Cell Tiss. Res, 179, 285-316 (1977)

Cell and Tissue Research 9 by Springer-Verlag 1977

Quantitation of Fiber Growth in Transplanted Central Monoamine Neurons* Ake Seiger and Lars Olson Department of Histology, Karolinska Institutet, Stockholm, Sweden

Summary. Nerve fiber production by central noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) neurons was studied using immature brain tissue containing locus coeruleus, substantia nigra, or ventro-caudal medulla oblongata respectively, homologously grafted to the anterior chambers of rat eyes. A method was developed for quantitation of the fiber growth that occurs on the sympathetically denervated host irides as observed in whole mounts using Falck-Hillarp fluorescence histochemistry and by the uptake of 3H-metaraminol into the irides. Survival and growth in oculo of the three different areas were characterized by direct observations through the cornea in vivo for a number of pre- and postnatal stages of development of the donors, and the findings correlated to the degree of monoamine nerve fiber production on the host irides. The growth of fetal locus coeruleus transplants on irides was quantified using both fluorescence microscopical measurements of innervated areas and uptake of 3H-metaraminol. The uptake was well correlated to the histochemical measurements on individual irides, thus validating the fluorescence microscopical measurements of fiber production. The fiber growth of fetal locus coeruleus grafts on irides was followed for 20 weeks. The nerves increased in number and uptake capacity approximately linearly for 6 weeks whereafter the increase rapidly levelled off. On average, the final amount of nerve production by fetal locus grafts did not cover more than 1/3 of the host iris surface, and the average uptake of aH-metaraminol by these nerves did not exceed 60% of that found in sympathetically intact control irides. The locus grafts produced a similar amount of fluorescent fibers in the host iris independent Send offprint requests to: Dr. ~. Seiger, Department of Histology, Karolinska Institutet, S-10401 Stockholm 60, Sweden

* Supported by the Swedish Medical Research Council (04X-03185), "Magnus Bergvalls Stiftelse" and "Karolinska Institutets Fonder". The skilful technical assistance of Miss Ingrid Str6mberg, Miss Maud Eriksson and Miss Gerd Bo~tius is gratefully acknowledged. Thanks are due to Swedish Pfizer tbr the generous supply of Nialamid|

286

A. Seiger and L. Olson of the crown-rump length stage of the donor fetus and the final size of the transplants in oculo. The survival and growth of NA, DA and 5-HT neurons grafted from various postnatal donor rats was also followed by fluorescence microscopy. Locus coeruleus grafts produced markedly more fibers than the two other types of grafts when the donor was one week old or less, and DA grafts produced the least fibers of the three. Even from one month old donors some MA neurons survived grafting. Also, using prenatal donars, the locus coeruleus grafts produced many more fibers on the irides than did the DA grafts. It was concluded that the intraocular transplantation technique is very suitable for quantitative studies of nerve fiber production by immature monoamine neurons, and that it should be possible to study many other neuron systems in similar ways with this technique.

Key words: Central monoamine neurons - Fiber growth - Locus coeruleus - Quantitation - Fluorescence histochemistry.

Introduction Understanding of the prenatal ontogeny of the central monoamine (MA) neurons in the rat (Olson and Seiger, 1972a; Seiger and Olson, 1973) has made it possible to prepare small well-defined pieces of brain tissue from animals at selected developmental stages which contain selected MA neuroblasts suitable for homotransplantation to the anterior eye chamber. The survival and proliferation of such transplants containing noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) nerves has been described in qualitative terms using Falck-Hillarp fluorescence histochemistry (Olson and Seiger, 1972 b, 1975; Seiger et al., 1976). Of special interest was the marked outgrowth of central MA fibers into sympathetically denervated host irides where the fluorescent fibers can be examined in great detail using whole-mount preparations. The uptake and field stimulation induced release of labelled transmitters from such central nerve fibers in a peripheral environment was found to be almost normal (Seiger et al., 1976). A particular advantage of the transplantation technique is the possibility of evaluating the total growth potential of given nerve cell groups such as the locus coeruleus, which is so diffusely distributed and so mixed with other MA neuron systems that measurements of total fiber production in situ become impossible. In the present report, which is the first of a series (Seiger and Olson 1977a, b, c), we describe a method that permits detailed quantification of central MA nerve fiber production by intraocular brain transplants using Falck-Hillarp histochemistry. The validity of this method was demonstrated by comparison with simultaneous aH-metaraminol uptake (cf. Olson et al., 1968). The technique makes it possible to compare nerve growth in different experimental groups. It will be shown that the time course of fiber production in irides by fetal locus coeruleus grafts is a strict expression of its inherent developmental program

Growth of Transplanted Central Monoamine Neurons

287

and ceases at a predetermined time, regardless of the degree of reinnervation of the sympathetically denervated host iris. The different relative abilities to survive transplantation shown by M A neurons obtained at different stages of development of the donor will be demonstrated. M A neurons can survive transplantation at least up to one m o n t h after birth.

Material and Methods Animals A total of about 510 adult female albino rats (Sprague-Dawley, 125 200 g, Anticimex, Stockholm) were used as recipients for transplantation of pieces of tissue containing central MA neuroblasts bilaterally into the anterior chamber of the eyes, see Table 1. All these rats had the sympathetic adrenergic nerves of the irides bilaterally removed by superior cervical ganglionectomy one or a few days prior to transplantation. Normal control irides were obtained from 25 rats. Donors of central MA neurons were: 354 fetuses, crown rump length ( C R L ) = 15-30 mm, 26 newborn rats, 17 one day old, 26 eight day old, 26 fifteen day old, 26 twenty two day old, 26 twenty nine day old and 9 adult rats. Adult rats were fed pellets and water ad lib., and were kept 5 to a cage. The cages were lighted between 0600 h and 1800 h.

Preparation of Tissues Used for Transplantations Pregnant rats were sacrificed by cervical dislocation under ether anaesthesia and the uteri rapidly dissected out. Fetuses were stored in utero and kept at r o o m temperature until dissection. A maximal time of 2 h before introduction of a dissected piece into the anterior chamber of the eye was allowed, within which time no decline in survival, growth or vascularization after transplantation could be seen. The head of the fetus was cut off. Removal of the whole brain from the skull was performed in two ways depending on the CRL of the donor; > 18 ram: the brain was easily removed holding the skull between the fingers. < 18 mm: the skull was so small that brain removal had to be performed under a stereoscopic dissection microscope. All dissections were performed using sterile instruments and sterile Ringer solution at room temperature. Dissected pieces were handled with modified Pasteur pipettes. They were stored in sterile Ringer solution until transplantation.

Table 1. Total numbers and mean experimental group numbers of irides carrying NA, DA and 5-HT grafts Type of analysis

In vivo observations Fluorescence microscopy 3H-metaraminol

NA

DA

5-HT

Total

total

group mean

total

group mean

total

group mean

651 389 150

17 14 15

238 82 .

24 14

70 70

14 14

.

.

.

