explants which were grown on various substrata including laminin(LN) , collagen gel(CG) , collagen I (Co), and human amniotic basement membrane(HABM). For comparison, neurite outgrowth of diencephalic explants were also examined. Preliminary results of this study have already been reported as short communication (Ghan et al, 1988) .

Materials and Methods

Five litters of newborn Sprague-Dawley albino rats obtained from the animal unit of the Chinese University of Hong Kong were used in this study. The animals were deeply anaesthetized by ether and then decapitated. The dorsal surface of the occipital cortex and the part of the dorsal thalamus containing the lateral geniculate body were dissected out in MEM culture medium under dissecting microscope. The tissues collected were cut into small pieces of 1 mm X lmm in size before they were transferred to culture dishes previously coated with laminin, collagen I, collagen gel, or a piece of HABM. The HABM was obtained from human placenta which were kindly provided by the Department of Paediatric at the Chinese University of Hong Kong. Details of procedures of coating culture dishes and preparation of HABM had already been described (Liotta et al, 1980. Davis et al, 1987. Zhao and Chau, 1989). As indicated in table 1, one piece of cortical(C) or diencephalic(D) explant was

Table 1. Numbers of culture dishes containing explants of various combinations at 1 cm apart on different substrata. C, cortical explant; G, diencephalic explant; HABM, human amniotic basement membrane; LN, Laminin; CC, collagen gel; Co, collagen I.

Substratum\Explants c + c, C + G, C + C + HXBM, C + G + HABM

LN 8 8 8 8 CG 7 8 8 G co G 6 4 4 HABM 7 4

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co-cultured with another piece of cortical or diencephalic explant with or without a piece of HABM positioned about 1 cm apart from each other in an 1 in. size culture dish containing MEM supplemented with 10% fetal calf serum, Penicillin G/Streptomycin(lOO units/ml) and f ungizone (2.5 ug/ml) . A total of 92 culture dishes with explants were prepared(Table 11, and the outgrowth of neurites of all explants were examined from day 2 to day 8. The extent of the neuritic outgrowth of the explants was measured according to their density and length under the inverted microscope or from photomicrographs and expressed as the neuritic growth indices(NG1) which has been used by other investigators in previous studies (Landreth and Agranoff, 1976. Schwartz et al, 1982).

RESULTS

In general, neurites of cortical explants obtained from occipital region grew extensively on all four different substrata used in this study, but significant growth of diencephalic explants was observed only in cases where culture dishes were coated with LN. In those cases with postive growth of neurites, the neuritic outgrowth was noticeable at the end of day 1 although significant growth or extension of neurites did not occur until day 2. By the end of day 5, the overall pattern of neurite outgrowth in all cases had already been established. There was no significant changes in the gross pattern of the outgrowing neurites after day 5 despite the length of the neurites in some of the cases continued to increase. For this reason, NGIs of cortical explants were computed up to the end of day 5 for comparision and quantification.

As shown in figures 1 and 2, several different patterns of neurite outgrowth of cortical explants were observed in this study. In cortical explants which were seeded on LN, the outgrowing neuropils could be divided into an outer fibrous zone with smooth and distinct frontier and an inner cellular zone with many cells intercalated between the neurites (Fig. 1 A & B). The neurites were arranged both radially and circumferentially forming an elaborated network which was frequently led by a series of growth cones. This pattern of a network formed by interweaving neurites was noticeable on day 2 in culture and was basically established by day 4. No obvious changes in the pattern and degeneration of the neurites were observed until day 8.

Cortical explants cultured on CG-coated plates had a different pattern as compared to those described above. Extensive outgrowth of slender neurites radiated from the explant without a demarcated circumferential boundary of frontier (Fig. 1 C & D). Growth cones were frequently located at the free ends of the growing neurites, and small fascicles of neurites (Fig. 1 D) were commonly observed. Although a few circumferentially or obliquely arranged fibers were found in some of the cases examined, they did not form a network as that observed in LN cases. The neurites continued to grow or elongate until the end of the culture experiments, some extended for a distance as long as 5 or 6 mm away from the explants.

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Fig. 1. Photomicrographs showing growing neurites of cortical explants (c) which were plated on laminin (A & B), and collagen gel (C & D). Note the different patterns of neurite outgrowth of esplants which were grown on different substrata. Arrows indicate growth cones. Scale bars: 200 urn.

Cell Biology International Reports, Vol. 14, No. 2, February 1990

Fig. 2. Photomicrographs showing growing neurites of cortical (c) and diencephalic explants (g) on different substrata. (A) shows neurite outgrowth of a cortical explant plated on collagen I. (BI shows neurite outgrowth of a diencephalic esplant plated on laminin. (C) and (D) show neurite outgrowth of cort.ical explants placed on human amniotic basement membrane (h). Scale bars: 200 urn.

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Fig. 3. Photomicrographs showing cortica,l and diencephalic explants plated on various substrata. (A) and (B) illustrate interaction between cortical and cortical explants, and between cortical and diencephalic explants which were plated on collagen gel. (C) and (D) show diencephalic explants which were grown on laminin for 2 and -I days respectively. Scale bars: 200 urn.

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Extensive outgrowth of neurites was also observed in cortical explants seeded on Co, although the neurites appeared to be organized as bundles or distinct fascicles radiating outward from the explant and the neurites were preceded by a cellular zone (Fig. 2 A). Although an interweaving network of neuropils was detected in some of the cases examined, it was restricted to certain regions of the explants and is certainly not a common pattern as encountered in cortical explants cultured on LN or CG.

