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I>EVELOPMENTAL BIOLOGY 74,401-408 (1980)

Cholinergic Neuronotrophic Factors

III. Developmental Increase of Trophic Activity for Chick Embryo Ciliary Ganglion Neurons in Their Intraocular Target Tissues

K. B. LANDA,* R. ADLER," M. MANTHORPE,** AND S. VARON*'"

• Department of Neuroscience and·· Department of Biology, University of California, San Diego, La JolLa, California 92093

Received March 30, 1979; accepted July II, 1979

Between stages 34 and 40 in the chick embryo, the ciliary ganglion (CG) undergoes a 50'1 loss of neurons. Such neuronal death is a common feature in neural development and it has been proposed that neurons are dependent for survival on trophic support from their target tissues. Using an ill vitro bioassay it was previously shown in this laboratory that trophic activity for CG neurons is highly concentrated in eye structures containing CG target tissues. In the present study we have found that trophic activity in the eye increases markedly between stages 37 and 39, the time when neuronal death in the ciliary ganglion is ending. Thus, a developmental increase in trophic activity within the eye may be involved in determining neuronal survival in the CG. Furthermore, this study provides the ftrst indication that the trophic content of target tissue is itself developmentally regulated.

INTRODUCTION

During normal development of many neuronal populations there is a defined pe­riod when large numbers of the previously produced neurons die (Hamburger and Levi-Montalcini, 1949; Hamburger, 1958, 1975; Cowan and Wenger, 1968; Prestige, 1970; Cowan, 1973; Rogers and Cowan, 1973; Landmesser and Pilar, 1974a,b; Clarke and Cowan, 1976; Clarke et aI., 1976; Hollyday and Hamburger, 1976; Narayanan and Narayanan, 1978; Varon and Bunge, 1978; Varon and Adler, 1979) . Neuronal survival has been shown to be proportional to the size of the territory innervated by the neurons, decreasing with removal of the target tissues and increasing with addition of extra target. It has been proposed that target tissues supply trophic support for the neurons which innervate them, perhaps via the delivery by the target of specific mole­cules necessary for the survival of the in­nervating neurons (cf. reviews by Varon and Bunge, 1978; Varon and Adler, 1979).

401

Such neuronotrophic factors (NTFs) I would be taken up by the neuronal termi­nals and transported retrogradely to the somas. Developmental neuronal death could result from an inability to secure an adequate supply of NTF, either because of insufficient production of the material by the target or because of some mechanism which restricts neuronal access to the sup­ply.

One NTF which has been purified is the nerve growth factor (NGF). NGF is a pro­tein which ensures the survival of sympa­thetic and dorsal root ganglion neurons, both in vitro and in vivo (Levi-Montalcini, 1966, 1976; Levi-Montalcini and Angeletti, 1968; Varon, 1975; Varon and Bunge, 1978; Varon and Adler, 1979). When labeled NGF is injected into the peripheral targets of sympathetic and dorsal root ganglion neu-

I Abbreviations used: CG, ciliary ganglion(ic); HBSS, Hank's balanced salt solution; HEBM, high­bicarbonate Eagle's basic medium; NGF, nerve growth factor; NTF, neuronotrophic factor.

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402 DEVELOPMENTAL BIOLOGY VOLUME 74,1980

rons, it is selectively transported to the neuronal somas involved (Stoeckel and Thoenen, 1975; Hendry, 1976; Brunso­Bechtold and Hamburger, 1978). Although the target origin of endogenous NOF has not yet been fully proven, all available in­formation on NOF is consistent with the working hypothesis presented above (Va­ron and Adler, 1979).

The chick ciliary ganglion (CO) is a sys­tem which has many advantages for the study of trophic relationships between neu­ron and target (Landmesser and Pilar, 1978). (1) The ciliary ganglion provides parasympathetic innervation to the intrin­sic muscles ofthe eye. The discreteness and accessibility of the CO and its innervation territory make experimental manipulations relatively easy. (2) The ciliary ganglion con­tains only two types of neurons, both of which are cholinergic and cholinoceptive. The ciliary neurons innervate the ciliary body and iris; the choroid neurons inner­vate the vasculature of the choroid coat. These neuronal types develop at the same time from similar precursors, and undergo equivalent biochemical differentiation. (3) Survival of these neurons is target de­pendent. Both populations undergo a 50% neuronal death between Hamilton-Ham­burger stages 34 and 40 (Landmesser and Pilar, 1974b) . As in other systems, neuronal death is increased by removal (Landmesser and Pilar, 1974a) and reduced by expansion (Narayanan and Narayanan, 1978) of the target tissue.