959 541 150

Note. Numbers for fluorescence microscopy and 3H-metaraminol are parts of the numbers for in vivo observations. The approx. 420 eyes analysed for in vivo observations additional to the numbers for fluorescence microscopy are from animals where other analyses will be dealt with elsewhere (Seiger and Olson 1977c)

288

A. Seiger and L. Olson

"Locus Coeruleus Transplants" In fetuses and early postnatal rats the locus coeruleus area was most easily reached from the dorsal aspect of the brain. The developing tectum and cerebellar anlage were cut in the midline leaving the floor of the forming IVth ventricle intact. Just caudal to the cerebellar anlage a transverse cut was made bilaterally in the roof of the ventricle all the way to its lateral edge, after which the cerebellum was unfolded. In the caudo-lateral part of the floor of the ventricle, just rostral to the pontine flexure, a small block of tissue was cut out. The aim was to remove as many of the locus coeruleus NA neuroblasts as possible, while minimizing the inclusion of other regions of the brain stem. In trying to get the same constituents of the piece to be transplanted regardless of the CRL of the donor, larger pieces were taken from older donors than from younger ones. The size range was 1 x 1 x 1 mm to 2 x 1.5x 1 mm. These pieces will be referred to as "locus coeruleus transplants". For postnatal donors of two weeks or older, the dissection technique had to be modified because of the rapid growth of cerebellum. In such animals the whole brain stem was divided in the midline from its dorsal aspect. The locus coeruleus area was then reached from the midline. Laterally in the floor of the IVth ventricle it was possible to recognize a very small inflection in whose caudo-lateral part the nucleus locus coeruleus is situated. Pieces dissected for transplantation from fetuses, young animals and adults were sometimes taken for fluorescence histochemical analysis of the content of green fluorescent locus coeruleus NA neurons, as a test of the reproducibility of the dissection technique. In most pieces the whole locus coeruleus was included. "DA Transplants". The region of the developing substantia nigra with its DA cell bodies was reached from the ventral surface of the brain stem. A small median wedge approx. 2-3 mm wide and 1.5-2 mm long was cut out at the level of the vertex of the mesencephalic flexure. The wedge reached dorsally halfway to the aqueduct. It was then divided sagittally in the midline to obtain unilateral ventro-medial tegmental pieces which will be referred to as " D A transplants". At older postnatal stages, the pieces were also trimmed ventrally at the surface, thereby removing the relatively thick layer of myelinated axons. "5-HT Transplants". 5-HT neurons were taken from the ventral surface of the brain stem, in a piece of tissue from the ventro-caudal part of the raph6 region in the medulla oblongata. This piece contained parts of the B1-B3 complex (according to the nomenclature of Dahlstr6m and Fuxe, 1964, see also Seiger and Olson, 1973). The midline tissue block was 2 3 mm wide and 1.5 mm long. It never reached dorsally more than halfway to the aqueduct. It was then cut in the midline to give two unilateral median pieces, which will be referred to as " 5 - H T transplants". It should be noted however that these transplants almost invariably also contained some catecholamine (CA) nerve cell bodies from the A1 group.

Transplantations to the Anterior Chamber of the Eye All recipient animals had their whiskers cut to avoid contamination of operating instruments and their eyes atropinized by topical application of one drop of a 1% solution of atropine a few minutes before transplantation. This caused maximal dilatation of the iris so that damage to the vascular network of the iris during transplantation could be avoided. Transplantations were performed under ether anaesthesia with sterile instruments using a stereoscopic operating microscope. A slit was made in the cornea. The transplant was sucked up in a modified Pasteur pipette together with some sterile Ringer solution, the pipette introduced into the anterior chamber through the slit, and the piece of tissue injected into the eye. Because the eye ball was punctured, the transplant could be placed in the desired position by applying gentle pressure to the cornea from the outside with a pair of forceps. It is to be noted that no blood vessels of the host are cut during the transplantation procedure so that there is no bleeding. For further details, see Olson and Malmfors (1970). Immediately after transplantation a first measurement of the size of the transplanted piece was made with the operating microscope. The largest diameter and its perpendicular counterpart was noted, as well as details on occasional suboptimal events (see below). About 12 animals could be bilaterally transplanted and measured per hour.

Growth of Transplanted Central M o n o a m i n e Neurons

289

Postoperative in vivo Observations All transplants were followed for postoperative survival, vascularization and growth by repeated inspection in vivo through the cornea during light ether anaesthesia using a stereoscopic operation microscope. Two diameters were measured together with a rough estimate of the thickness of each transplant. The product of the two measured diameters was taken as an estimate of the surface of a transplant. Variations in thickness of transplants matured in oculo were limited. Thus, the product of the diameters was also a rough estimate of the volume of each transplant, here called "transplant size". When, occasionally, transplants were very thin, the product of the diameters was divided by two to obtain a better estimate of transplant size. Other features of the operated eyes such as corneal vascularization or opacity, bleeding or debridements on the iris, cataract formation, macro- or micro-ophthalmia, intraocular infections, prolapses etc. were also noted. All these complications were rare, One important purpose of the in vivo observations was to be able to exclude transplants a priori from a group because of occasional technical failures, and to exclude eyes where, for u n k n o w n reasons, the transplant did not take.

3H-metaraminol Incubations and Fluorescence Histochemistry All recipients with " 5 - H T transplants" were pretreated 4 h before sacrifice with Nialamid (Pfizer) 500 mg/kg i.p. to increase the intraneuronal 5-HT levels, and thereby increase the visualization of 5-HT containing fibers in the iris. Animals were sacrificed by neck dislocation under ether anaesthesia. The iris with its attached CNS transplant was freed from the rest of the eye. Most of the choroid m e m b r a n e was removed. The transplant was removed from the iris by cutting along its periphery. It was important to ensure that no transplant tissue was left on the iris since such remnants would have influenced the 3H-metaraminol uptake. The transplants were rapidly frozen in liquid propane, cooled by liquid nitrogen, freeze-dried (Olson and Ungerstedt, 1970) and further processed for visualization of biogenic m o n o a m i n e s according to Falck and Hillarp (Falck et al., 1962 ; see also Corrodi and Jonsson, 1967; Jonsson, 197l). The irides containing locus coeruleus derived nerves and irides from intact control rats (32) were incubated in pairs in 1 ml Krebs-Ringer bicarbonate buffer saturated with CO2 (6.5%) in O2 at 37 ~ C (Hamberger, 1967). To the incubation buffer had been added 3H-metaraminol (dl-metaraminol-7-3H(N)-5,10 Ci/ mmole, New England Nuclear, Boston, Mass.) at a concentration of 10 7 M, irides were then rinsed in 5 ml of buffer at 0 ~ for 5 min. 3H-metaraminol was preferred to 3H-NA because of its resistance to m o n o a m i n e oxidase and becaused the stability of the isotope. Its relative affinity for noradrenergic uptake sites is approx. 9-fold that of N A (see Iversen, 1967). All irides were stretch-prepared as whole m o u n t s and reacted with gaseous paraformaldehyde. After the fluorescence microscopical analysis all irides were scraped off the slides, dissolved in Soluene (Packard) and the radioactivity determined (Olson et al., 1968).

Quantitative Fluorescence Microscopy A schematic drawing was made of each iris where all fluorescent fibers were depicted (Fig. 1). The area of the iris covered by central M A nerves outside the site of attachment of the transplant was calculated in the microscope using the total field of view with an objective magnification of 10 times as a measuring unit. The measuring unit was 2.4 m m 2. The total area of a whole m o u n t of an iris was approx. 30 m m 2. The total iris area covered by M A nerves was obtained by adding the size of the hole in the iris after the transplant ( = t h e final size of the transplant as measured in vivo) to the innervated area around the hole (cf. Fig. 1). The density of innervation, which varied from very dense to very sparse was subjectively estimated and noted together with the drawing. All these measurements were carried out on a blind basis. Several normal irides were included in each experiment. Their sympathetic ground plexus was used as a control of the histochemical reaction and as an internal reference of nerve density. Still working blind, the area covered by fluorescent nerves and their density in the experimental irides were then converted into an expression of total nerve fiber content using a 0-5 scale. In each experiment this last conversion of the primary measurements was started by ranking all irides for number of fluorescent

290

A.. Seiger and L. Olson LOCUS COERULEUS IN OCULO:

I Fig. 1. A sample of semischematic drawings of 8 individual stretch prepared irides, subjected to fetal locus coeruleus transplantations > 6 weeks before. The fluorescent fibers, here in black, are densely packed around the hole formed by removal of the transplant. The area covered by outgrowth is slightly above average in this sample. All irides were sympathetically denervated before transplantation. Coupled to each drawing was an estimation of nerve density and a measurement of the area covered by fluorescent fibers. Actual drawings were 10 x 2 cm. Approx. magnification in Figure 2,5 times

nerve fibers. The iris with most nerves was rated as the end point of the semiquantitative scale, that is 5. All the other irides were then compared with this iris and with each other. These estimated measurements can therefore only be compared within one experiment and not between different experiments, as the scale will vary depending on the a m o u n t of nerves present on the iris with the most nerves. The aim was to use this scale as linearly as possible in every experiment. In order to compare numbers of fibers between different experiments, ranking was redone using new copies of the drawings of individual irides with their attached area and density measurements, but now mixing the populations of irides from different experiments that were to be compared. Again, the ranking was performed on a blind basis. Using fetal locus coeruleus transplants, there was a close correlation between the density of nerves and the innervated area on individual irides. The correlation within transplanted groups between the estimated number of fibers (making use also of the density estimations) and the corresponding area covered by these nerves was high (r=0.96). Therefore, for whole groups, the innervated area was considered a good measurement of total outgrowth. In one initial experiment to test the validity of the histochemical analysis, locus coeruleus pieces were transplanted from one day old rats. After 31/2 m o n t h s in oculo all 34 irides with transplants on were incubated in 3H-metaraminol and stretch-prepared. The fluorescence estimate of the a m o u n t of innervation and the radioactivity in each iris were strongly correlated to each other (r=0.86, Fig. 2). The mean neuronal uptake of radioactivity in the transplanted irides was roughly 1/3 of that in normal control irides.

Time Course Experiment Locus coeruleus pieces, obtained from five pregnant rats, were grafted bilaterally into 60 recipients with sympathetically denervated eyes. The C R L range was 24-30 m m and the recipients were randomly assigned to groups to be sacrificed at postoperative times 1, 2, 4, 6, 10 and 20 weeks so that five donor litters were equally represented in each time group. Fiber production on the host irides were evaluated by 3H-metaraminol uptake and fluorescence histochemistry. At each time point a series of normal irides from 200 g rats were included as a reference for the uptake values and control of the histochemical reaction. Calculations of uptake values as percentages of values derived from the normal irides at each time point permitted comparisons between the different time points.

Growth of Transplanted Central Monoamine Neurons

291

18

16 14

E o. v

8

0 015 1

2

;

4

fl ..........

estimate

Fig. 2. Correlation of subjective fluorescence estimate with radioactivity determinations of the same irides after 3H-metaraminol incubations (r=0.86). Locus coeruleus transplants from one day old donors. Postoperative time: 31/2 months, n =4-8. Control uptake in sympathetically intact normal irides was approx. 17 • 103 cpm

Results

In vivo Observations 90% or more of the transplants from fetal donors gave rise to surviving, proliferating pieces of brain stem in oculo that became vascularized within a few days. The transplants were mostly rounded and well demarcated and attached to the iris dilator plate. No signs of an immunological reaction to the transplanted tissue could be recognized. Other adverse reactions were very rare but occasionally there were corneal vascularizations, cataract formation or infections. Macro-ophthalmia was never found. The brain stem pieces seldom grew to more than twice their initial size. During the first week all transplants decreased in size and thickness, often to less than half their initial size (Fig. 3). After the first week the transplants increased in size for at least one month, after which their size was unchanged for 10 months (the longest postoperative time studied). For locus coeruleus transplants from fetal donors in sympathetically denervated eyes, four growth parameters were followed in more detail. The parameters were for comparisons between groups: mean C R L of donors; mean size of the dissected piece at transplantation; " f i n a l " mean size of transplant (that is, after at least 6 weeks in oculo); and surface area of host iris covered by central N A fibers. The mean C R L of transplanted groups was negatively correlated to the final size of transplants. Pieces from larger C R L stages increased their size less in oculo than did pieces from smaller C R L stages ( r = - 0 . 7 7 , Fig. 4a). The variation of initial size in individual groups and between groups

292

A. Seiger and L. Olson

m e a n size of 2 transplants (mm)

1.5'

160) (120)

0.5

weeks in oculo

Fig. 3. Mean size of fetal locus coeruleus transplants (donor CRL: 24-30 ram) in oculo at different times after transplantation. The size was calculated as the product of the largest diameter and its perpendicular counterpart, with compensatory reduction for very thin transplants (see Material and Methods). There is a large decrease in size of the transplants during the first week, followed by a recovery phase. It is typical for this CRL stage that the final size is similar to the initial size. Number of observations within parentheses. The points at approx. 2 and 6 weeks have 100 and 60 observations respectively, because some irides have randomly been picked for analysis in the fluorescence microscope at earlier stages

was small, for the f o r m e r m o s t l y < 5 % , whereas the v a r i a t i o n o f final size in i n d i v i d u a l g r o u p s a n d between g r o u p s was clearly larger. L o c u s coeruleus t r a n s p l a n t s f r o m postnatal donors survived a n d p r o l i f e r a t e d to a d e c r e a s i n g extent with increasing age o f the d o n o r s (Fig. 4a). T h e initial d e g e n e r a t i o n phase, also seen with fetal locus coeruleus t r a n s p l a n t s , was not f o l l o w e d by a recovery p h a s e in these groups. T h e size decreased in oculo until a steady state was reached with very thin r e m n a n t s o f the original pieces for all g r o u p s b u t the n e w b o r n d o n o r s , where a s u b s t a n t i a l piece o f b r a i n tissue survived. U s i n g fetal donors for D A t r a n s p l a n t s a decrease in final size was n o t e d with increasing C R L stage o f d o n o r s ( r = - 0 . 9 2 , Fig. 4b). I n the postnatal age r a n g e for d o n o r s o f D A t r a n s p l a n t s the survival was very limited. The final size was very small in all groups. 5 - H T t r a n s p l a n t s were only o b t a i n e d f r o m p o s t n a t a l d o n o r s (litter m a t e s o f the a n i m a l s used for D A transplants). A l l these t r a n s p l a n t s t o o k p o o r l y , a n d the final size was for all g r o u p s very small.

Correlations of Fluorescence Estimate with in vivo Observations T h r o u g h o u t the p r e n a t a l p e r i o d o f d o n o r age, locus coeruleus t r a n s p l a n t s p r o d u c e d a p p r o x i m a t e l y the same a m o u n t o f fluorescent fibers a n d the same

3

2.5

2

&E

Q o. e9

21.5 e N

0

c

g

0.5

a

0

t(

~'5

2~o

2~

3'o

3's "

crl (turn)

,

,

.,,*

i

born

~

3

1

h

weeks

i/Or

9

d o n o r stage

od,~l~

~c 2 o

| N 0

Z~

, 1'0

, 20

,

, 30 crl(mm)

, new

born

, T

, 2

, 3

, we4eks

d o n o r stage

Fig. 4. a Plot to show the negative correlation between final size (prenatal d o n o r s - a t least 6 weeks; postnatal d o n o r s - 4 weeks) of locus coeruleus transplants in oculo and donor stage, n = 4 - 2 5 , total number of observations approx. 400. (For the prenatal part: r = - 0 . 7 7 ) . h Decrease in survival and growth (expressed as mean final size) of D A transplants with increasing age of donor (for the prenatal part r = -0.92). However, for the prenatal donors initial size is also negatively correlated to final size, making the relative importance of C R L and initial size for final size unclear. In the postnatal part of the curve there is no negative correlation between final size and donor age, but here initial sizes are larger than the prenatal ones. n = 12-54, total number of observations 238

294

A. Seigerand L. Olson

total area covered by fluorescent fibers on sympathetically denervated host irides independent of CRL stage, initial size or final size of the transplants. There was no substantial correlation between fluorescent area and initial size or final size of individual transplants within any of the 15 groups tested (n= 4-23). Using postnatal donors for locus coeruleus transplants there seemed to be a positive correlation between the surface area covered by fluorescent fibers and the final size of the transplants, and thus also a negative correlation with the stage of development of donors. The corresponding groups of DA transplants and 5-HT transplants having postnatal donors showed no correlation between fluorescent area and final size of the transplants, but strong negative correlation with developmental stage of donors.