In cases where cortical explants were placed on HABM, the outgrowing neurites tended to group together to form distinct fascicles extending radially (Fig. 2 C & D), except that a cellular frontier was not clearly observed. Also, the neurites and fascicles were obviously much more robust and shorter in length in comparison with those grown on CG. The presence or absence of another piece of explant on HABM did not seem to change significantly the patterns observed.

By comparing the NGIs of cortical explants cultured on different substrata, it was found that cortical explants grew better on CG than other substrata such as LN (Fig. 4, A and B). In fact, the NGIs of cortical explants at day 3, day 4, and day 5 cultured on CG were about twice of those cultured on LN. However, one of the major factors contributing to the high NGI is the length of the outgrowing

4 Laminin B Collagen Gel

I

012345 012345012345

DAY IN CULTURE

Fig. 4 Graphs illustrating neuritic growth indices (NGIs) of cortical (0 explants plated on laminin (A), collagen gel (B), and human amniotic basement membrane (HABM) on collagen gel (Cl from day 2 to day 5 (X-X-X). The NGIs of explants in cultures containing an additional piece of HABM are indicated by triangle sign (A-A-A ). Note that the NGI of cortical explants plated on collagen gel is much higher than that of explants plated on laminin.

Cell Biology International Reports, Vol. 14, No. 2, February 1990

neurites from explants which was considerably longer on CG than that on other substrata. Some of the neurites growing on CG had reached 5 mm in length by the end of day 4, which were much longer than those growing on other substrata and still seemed to be elongating,

The patterns of neurite outgrowth of diencephaic explants were clearly different from those of cortical explants. In fact, neurite outgrowth was observed only in diencephalic explants which were plated on LN, and the pattern appeared to be similar to that of cortical explants grown on Co (Fig. 2 B). Neurite outgrowth was not observed in diencephalic explants plated on other substrata. Only cellular elements (Fig. 3 C and D), many of which appeared to be fihroblasts, were observed in the cultures.

While growing neurites of a cortical explant appeared to intermingle well with neurites of another piece of cortical explant (Fig. 3 A), no significant mixing of neuropils between cortical and diencephalic explants was detected (Fig. 3 B).

DISCUSSION

One of the most important findings in this study is that the growing neurites of cortical explants plated on laminin not only grow extensively but also form a very complex and elaborated network consisting of interweaving fibers running in different direction, a pattern similar to that observed in cortical tissues in vivo. The lack of cellular elements in the frontier of the expanding network of interweaving fibers also suggests that the substratum laminin is by itself sufficient to induce or promote specifically the extension of neurites of cortical explants, thus providing further support to the notion that the laminin is not only an adhesive substratum but also acts as a neuronotrophic factor for the growing neurons of explants from developing or mature nervous tissues (Edgar et al, 1984. Manthorpe et al, 1983).

While extensive neurite outgrowth has also been observed in cortical explants plated on CG, the pattern of the growing neurites was clearly different in that the neurites tend to be radially arranged, and in many cases grouped into small fascicles. The lack of an interweaving pattern implies that CG can not replace the relatively pure laminin with regard to the formation of a complex neural circuitry. However, the remarkable extent of neurite outgrowth of cortical explants on CG, judging by the length and NGI, indicates that CG is a better substratum than laminin as far as promoting extension or elongation of neurites is concerned.

The Co appears to be less effective substratum in promoting neurite outgrowth comparing with laminin and CG. The limited outgrowth of neurites of cortical explants and the presence of acellular frontier suggest that the neurites were not able to extend over long distance on the Co. Therefore, the Co which is a purified chemical substance does not seem to be a favorable substratum for the cortical explants as compared to the CG which is a mixture of chemical subst antes, although the neurons can probably be maintained on Co surface and their neurites do appear to persist for a long

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period of time. Another interesting finding in this study is that explants

obtained from the occipital cortex can be maintained and can extend neurites on HABM which is a naturally existing biological membrane. This is supportive to previous study using similar approach with the cholinergic neruons (Davis et al, 1987). Since it has also been reported that the HABM is rich in laminin which may not be evenly distributed on the membrane (Davis et la, 1985, 19871, the finding of neurite fascicles which apparently changed their directions abruptly is not totally surprising. In other words, the growing neurites may extend on laminin rich regions on the surface of the HABM, which is reflected by the irregularly organized pattern of the growing neurites.

In contrast, the relatively limited or lack of neurite outgrowth of diencephalic explants indicate that the diencephalic neurons, unlike the cortical explants, do not grow well in the culture system with the substrata provided in this study. Also, the presence of another piece of tissue such as a cortical explant does not seem to improve neurite outgrowth of the diencephalic explant in the co-culture system used in this study.

Taken together, results obtained in this study indicate that developing nervous tissues from different sources interact with certain substrata in different manners in culture system, and the same type of tissues may express different behaviours depending on the substrata they were plated on. These suggest strongly that the pattern and extent of neuritic outgrowth are determined primarily by both intrinsic factors which are unique to the tissue types and extrinsic factors such as the substrata which determine the micro-environment where the developing neurons are growing.

Acknowledgements. This study was supported by UPGC research grant 221400030 and Croucher Foundation Grant 360-031-0792-4F.

References

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Paper received 7.8.89 Revised paper accepted 23.12.89.