In vitro, dissociated CO neurons fail to survive for 24 hr when cultured in medium supplemented only with serum. CO neurons do survive, however, when the culture me­dium is supplemented either with medium conditioned over heart cells or with aqueous extract from chick embryos (Hel­fand et al., 1976, 1978; Tuttie, 1977; Nishi and Berg, 1979; Varon et at., 1979). Using a bioassay developed on the basis of this differential in vitro behavior of the neurons, we examined the distribution of trophic

activity in stage 37 embryos (Adler et al., 1979). The trophic activity was found to be highly concentrated in the eye and, more specifically, in a fraction of the eye contain­ing the target tissues of the CO neurons. The presence of trophic activity in embryo extract suggests that there is a soluble ma­terial capable of supporting CO neurons. The selective localization of trophic activity in target tissues of CO neurons supports the view that CO neurons may derive trophic material from their target tissues during normal development. In the present study, we describe the finding of a devel­opmental increase in the amount of trophic activity in the eye which occurs at a time when survival of CO neurons is being de­termined.

MATERIALS AND METHODS

Tissue Extracts White Leghorn chicken eggs were incu­

bated at 37.5°C in a forced-air incubator. After staging the embryos (Hamburger and Hamilton, 1951) whole eyes were removed and cleaned of surrounding tissues. In some cases different eye components were fur­ther dissected following a procedure given below. Whole eyes or eye components were kept in ice-cold Hank's balanced salt solu­tion (HBSS) for no more than 3 hr before extraction. The tissues were homogenized in 6 m1 of cold distilled water per gram of wet weight, using 20 strokes of a Potter­Elvehjem homogenizer. Homogenates were centrifuged at lOO,OOOg for 2 hr and the supernatants collected and stored at 0-4°C overnight for trophic bioassay the next day.

Dissection of Eye Components

Eyes from three to five embryos were placed in a large petri dish containing HBSS and freed of surrounding mesen­chyme and extraocular muscles. The first step in the dissection was the removal of the cornea, the external edge of which is easily recognized. One of the arms of a fine watchmaker's forceps was then introduced

LANDA ET AL. Ciliary Nellronotrophic Fa ctor 403

through the pupil to facilitate immobiliza­tion of the eye. Using iridectomy scissors, a coronal section was made, following a line posterior and parallel to the external border of the iris. The eye was thus divided into two components, identified for convenience as "frontal ring" and "fundal cup." The vitreous and lens were removed from these two eye fragments, some caution being re­quired to cleanly separate these materials from the iris. The neural retina was then peeled from both fundal cup and frontal ring. The only fIrm attachment of the neural retina to the remaining eye struc­tures is at the choroid fIssure and the ora serrata. In some cases, dissection culmi­nated with the separation of the iris from a fraction containing choroid, pigment epi­thelium, ciliary body, and sclera. The ciliary body can be recognized as a thick, white ring that requires careful dissection from the iris. In other cases, a further separation of the sclera from the choroid-pigment ep­ithelium-ciliary body complex was per­formed. The choroid-pigment epithelial layer and the sclera were each grasped with a pair of fInd watchmaker's forceps and gently pulled apart, beginning at the free edge created by the initial coronal section. The continuity of the layers at the choroid fIssure had to be severed before they could be completely separated.

Attempts to separate pigment epithelium from choroid-ciliary body, either mechani­cally or after enzymatic treatments, have so far been unsuccessful.

Bioassay for Trophic Activity

The bioassay for trophic activity is based on an in vitro culture system which allows the quick measurement of trophic activity in many extracts at a time. Ciliary gangli­onic cells are dissociated and cultured as previously reported (Varon et at., 1979) on highly adhesive collagen in high-bicarbon­ate Eagle's basic medium (HEBM), supple­mented with 10% horse serum and tissue extract. This system has been scaled down

to lS-mm microwell plates (5000 CG cells per well, approximately half of which are neurons) to allow assaying large numbers of extracts (Adler et at., 1979). Under these scaled-down conditions maximum survival of large-bright, neuronal cells is approxi­mately 30%. Up to 100% survival can be obtained by increasing the seeding density of the CG cells (Varon et ai., 1979), but it was felt appropriate to use the lower seed­ing density in order to maximize the num­ber of assays. Also, penicillin, streptomycin, and fungizone have been added to the cul­ture medium in order to avoid time-con­suming sterilization of a large number of extracts. Pilot experiments indicated that these materials did not affect neuronal sur­vival and maintained sterility during the 24-hr assay.