Time Course of Fiber Production on Host Iris by Fetal Locus coeruleus Grafts At one week the CNS transplant was rather loosely attached to the iris and could easily be pulled off without damaging the iris. In that way all fibers produced outside the CNS tissue piece could be seen. From 2 weeks posttransplantation on, the transplants were more rigidly attached to the iris and they could no longer be pulled off. The transplants in the five following groups had therefore to be cut away from the host iris, leaving a hole in the iris. The one week irides were therefore not directly comparable to the others regarding fluorescent surface area, amount of fibers and radioactivity, and will not be presented in Figure 5a and b. After one week the fluorescent fibers were very irregular, both in pattern and thickness (Fig. 6). Many thick short swellings reminiscent of growth cones could be seen on the part of the iris surface that had been in contact with the transplant. Very few fibers had grown beyond this area of the iris. No bundles of axons had formed. The fibers were weakly fluorescent at this stage of intraocular development. At two weeks small plexuses of fibers could be seen around the site of attachment of the transplant, now less irregular than the week before. Near the transplant the fibers were partly densely aggregated but at a short distance from the transplant they were more sparse (Fig. 7). The mean area covered by fluorescent nerves was 2.8 mm 2 (Fig. 5b). At 2 weeks both the 3H-metaraminol uptake and the fluorescent area were about 1/3 of the final maximal values (Fig. 5a and b). By four weeks post-transplantation most irregular swellings in the corona of fibers around the transplants had disappeared. The fluorescent area had increased moderately to a little less than half the final values (Figs. 5 b and 8). The increase in radioactivity was more marked and had doubled during the previous two weeks, reaching approx. 2/3 of the final values (Fig. 5a). Six weeks postoperatively both the fluorescent area and the uptake had reached final maximal values. No significant increase or decrease was encountered at 10 weeks or at 20 weeks (Figs. 5a and b, 9, 10). The fibers were now very densely aggregated close to the border of the transplant, and radiated

Growth of Transplanted Central M o n o a m i n e Neurons

295

LOCUS COERULEUS IN OCULO

70-

60.

50

=o 40

~" 3 0

n= 14-18 a 2(I i

I0'

locus tp gest. age

2

4

6

1'0

20 weeks in oculo

20

6"

-g E ~4 i3

n- 13-20

I

2i

IocusTp gest. age

2 20

4

6

1'0

2'0 weeks in oculo

Fig. 5. a Total mean uptake of aH-metaraminol by locus coeruleus fibers (CRL of donors : 24-30 mm) in sympathetically denervated irides at various times after transplantation. Uptake expressed as percent of corresponding uptake capacity in normal irides is linear during the first 6 weeks in oculo whereafter the uptake levels off at 60% of the uptake seen in normal irides, b Mean area of host irides covered by locus coeruleus fibers (CRL of donors: 24-30 m m same material as Fig. 5a) in sympathetically denervated irides at various times after transplantation. The area increases for 6 weeks, although slowly between 2 and 4 weeks postoperatively, after which it levels off at approx. 7.2 m m : . This means that the innervation will never be more than partial from these locus transplants

296

/~. Seiger and L. Olson

Fig. 6. The site of attachment of a fetal locus transplant one week postoperatively (CRL 27 30 mm). The iris tissue is intact beneath the removed CNS tissue. The fluorescent on the iris are mostly irregularly formed processes and a few beaded short processes. varicose fibers, plexuses or axon bundles can be seen. Sphincter margin to the left. mount. Montage of fluorescence microphotographs x 104

of donor: structures Few truly Iris whole

Fig. 7. Locus coeruleus fibers in a host iris 2 weeks after transplantation. The pattern of outgrowth is still irregular with beaded fibers and accumulations. Some varicose fibers can be seen, although not forming a true plexus yet. CRL of donor fetus: 27-30 mm. Fluorescence microphotograph of an iris whole mount x 135

Growth of Transplanted Central Monoamine Neurons

297

Fig. 8. Outgrowth from a locus coeruleus transplant 4 weeks postoperatively. A rather dense plexus of varicose fluorescent fibers can be seen, getting more sparse at a short distance from the transplant edge (not seen). Some strongly fluorescent accumulations are intermingled with the thin fibers and in the periphery a few smoother, more weakly fluorescent non-terminal axons are seen (above). CRL of donor fetus: 24 26 mm. Montage of fluorescence microphotographs of an iris whole mount • 135

298

A.. Seiger and L. Olson

Fig. 9. Branching axon bundles from a locus coeruleus transplant in a host iris far away from the transplant attachment. The bundles give rise to a sparse rather "sympathetic-like" network with only very few accumulations. Postoperative time: 6 weeks. C R L of donor fetus: 24-26 ram. Iris whole mount. Montage of fluorescence microphotographs x 135

Growth of Transplanted Central M o n o a m i n e Neurons

299

Fig. 10. Survey of the total outgrowth from a locus coeruleus transplant in a host iris. Close to the hole left by the extirpated transplant the fluorescence has a diffuse appearance, indicating CNS tissue remaining on the iris in a narrow zone. Axon bundles intermingled with fiber plexuses radiate out from this zone and become sparser at short distances from it. Postoperative time: 20 weeks. C R L of donor fetus : 27-30 ram. Iris whole mount. Montage of fluorescence microphotographs • 22

as a corona that became sparser at a short distance from that border. Sometimes large fluorescent axon bundles could be seen to follow the normal iris axon bundles. Occasionally these locus bundles grew for long distances (4-5 mm) parallel to the sphincter margin in the iris dilator plate. The mean final area covered by these locus coeruleus fibers was ~ 7 mm 2. Thus, the outgrowth of the locus coeruleus fibers was always restricted to an average area of 20-25% of the host iris, leaving the remaining 75 80% chronically adrenergically denervated. Although the range of fluorescent area covered by NA fibers was large, the pattern of growth was strikingly similar between individual irides. This corona-like pattern was reminiscent of the pattern of growth seen in vitro in ganglion explants. No late signs of degeneration of these fibers on the irides were recognized after up to I0 months in oculo. The correlation between radioactive label uptake and fluorescent area was high (r=0.8). Mean final uptake of 3H-metaraminol after 6, 10 and 20 weeks in oculo was 60% of that in the normal irides (Fig. 5 a). However, due to the wide range, individual extreme values clearly higher than those of normal irides were found (125-150%). The amount of fibers was not directly comparable to that seen in a normal sympathetically intact control iris, because of the discrepancy in distribution and density.

i

a

0

o.

0.5-

1.0-

1.5-

"~ 2.o-

2.5-

3,o-

35-

p o s t n a t a l age of d o n o r tissue

25

50

~. so

25-

75

100

75

SURVIVAL OF LOCUS COERULEUS IN O C U L O

0

2

I 2 p o s t n a t a l a g e of d o n o r

i

3 tissue

3

SURVIVAL OF DA-NEURONS IN O C U L O

4 leeks

4 weeks

C

0

01"

02"

03-

0.4

0.5

0.6

0.7

0.8

0.9

t

0

25"

50"

75-

postnatal

1

3

3

IN OCULO

age of d o n o r tissue

2

I

2

I

1

S U R V I V A L OF 5 - H T N E U R O N S

I

4 weeks

4 weeks

Growth of Transplanted Central M o n o a m i n e Neurons

301

Some transplanted irides had a larger fluorescent area than average, reaching 50% of the total iris surface. There was a close resemblance between the plots of areas and uptake over time (Fig. 5 a and b).

Growth of Locus Coeruleus Transplants as a Function of Postnatal Donor Age The survival and outgrowth from newborn locus coeruleus neurons was surprisingly good (Figs. l l a , 12 and 20). After 1 month in oculo many irides in this group had fuorescent areas close to the average areas found in experiments with fetal donors. With increasing postnatal age of donors the number of irides with surviving locus neurons rapidly decreased and the average amount of fiber growth decreased even faster (Fig. 11 a). Locus coeruleus transplants from 8 day old donors occasionally formed rather dense plexuses however, the amount of fibers averaging about half of prenatal transplant levels (Fig. 13). Only sparse plexuses were formed by the surviving locus transplants from donors of 15 or 22 days postnatal age (Fig. 14). In one eye out of 24 a few locus neurons taken from a 29 day old donor survived transplantation for 1 month, although there was a very limited outgrowth on the iris (Fig. 15). In a separate experiment 9 recipients were bilaterally transplanted with locus coeruleus derived from adult (200 g) donors. None of the 18 irides contained surviving locus neurons 21/2 months after transplantation. Macroscopically, most transplants from most postnatal donors disappeared altogether. Microscopically, the only remaining sign of the degenerated transplant was a concentration of autofluorescent macrophages at one spot, sometimes intermingled with a few monoamine neurons (e.g. Fig. 15). Sometimes the background fluorescence of the iris around the monoamine cell bodies was as low as that of the rest of the iris, indicating that no substantial CNS tissue survived except the monoamine neurons. It was clear t h a t the decline in final transplant volume occurred much earlier and proportionally faster with increasing donor age than the decline in the amount of adrenergic nerves formed on the host irides.

Fig. l l a - e . The decline in proportions of positive ( = M A nerve containing) irides (above) and mean relative a m o u n t s of fibers (below) produced by m o n o a m i n e neuron containing transplants obtained from donors with increasing postnatal age. Postoperative time 25-29 days. The mean relative a m o u n t s of fibers were obtained by pooling all individual irides and evaluating them together on a blind basis and are therefore comparable between the three graphs. N A , D A and 5-HT transplants from one week old donors all show a more marked decline in fiber production than in proportion of positive irides. Two week old and older donors give rise to very few fluorescent fibers, a N A fibers from locus coeruleus containing transplants. Initial sizes for the five different donor ages were (from youngest to oldest) approx. 1.25, 1.75, 2.25, 2.25 and 2.25 m m z. n = 2 4 at each point, b D A fibers from substantia nigra transplants. The initial sizes for the five different donor ages were (from youngest to oldest): approx. 3.5, 4.25, 4.25, 4.25 and 4.25 m m 2. n = 1 4 at each point, c 5-HT fibers from lower brain stem transplants. The initial sizes for the five different donor ages were (from youngest to oldest): approx. 3.25, 4.25, 4.25, 4.5 and 4,5 m m 2 respectively, n = 14 at each point

302

A.. Seiger and L. Olson

Fig. 12. Locus coeruleus transplant from newborn donor. Outgrowth of fluorescent fibers on a host iris after four weeks in oculo. The pattern and density of innervation around the edge after the extirpated transplant is very similar to that obtained after transplantation of fetal locus coeruleus to the iris. Some transplant tissue is left in a narrow zone closest to the hole. Sphincter margin to the left. Montage of fluorescence microphotographs of an iris whole mount x 75

Growth of DA and 5 - H T Transplants as a Function of Postnatal Donor Age ( F i g . 11 b a n d c). D A t r a n s p l a n t s f r o m n e w b o r n d o n o r s s u r v i v e d v e r y p o o r l y m a c r o s c o p i c a l l y . I n s p i t e o f t h i s , m o s t i r i d e s h a d s u r v i v i n g f l u o r e s c e n t cells

Growth of Transplanted Central Monoamine Neurons

303

Fig. 13. Regular plexus formation just outside the transplant border (upper left corner) four weeks

after grafting the locus coeruleus region from an 8 day old donor. Single varicose fibers in sympathetic-like pattern are intermingled with small axon bundles. Montage of fluorescence microphotographs of an iris whole mount x 84

densely packed in a small aggregate with m a n y terminal-like dots around the cells. There was a disproportionally p o o r outgrowth onto the irides from these small, often intensely fluorescent transplants (Fig. 16). This was even more marked with transplants from 8 day old donors. In the 15 day group only a few irides contained surviving fluorescent cells, now almost without processes, and all 14 irides in the 22 day group were without fluorescent cells (Fig. 11 b). In one of the irides with 29 day old donor-derived grafts, one surviving cell could be detected (Fig. 17). There was strong negative correlation between fluorescent area and age of donors for postnatal D A transplants whereas there was no correlation between fluorescent area and final size of these transplants. 5-HT transplants from newborn donors also survived very poorly macroscopically. However, the fiber production on the host irides was clearly greater than for the D A transplants. Often a small group of yellow fluorescent cells could be seen without very much additional CNS tissue. Plexus formation resembling that of newborn locus transplants could be seen, although mostly fibers were not as densely aggregated (Fig. 18). The fluorescence intensity of the cell bodies was mostly strong, but the delicate varicose fibers in the plexuses were weakly fluorescent in spite of the nialamid pretreatment. The proportion of irides with surviving 5-HT cells as well as the mean fluorescent area and total amount of fibers in the positive irides decreased rapidly with increasing age of donors, as with the locus coeruleus- and DA-transplants (Fig. 11 a - c and 20). In one iris with a 5-HT transplant from a 29 day old donor a few 5-HT cells survived transplantation. When dissecting the 5-HT transplants from the ventral part of the lower

304

A.. Seiger and L. Olson

Fig. 14. Remnant of a locus coeruleus transplant close to the sphincter margin (right) from a 22 day old donor after four weeks in oculo. Sparse plexuses can be seen surrounding the intensely fluorescent transplant and intermingled with a moderate number of macrophages (white dots). Montage of fluorescence microphotographs of an iris whole mount x 75

brain stem, some C A cells f r o m g r o u p A1 were almost invariably included, and therefore surviving C A cells were seen in all the 5-HT groups studied. In 10/14, 8/14, 3/14, 1/14 and 2/14 irides f r o m newborn, 8 day, 15 day, 22 day and 29 day old d o n o r s respectively, small groups or single C A cells with surr o u n d i n g processes were recognized. The C A cells were partly present on the same irides as the 5-HT neurons but were easily differentiated f r o m them because o f fluorescence colour and intensity. The C A cells were strongly fluorescent and p r o d u c e d strikingly few fibers which never formed plexuses (Fig. 19). In this respect they resembled the D A cells more than the 5-HT ones or the N A cells of locus coeruleus. These C A fibers also decreased in n u m b e r with increased d o n o r age. A c o m p a r i s o n o f the total fiber a m o u n t p r o d u c e d by the postnatal N A , D A and 5-HT transplants can be made f r o m Figures 11 a, b and c. The innervated areas obtained by these different transplants are directly c o m p a r e d to each other in Figure 20. It follows that N A transplants p r o d u c e d most fibers, 5-HT transplants less and D A transplants least.

Growth of Transplanted Central Monoamine Neurons

305

Fig. 15. Survival of a few fluorescent locus coeruleus neurons grafted from a 29 day old donor, after four weeks in oculo, The few fibers with CA fluorescence (small arrows) are intermingled with a large mass of macrophages at the site of the almost totally degenerated transplant. A couple of weakly fluorescent fibers can be seen outside the macrophage group (large arrows). Montage of fluorescence microphotographs of an iris whole mount x 135

Comparison of Fiber Production between Fetal DA and NA Transplants In o r d e r to o b t a i n a better c o m p a r i s o n b e t w e e n the a m o u n t o f fibers p r o d u c e d by N A a n d D A t r a n s p l a n t s , such pieces were p r e p a r e d f r o m the s a m e fetuses ( C R L 15-16 ram). I n this w a y the t r a n s p l a n t s c o u l d be t r i m m e d to exactly the s a m e initial size, they were o b t a i n e d f r o m exactly the s a m e stage, t r a n s p l a n t e d at the same time a n d f u r t h e r a n a l y s e d t o g e t h e r on a b l i n d basis. In vivo o b s e r v a tions r e v e a l e d t h a t the p r o l i f e r a t i o n was very g o o d a n d similar, giving, in the two g r o u p s , m e a n final sizes n o t differing f r o m each other. T h e locus grafts p r o d u c e d a 3-times larger m e a n fluorescent a r e a t h a n the D A grafts, a n d the m e a n d e n s i t y o f fibers in the locus irides was m u c h higher t h a n the m e a n d e n s i t y in the D A irides, giving a 6-fold difference in the m e a n a m o u n t o f fibers p r o d u c e d on the different irides ( N A : 3 . 6 + 0 . 3 , D A : 0.6_+0.2, 12 o b s e r v a tions in each group). O n the D A - g r a f t e d irides there was never a large density