Extracts to be assayed for trophic activ­ity were serially diluted in HEBM. Two hundred-microliter aliquots of these dilu­tions were added to the collagen-coated microwells, followed by 200 1'1 of the CG cell suspension in HEBM with 20% horse serum. The cultures were incubated for 24 hr at 37°C in an atmosphere of 5% CO2 in air. The culture medium was then removed and the cells remaining were fIxed with a 2% glutaraldehyde solution. The number of large-bright neuronal cells per well was de­termined and graphed as a function of the concentration of tissue extract in the cul­ture medium.

In initial experiments to define the con­ditions of the bioassay, cultures were main­tained for 2 and S hr, then rinsed and fIxed as described above. It was found that at these shorter assay times, neuronal number was the same in the presence or absence of tissue extract, and that only after longer assay times (e.g., 24 hr) did differences in neuronal number develop. Thus, the differ­ential survival of CG neurons in the pres­ence of tissue extract, as presented below, cannot be explained by selective attach­ment of the cells to the collagen substra­tum.

404 DEVELOPMENTAl. BIOLOGY VOLVME 74, 1980

When the culture contains only HEBM and horse serum, no large-bright, neuronal cells remain after 24 hr, either attached to the wells or floating in the medium. As the concentration of tissue extract increases (that is, as the dilution factor decreases) , there is an increase in neuronal survival until a plateau value is approached, where the survival tends to level off. That dilution of extract which allows survival of 50% of the plateau number of neurons is defined as having 1 unit of trophic activity per mi. The trophic units per milliliter (titer) of the original extract can be determined on the basis of the dilution necessary to obtain a 1 unit per mI response. Total trophic con­tent of a tissue was calculated from the titer and volume of extract obtained from the tissue. Activities are expressed as the mean and standard deviation of from 3 to 7 ex­tracts assayed independently.

Protein Determinations

Protein concentrations of the extracts were measured using the method of Lowry et al. (1951). Specific activities of the ex­tracts were expressed in terms of trophic units per milligram protein.

Histology

A series of tissue samples was collected for histological examination. Tissues were fixed in Bouin's fixative, dehydrated to 95% ethanol, and embedded in JB-4 embedding medium (Polysciences). Sections of 3-4 11m were stained with Gill's modified hematox­ylin and eosin/light green (Polysciences).

RESULTS

Development of Trophic Activity in the Eye

Total soluble protein and total soluble trophic activity were measured in whole eyes from stages 30 to 44. These results, along with the resulting specific activities, are shown in Fig. 1.

The protein content of the eye (dotted line) increases steadily throughout this pe­riod of development, reflecting the general

"

" . EM811YONIC STACX

FIG. 1. Development of protein and trophic activ­ity for ciliary ganglionic neurons in chick eyes from slages 30 to 44. Vertical brackets indicate the standard deviation of from 3 to 7 extracts assayed independ­ently. ( .. ... ) = Soluble eye protein per embryo. (-) = Soluble trophic activity per embryo. (- --) = Specific activity.

growth of the eye. From an initial value of approximately 1.2 mg per embryo, eye pro­tein increases more than 7-fold, to 8.5 mg per embryo.

In contrast to eye protein, there appears to be a discontinuous development of trophic activity. Total trophic activity (solid line) initially increases slowly, with a slope similar to that of the increase in eye protein. Specific activity (dashed line) dur­ing this period ranges between 500 and 700 units per mg protein. Trophic activity then begins to increase markedly. During the 2-day period between stages 37 and 39, the amount of total trophic activity in the eye almost triples, from 2500 to 6700 units per embryo. This rapid increase is reflected in a doubling of the specific activity. Finally, specific activity appears to plateau after stage 39 at 1400 to 1500 units per mg. Total activity continues to increase during this period, however, as the eye continues to increase in size and protein content.

Intraocular Distribution of Trophic Actit'ity

It was previously found that, at stage 37, 80% of the intraocular trophic activity could be localized in a fraction of the eye contain­ing the choroid coat, ciliary body, pigment

LANDA ET AL. Ciliary Neuronotrophic Factor 405

epithelium, and sclera (Adler et ai., 1979). Other structures, such as the retina, vitre­ous humor, lens, or cornea, contained little trophic activity, although they contributed over 70% of the total eye protein.