306

A. Seiger and L. Olson

Fig. 16. Survival and outgrowth from a " D A transplant" obtained from a newborn donor after one m o n t h in oculo. The transplant, attached to the sphincter margin of the host iris, is intensely fluorescent and contains both cell bodies and processes. The outgrowth is very moderate and irregular without substantial plexus formation. Montage of fluorescence microphotographs of an iris whole m o u n t x 75

Fig. 17. Examples of isolated D A nerve cell bodies remaining on host irides 4 weeks after transplantation of the substantia nigra area from 8 day old (left and center) and 29 day old donor (right). These cells sometimes have irregular protrusions. The thin fluorescent fibers (right) are remaining sympathetic fibers, due to an incomplete sympathectomy and do not derive from the DA cell body. Fluorescence microphotographs x 135 (left), • 340 (center and right)

Growth of Transplanted Central M o n o a m i n e Neurons

307

Fig. 18. Sparse weakly fluorescent plexus of 5-HT fibers radiating out over the iris dilator plate from its transplant origin (lower left corner). Most fibers have a rather smooth appearance probably because of the M A O inhibition. Graft obtained from a newborn donor, postoperative time four weeks. Pretreatment with the M A O inhibitor Nialamid | 500 mg/kg 4 h before sacrifice. Montage of fluorescence microphotographs of an iris whole m o u n t x 135

Fig. 19. Survival and moderate outgrowth on a host iris of catecholamine neurons included in a "' 5-HT t r a n s p l a n t " from ventro-caudal medulla oblongata of a newborn donor. The CA neurons probably come from the A1 group of CA cells. Fluorescent accumulations and irregularly beaded fibers with very little plexus formation can be seen. Postoperative time: four weeks. Pretreated with the M A O inhibitor Nialamid | 500 mg/kg, 4 h before sacrifice. Montage of fluorescence microp h o t o g r a p h s of an iris whole mount x 104 6" =NA = 5"HT "I-" : D A

a

~= 2 -

new born

1

2

3

4

weeks

postnatal age of donor tissue

Fig. 20. Comparison of mean fluorescent areas covered by locus coeruleus N A neurons, DA neurons and 5-HT neurons obtained from 5 different postnatal donor ages after approx, four weeks in oculo. For NA, n = 2 4 , for DA and 5-HT, n = 1 4 . The outgrowth fiom locus coeruleus grafts covered the largest area on a sympathetically denervated host iris. DA transplants covered clearly the smallest area at all occasions. The 5-HT transplants were in between two the catecholamine types (see also Fig. 11)

Growth of Transplanted Central Monoamine Neurons

309

Fig. 21. Growth of DA fibers from a fetal graft (CRL of donor: 23-26 ram) on the sympathetically denervated host iris close to the intensely fluorescent transplant tissue (below). The distribution in the radiating zone is typical of fetal DA transplants with a relatively sparse plexus from the transplant edge. Postoperative time: 3 months. Montage of fluorescence microphotographs of an iris whole mount • 59

gradient from the transplant border towards the periphery (Fig. 21). Also large D A transplants with intense fluorescence in the transplant tissue showed an outgrowth consisting of relatively sparse plexuses growing from the transplant border. The individual D A fibers on the irides also looked smoother and had a lower fluorescence intensity than locus fibers. A more limited fiber production from D A transplants has also been observed at other prenatal donor stages. Thus, at all pre- and postnatal stages of donor age tested, the locus transplants produced markedly more fibers on a sympathetically denervated host iris than did D A transplants. Of particular interest were the occasional opportunities, probably caused by early fragmentation of transplants, to study in detail the total ramifications of single isolated M A neurons on the irides. Figure 22 illustrates such a case, where one locus coeruleus derived neuron was found at a considerable distance from the main transplant in an area not reached by fibers coming from the main transplant. As far as could be judged in the fluorescence microscope, the fluorescent nerve cell body was completely isolated, and no accompaning brain tissue could be observed. By one month, a rich network of varicose fibers was found close to the single fluorescent cell body, as well as many fibers leaving the cell body in various directions following the non-fluorescent axon bundles of the host iris.

Fig. 22. Single NA neuron derived from a fetal locus coeruleus transplant (CRL 22-24 mm). The strongly fluorescent nerve cell body on the host iris is situated well outside the area innervated by the main transplant, and therefore all fluorescent fibers that can be seen stem from this cell. No additional brain tissue was found. Arborizing varicose fibers form a small plexus around the nerve cell body and several axons run for long distances (also outside the figure) partly following the non-fluorescent axon pathways of the host iris (arrows). The main blood vessel seems to offer no particular guidance for the growing axons. Probably, this neuron is still growing since the postoperative time is only one month. Montage of fluorescence microphotographs of an iris whole mount x 122

Growth of Transplanted Central MonoamineNeurons

311

Discussion

Rat fetal NA, DA and 5-HT neurons survive homotransplantation to the eye, where they innervate the surrounding brain tissue transplant as well as nearby areas of the host iris (Olson and Seiger 1972b, 1973, 1974, 1975, 1976; Seiger et al., 1976). The present work offers methods of quantitating this type of nerve growth and describes, in detailed quantitative and qualitative terms, the fiber growth characteristics of central monoamine neurons at different stages of development after intraocular transplantation. The NA cell group of the locus coeruleus, the 5-HT cell groups in the raph6 and the DA cell groups of the substantia nigra in the rat all seem to go through their final divisions (Lauder and Bloom, 1974) before or around the time at which they can first be visualised with the fluorescence microscope (Olson and Seiger, 1972a). This suggests that mitotic activity does not substantially contribute to the rapidly increasing number of neuroblasts visualised by fluorescence microscopy after the 15th day of gestation. At day 15 all MA cell groups to be used for transplantation have already formed prominent long axon bundles (Olson and Seiger, 1972a; Seiger and Olson, 1973; Lauder and Bloom, 1975). The earliest stage used in the present study for preparation of transplants was the 16th day of gestation and therefore it is probable that most MA neurons in the dissected pieces were axotomized close to their cell bodies. Thus, the fiber formation in oculo by the MA neurons can be regarded as a combination of regenerative and developmental growth. A major obstacle to successful axonal regeneration after mechanical lesions in situ seems to be the extensive glial scar formation (Clemente, 1964; Guth, 1974), which is lacking in the anterior chamber transplant system. Also, probably contrary to the adult state, the neurons are rapidly growing when axotomized around birth. They are in the beginning of a genetically programmed phase of growth and demonstrate a large potential for growth upon explantation (Crain, 1966; Coyle et al., 1973; Sobkowicz et al., 1974a, b) as well as after transplantation to the anterior eye chamber (Olson and Seiger, 1972b, 1973; Hoffer et al., 1974, 1975, 1977; Seiger and Olson, 1975; Seiger et al., 1976; Olson et al., 1977), or to the brain (Das, 1974). More immature brain tissue showed a better survival and volume increase in oculo than older tissue. Around day 19-20 of gestation, there was just enough of an increase to make up for the initial volume loss that always occurred during the first few days after transplantation. Brain pieces from newborn and older donors decreased in volume in oculo. Using tissue from 4 week old donors, only thin, flattened granular remnants of the transplants remained in the eyes after longer time periods.

Nerve Growth Quantitation A meaningful evaluation of factors that influence central MA nerve fiber growth under various experimental conditions requires methods for quantitation of the nerve growth, yet surprisingly few attempts have been made to obtain

312

~. Seiger and L. Olson

quantitative data using Falck-Hillarp histochemistry. In the brain, the diffuse and widespread distribution of many of the MA neuron systems, makes the evaluation of total fiber production by a given set of neurons almost impossible. By transplanting small, well defined brain areas at precisely selected developmental stages to the anterior chamber of the eye it is possible to isolate specific groups of MA neurons from other interfering MA neuron systems. A large part of their nerve fiber production in oculo will occur in the iris of the host eye, that is in an almost 2-dimensional fashion, where quantitation of growth can be made by direct observation. The validity of the results presented here and in our forthcoming reports on factors that regulate fiber production by grafted central MA neurons (Seiger and Olson 1977a, b, c) rests on the method for growth quantitation presented here. It is based on three primary observations made in the fluorescence microscope on coded slides: 1. a schematic scale drawing of the innervated area on each iris indicating the density and distribution of fluorescent fibers around the transplant. A schematic drawing can be made almost to scale by relating the zone of outgrowth to the borders of the iris and to the hole after the transplant. This is the only practical way to obtain such pictures. Drawing using a projection unit or montages of microphotographs of each iris become impossible to use at the present state of development of fluorescence microscopical optics when large series of irides are involved. 2. A measurement of the area on the host iris covered by MA nerves. This measurement was made in the microscope using the field of view of the 10 x objective as measuring unit, and transformed into mm 2. One problem with this kind of measurement can be the precise delineation of the outer border of an innervated area. In the present case this source of variation was found to be minimal since the innervated area was usually well demarcated and therefore the zone of a dubious outer limit small in comparison to the total area. Moreover, independent area measurements of the same irides by different investigators yielded almost identical values. 3. The density of innervation in different zones of the innervated area, estimated using a semiquantitative scale. The density of the sympathetic ground plexus in normal irides served as an internal reference standard for the density estimations in each experiment. The validity of nerve density estimations by Falck-Hillarp histochemistry has been repeatedly demonstrated by comparisons with other methods of estimating the amount of nerve terminals (Olson et al., 1968; Olson and Malmfors, 1970; Lidbrink and Jonsson, 1971; Agnati and Fuxe, 1974; Nygren et al., 1974). Using a standard procedure to obtain the three growth parameters described above and some additional notes on special individual growth features, fluorescence intensities etc., some 6-10 irides could be processed per hour. One advantage of the procedure described here is that sets of irides from different experiments can be quantitatively compared to each other and any differences tested statistically. Copies of the original protocols are then cut and the drawings of all individual irides, incorporating area and density estimations, mixed at random and ranked for nerve amount on blind basis. It was found that area and density were so closely related (r = 0.98) that ranking could be made using area measurements alone. Decoding produced means of innervated areas and visual information on group variability.

Growth of Transplanted Central Monoamine Neurons

313

The fluorescence histochemical quantitation was tested by measuring the in vitro uptake of a labelled transmitter analogue, 3H-metaraminol, into the same irides (Olson et al., 1968). It has been demonstrated that the central MA fibers on irides possess essentially normal mechanisms for transmitter uptake and release (Seiger et al., 1976) and therefore the 3H-metaraminol uptake can be regarded as a second independent estimate of the amount of nerve fibers formed. The correlation between uptake and fluorescence histochemical data was found to be strong. Being a more indirect method, however, 3Hmetaraminol uptake has some disadvantages. High values caused by incomplete sympathetic denervations of host irides or transplant tissue remaining on the iris cannot be detected, and the presence of very few nerves (as was the case with older donor material, single cells and fibers) cannot be detected because the neuronal uptake becomes masked by the non-neuronal uptake.

Development of Iris Innervation The innervation of the sympathetically denervated iris by the grafted fetal central MA neurons seemed to start with no, or only a very short latency, as a large number of short irregular fibers were already seen one week after grafting. The number of fibers in the iris then increased almost linearly for another 4 to 5 weeks after which it rapidly levelled off. The morphology of the fibers also changed with time in oculo so that the irregularly beaded fibers with growth cone-like structures and accumulations typical of the one and two week survival times matured into a plexus of varicose fibers intermingled with axon bundles after longer survival times (up to 10 months). There was no net change in number of fibers at very long survival times and no change in morphology of the individual fibers, indicating neither substantial degeneration nor growth of the MA neurons after 6 weeks in oculo. On the host irides, there were never signs of immunological rejections comparable to the late fiber degenerations described by Svendgaard et al. (1975) in irides grafted to the brain. The final mean amount of nerve fibers growing on host irides using transplants from donors with similar CRL's was found to be constant and predictable. Although the range in the amount of nerve growth within a given experiment was large, there was a striking similarity in the basic pattern of outgrowth between individual irides. Several very characteristic features were found in all irides that had more than scattered fibers produced by the locus transplant. Possibly the most important of these features was the restriction of the outgrowth on the host iris to an area centered around the transplant. This area almost never exceeded one third of the total area of the iris, thereby leaving two thirds non-innervated. This indicates either that the sympathetically denervated host eye per se does not constitute a specific growth promoting stimulus for the immature lesioned central MA neurons, or less likely, that there is such a growth promotion, but that it ceases after 6 weeks since the neurons are thereafter "resting". This possibility is further supported by recent studies, where growth of such " r e s t i n g " intraocular MA neurons on the host iris has been re-initiated by the addition of iris grafts to the same eyes (Seiger and Olson 1977a, b). Interestingly enough, the reinnervation of such an iris graft by

314

A. Seiger and L. Olson

an intraocular mature locus coeruleus graft, will proceed until this second iris is fully covered with nerves although the innervation of the host iris by the same locus coeruleus graft will remain restricted. A similar widespread distribution of locus coeruleus-derived fibers is also obtained in irides which are grafted directly to the adult rat brain (Svendgaard et al., 1975). The various mechanisms which could underlie these distribution differences will be discussed in the following papers of this series. The restricted type of outgrowth from central fetal MA neurons on sympathetically denervated host irides found in the present experiments also contrasts markedly with the very organotypical reinnervation obtained after transplantation of sympathetic ganglia to the eye chamber (Olson and Malmfors, 1970; see also Silberstein et al., 1971). In the latter case the iris acts as a highly growth promoting and growth regulating stimulus and the growth response of the ganglion cells is shut off when the whole iris is covered by newly formed fibers of normal density. The immature central MA neurons are therefore evidently not sensitive to the same neurotrophic factors that causes the sympathetic adrenergic neurons to reinnervate a sympathetically denervated iris. It seems instead as if the time-course of growth by the grafted central MA neurons very closely follows the normal in situ time table, and this further underlines the strong inherent autonomous regulation of growth and differentiation present in various isolated CNS areas (De Long, 1970; Berry and Hollingworth, 1973; Sobkowicz et al., 1974a, b; Hoffer et al., 1974, 1975, 1977; Seiger and Olson, 1975; Olson et al., 1977).