To determine whether the rapid increase in tropic activity seen between stages 37 and 39 was accompanied by changes in intraocular distribution of trophic activity, a similar eye subdissection was carried out using stage 40 embryos. As can be seen in Table 1, the same type of distribution is present at stages 37 and 40. The choroid­ciliary body-pigment epithelium-sclera

fraction still contains about 80% of the trophic activity but only 25% of the protein, and accounts for most of the increase in trophic activity seen between stages 37 and 39.

The fraction containing most of the ac­tivity at stages 37 and 40 comprises not only CG targets such as choroid and ciliary body, but also sclera and pigment epithe­lium which do not receive CG innervation (Fig. 2a). In an attempt to further localize the source of trophic activity, this trilami­nar structure was split into two subfractions in stage 40 eyes by mechanically separating

TABLE I

DISTRIBUTION OF TROPHIC ACTIVITY AND PROTEIN IN STAm~ 37 AND STAGE 40 EVES"

Tissue Stage 37 eyes (Days 11 to 12) Stage 40 eyes (Days 14 to 15)

Activity/ Protein/ Specific Activity/ Protein/ Specific embryo embryo activity embrvo embryo activitv (units) (mg) (units/ (units) (mg) (unitsi

mg) mg)

Whole eye 2500 2.88 880 nOD 5.35 1300 ±66O ±0.57

Choroid, ciliary body, sclera, pig- 2000 0.82 2400 6200 l.lS 5400 ment epithelium ±890 ±0.08

Remaining eye tissues 270 1.97 140 2100 3.20 660 ±430 ±O.19

" The values for stage 37 eyes are taken from AdJer et af. (1979).

_2~ _ __~ ~_ -- ----~- -- ... ".; -....... ~ ... . ........................ oa ..,···l" .... " .. , ..... t\ . • ...... '. ..•••• .. •••. ., ...... (: .,,, "'''''#

~ : c._ ~I.','.,!: ........ : •• ~ .. ~ ... • l~t.·., .. , I~. ; •. "'''1&:.. , l'I# •••• f ...... ~ .... • J# ..... l .. S ...... .

.. ' •• " ',,. .t' •• _ ..... •• _ I. ' . . _ / f I. .. • ..... - .... - .. .. ::: ... ': ... ;:.~.:... . ~ 'L '_ ... ,: ...... , ... ' ~ .. ,:' •• ··,··.0 .·,·

••• '~'. I ",. •. • ... o' :',':' '. ,

~;ItP;.;;..

O.2mm \ 2c

FIG. 2. Photomicrographs of eye subcomponents dissected from stage 40 embryos, (a) Fraction containing sclera (8), choroid (C), and pigment epithelium (PE). (b) Issolated sclera. Note the absence of choroid tissue. (c) Isolated choroid-pigment epithelium. The choroid layer tends to shrink during histoiogicaJ fixation when not supported by the sclera, leading to the rumpled appearance of this fraction.

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406 DEVELOPMENTAL BIOLOGY VOLUME 74, 1980

the sclera from the choroid-ciliary body­pigment epithelium complex. In histologi­cal sections of dissected material, the sclera appears as a wide band of tissue. comprised at this stage mainly of cartilage and dense connective tissue (Fig. 2b). Only infre­quently does any choroid tissue remain at­tached to the sclera. The choroid (Fig. 2c) appears as a highly vascular tissue, which cleaves from the sclera along a surprisingly smooth surface. The pigment epithelium remains firmly attached to the choroid (Fig. 2c) . The ciliary body (not shown) remains as a forward projection of the choroid-pig­ment epithelium complex. Most of the ac­tivity, but only one-third of the protein, is recovered in the choroid-pigment epithe­lium-ciliary body fraction (Table 2), which has a total activity of 3600 units per embryo and a specific activity of 10,000 units per mg protein. The sclera has little total activ­ity (660 units per embryo), with a specific activity of 830 units per mg.

DISCUSSION

It has been hypothesized that neurons are able to derive trophic material from the tissues they innervate, and that this sup­port may be involved in determining neu­ronal survival during development (Varon and Bunge, 1978; Varon and Adler, 1979). In terms of this hypothesis a putative neu­ronotrophic factor should at least: (i) be capable of supporting the survival of the

TABLE 2

FURTHER LOCALIZATION OF TROPHIC ACTIVITY

WITHIN STAGE 40 EYES"

Tissue

Choroid, ciliary body, sclera. pigment epi­thelium

Choroid, ciliary body, pigment epithelium

Sclera

Activ-ity!em-

bryo (units)

6200 ±890

3600 ±460

660 ± l OO

Protein Specific /ern- activity bryo (units! (mg) mg)

1.15 5,400 ±O.OB

0.35 10,000 ±O.O5

O.BO 830 ±O.17

"The histology of the fractions presented here is shown in Fig. 2.

pertinent neurons, (ii) be present in the target t issues of the neurons, and (iii) be available to neurons in significant amounts at developmental stages when their survival is target dependent. Our current findings will be discussed with respect to these three properties.

(1) Survival Factor for CG Neurons

The bioassay described in detail in this report is based on the 100% disappearance of CG neurons within 24 hr when cultured in HEBM supplemented with horse serum alone. That this disappearance is due to cell death rather than to a lack of attachment of the neurons to the substratum is indi­cated by the fact that neurons are not ob­served either attached to the dish or float­ing in the culture medium, as well as by the presence in these cultures of a substantial amount of cell debris. CG neurons are main­tained, however, when appropriate amounts of selected tissue extracts are added to the cultures. Also, in the sup­ported cultures, many of the neurons ex­tend neurites and there is a substantial amount of choline acetyltransferase activ­ity present (Varon et al., 1979).

(2) Localization of Trophic Activity in CG Targets

It has been reported (Adler et al., 1979) that the embryonic eye has a much higher specific trophic activity than any other re­gion of the chick embryo, and that most of this intraocular activity is localized in a dissected fraction of the eye containing sclera, choroid, pigment epithelium, and cil­iary body. It has now become possible to carry out a more refined dissection of this fraction, allowing the separate extraction of the sclera and the choroid-ciliary body-pig­ment epithelium complex. The latter frac ­tion, containing most of the peripheral tar­get territory of CG neurons, shows the high­est specific activity so far encountered in any tissue. In contrast, the sclera, which

LANDA ET AL. Ciliary NeUI'OflOtl'ophic Factor 407

does not receive CG innervation, shows low activity comparable to other eye structures not innervated by CG neurons. Techniques such as immunocytochemistry or the iso­lation of specific cell types, however, will be necessary for the direct identification of the source of trophic factor in the eye. Present efforts are directed toward purification of the active material (Manthorpe et al., 1979) and the development of antisera against it.

(3) Developmental Behavior of Intraocular Trophic Activity

Some trophic activity can already be de­tected in eyes from young embryos, before survival of CG neurons in vivo becomes obviously target dependent. Both total and specific trophic activities, however, increase most markedly at developmental stages co­inciding with those shown by Landmesser and Pilar (1974b) to represent the end of the neuronal cell death period and the onset of stable neuronal survival in the ciliary ganglion. The higher trophic activity at that developmental stage may be what is required for the survival of those ganglionic neurons that normally escape cell death.

Eyes from older embryos continue to show high levels of trophic activity. A sus­tained supply of trophic material may be required for the continued maintenance of CG neuronal survival. With the stabiliza­tion of neuronal number in the ciliary gan­glion, however, the continued increase in total trophic activity brings about an in­crease in the absolute amount of trophic activity per neuron. It has been suggested that concentrations of trophic factors greater than those required for neuronal survival may be involved in the maturation of important neuronal behaviors, by provid­ing a source of trophic "drive" (Varon, 1975; Varon and Bunge, 1978; Varon and Adler, 1979). In the case of the CG neurons, trophic factor derived from their targets in excess of survival needs could, for instance, support the increase in choline acetyl trans­ferase known to occur in CG neurons ter-

minals during this period (Chiappinelli et al. , 1976).

That the trophic content of the targets of the CG neurons is not constant, but shows a marked increase during a specific devel­opmental period, is an important finding. This is the first study of which we are aware that points to a developmental regulation of the very competence of a target tissue to supply trophic support. It is intriguing that this developmental increase occurs at a time when CG neurons are establishing functional relationships with their targets. It could be that the intraocular target tis­sues are responding to a signal from the incoming CG fibers to increase their con­tent of trophic activity. Alternatively, this developmental increase may be regulated independently of CG innervation, perhaps via hormonal or intraocular cell-cell sig­nals. The CG-eye system provides unique opportunities to study these types of ques­tions and work along these lines is now being undertaken in our laboratory.

This work was supported by USPHS Grant NS-07606 from the National Inst itute of Neurological and Communicative Disorders and Stroke. K.L. is a Grad­uate Fellow of the National Science Foundation.

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