Survival of Postnatal Grafts A remarkable and potentially important finding in the present study was that the central MA neurons could survive homotransplantation as late as one month after birth. Although survival and fiber production at this stage was minimal, the fact that some neurons did survive justifies new efforts to improve survival of more mature neurons. One critical factor may be the size of the graft. Multiple " s m a l l " pieces may receive better nutrition and give better growth results than the single " l a r g e " pieces used here. It is unclear why there was a certain survival of neurons from one month old donors, but no survival from three month old donors. One possible difference may be that MA neurons in one month old rats still exhibit some developmental growth which has ceased at three months. It is not impossible, however, to have fully mature 3 month old MA neurons surviving transplantation: Provided that they have matured in oculo, they will survive retransplantation if the whole host iris with its CNS graft is transferred to a new eye (Seiger and Olson, 1977a). In this case it was found that small, thin mature CNS grafts survived regrafting, while thicker larger grafts died. F r o m the results of this and the forthcoming report (Seiger and Olson 1977a) it would seem that transplantation of mature mammalian central nervous tissueonce regarded as impossible-should be looked upon as a technical problem, possibly a problem of nutrition and oxygenation. Our studies show that at least the

Growth of Transplanted Central Monoamine Neurons

315

central MA neurons have the ability to survive complete proximal axotomy and produce new fibers upon grafting from almost mature individuals. This is to our knowledge the latest stage of brain tissue grafting with partial neuronal survival that has been reported in mammals. References Agnati, L.F., Fuxe, K,: Quantitative comparisons of amine fluorescence in cortical noradrenaline terminals using smear preparations. J. Histochem. Cytochem. 22, 1122-1127 (1974) Berry, M., Hollingworth, T.: Development of isolated neocortex. Experientia (Basel) 29, 204-207 (1973) Clemente, C.D.: Regeneration in the vertebrate central nervous systems. Int. Rev. Neurobiol. 6, 257-301 (1964) Corrodi, H., Jonsson, G. : The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem. 15, 65-78 (1967) Coyle, J.T., Jacobowitz, D., Klein, D., Axelrod, J. : Dopaminergic neurons in explants of substantia nigra in culture. J. Neurobiol. 4, 461-470 (1973) Crain, S.M. : Development of "organotypic" bioelectric activities in central nervous tissues during maturation in culture. Int. Rev. Neurobiol. 9, 1 43 (1966) DeLong, G.R. : Histogenesis of fetal mouse isocortex and hippocampus in reaggregating cell cultures. Develop. Biol. 22, 563-583 (1970) Falck, B., Hillarp, N.-A., Thieme, G., Torp, A.: Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354 (1962) Guth, L. : Axonal regeneration and functional plasticity in the central nervous system. Exp. Neurol. 45, 606-654 (1974) Hamberger, B. : Reserpine-resistant uptake of catecholarnines in isolated tissues of the rat. A histochemical study. Acta physiol, scand., Suppl. 295, I 56 (1967) Hoffer, B., Olson, L., Seiger, A., Bloom, F. : Formation of a functional adrenergic input to intraocular cerebellar grafts: ingrowth of inhibitory sympathetic fibers. J. Neurobiol. 6, 565-585 (1975) Hoffer, B., Seiger, A.,'Freedman, R., Olson, L., Taylor, D.: Electrophysiology and cytology of hippocampal formation transplants in the anterior chamber of the eye. II. Cholinergic mechanisms. Brain Res. 119, 107-132 (1977) Hoffer, B., Seiger, A., Ljungberg, T., Olson, L.: Electrophysiological and cytological studies of brain homografts in the anterior chamber of the eye: maturation of cerebellar cortex in oculo. Brain Res. 79, 165 184 (1974) Iversen, L.L. : The uptake and storage of noradrenaline in sympathetic nerves. London: Cambridge Univ. Press 1967 Jonsson, G. : Quantitation of fluorescence of biogenic monoamines. Progr. Histochem. Cytochem. 2, 299 334 (1971) Lauder, J.M., Bloom, F.E. : Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. I. Cell differentiation. J. comp. Neurol. 155, 469 482 (1974) Lauder, J.M., Bloom, F.E.: Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. II. Synaptogenesis. J. comp. Neurol. 163, 251 264 (1975) Lidbrink, P., Jonsson, G.: Semiquantitative estimation of formaldehyde-induced fluorescence of noradrenaline in central noradrenaline nerve terminals. J. Histochem. Cytochem. 19, 747-757 (1971) Nygren, L.-G., Fuxe, K., Jonsson, G., Olson, L. : Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following 5,6-dihydroxytryptamine induced degeneration. Brain Res. 78, 377-394 (1974) Olson, L., Freedman, R., Seiger, A., Hoffer, B.: Electrophysiology and cytology of hippocampal formation transplants in the anterior chamber of the eye. I. Intrinsic organization. Brain Res. 119, 87-106 (1977) Olson, L., Hamberger, B., Jonsson, G., Malmfors, T.: Combined fluorescence histochemistry and 3H-noradrenaline measurements of adrenergic nerves. Histoehemie 15, 38-45 (1968)

316

/~. Seiger and L. Olson

Olson, L., Malmfors, T. : Growth characteristics of adrenergic nerves in the adult rat. Fluorescence histochemical and 3H-noradrenaline uptake studies using tissue transplantations to the anterior chamber of the eye. Acta physiol, scand., Suppl. 348, 1-112 (1970) Olson, L., Seiger, A.. : Early prenatal ontogeny of central monoamine neurons in the rat : Fluorescence histochemical observations. Z. Anat. Entwickl.-Gesch. 137, 301 316 (1972a) Olson, L., Seiger, A.: Brain tissue transplanted to the anterior chamber of the eye. 1. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons reinnervating the rat iris. Z. Zellforsch. 135, 175 194 (1972b) Olson, L., Seiger, A.: Development and growth of immature monoamine neurons in rat and man in situ and following intraocular transplantation in the rat. Brain Res. 62, 353-360 (1973) Olson, L., Seiger, A.: Nerve growth specificity and regulation as revealed by intraocular brain tissue transplants, ln: Dynamics of degeneration and growth in neurons (K. Fuxe, L. Olson and Y. Zotterman, eds.), pp. 499 508. Oxford and New York: Pergamon Press 1974 Olson, L., Seiger, /k. : Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens. Cell Tiss. Res. 158, 141 150 (t975) Olson, L., Seiger, ,&..: Locus coeruleus: fibre growth regulation in oculo. Med. Biol. 54, 142-145 (1976) Olson, L., Ungerstedt, U.: A simple high capacity freeze-drier for histochemical use. Histochemie 22, 8-19 (1970) Seiger, A., Olson, L. : Late prenatal ontogeny of central monoamine neurons in the rat : fluorescence histochemical observations. Z. Anat. Entwickl.-Gesch. 140, 281-318 (1973) Seiger, A., Olson, L. : Brain tissue transplanted to the anterior chamber of the eye: 3. Substitution of lacking central noradrenaline input by host iris sympathetic fibers in the isolated cerebral cortex developed in oculo. Cell Tiss. Res. 159, 325-338 (1975) Seiger, ~., Olson, L.: Reinitiation of directed nerve fiber growth in central monoamine neurons after intraocular maturation. Exptl. Brain Res. (1977a) (in press) Seiger, A., Olson, L. : Innervation of peripheral tissue grafts by lans coeruleus in oculo : Only partial correspondence with degree of sympathetic innervation. Brain Res. (1977b) (in press) Seiger, A., Olson, L. : Growth of locus coeruleus neurons in oeulo independent of simultaneously present adrenergic and cholinergic nerves in the iris. Medical Biol. (1977c) (in press) Seiger, A., Olson, L., Farnebo, L.-O.: Brain tissue transplanted to the anterior chamber of the eye. 4. Drug-modulated transmitter release in central monoamine nerve terminals lacking normal postsynaptic receptors. Cell Tiss. Res. 165, 157-170 (1976) Silberstein, S.D., Johnson, D.G., Jacobowitz, D.M,, Kopin, 1.J.: Sympathetic reinnervation of the rat iris in organ culture. Proc. nat. Acad. Sci. (Wash.) 68, 1121-1124 (1971) Sobkowicz, H.M., Bleier, R., Bereman, B., Monzain, R.: Axonal growth and organization of the mamillary nuclei of the newborn mouse in culture. J. Neurocytol. 3, 431 447 (1974) Sobkowicz, H.M., Bleier, R., Monzain, R.: Cell survival and architectonic differentiation of the hypothalamic mamillary region of the newborn mouse in culture. J. comp. Neurol. 155, 355-376 (1974) Svendgaard, N,A., Bj6rklund, A., Stenevi, U. : Regenerative properties of central monoamine neurons. Advances Anat. Embryol. Cell Biol. 51, 1-77 (1975)

Accepted September 6, 1976