/. Embryol. exp. Morph. Vol. 19, 2, pp. 121-35, April 1968
Printed in Great Britain
121
Analysis of the development of the nervous system
of the zebrafish, Brachydanio rerio
II. The effect of nerve growth factor and its antiserum
on the nervous system of the zebrafish
By JUDITH SHULMAN WEIS 1
From the Department of Biology, New York University
INTRODUCTION
Bueker (1948), investigating the effects of implanted tumors on the development of the nervous system of the chick embryo, found that sarcoma 180 caused
enlargement (hypertrophy and hyperplasia) of the spinal ganglia in the segments
adjacent to it. The motor columns, however, were not enlarged. He suggested
that the sarcoma provided a selective periphery with mechanical and histochemical properties which favored sensory innervation. Subsequently other
mouse sarcomas were found to have similar effects. Levi-Montalcini & Hamburger (1951) found that the sarcoma stimulated growth of the sympathetic
ganglia to a greater extent than the spinal ganglia. Similar results were obtained
when the tumor was grafted on to the chorioallantoic membrane, where the
tumor and embryo share circulation but establish no direct contact, so it was
hypothesized that a humoral factor from the tumor was responsible for the
enlargement of the ganglia (Levi-Montalcini & Hamburger, 1953). Subsequently
this group isolated a 'nerve growth stimulating factor' (NGF) from the sarcoma,
and devised an in vitro bioassay in which NGF caused outgrowth of fibers from
explanted embryonic spinal and sympathetic ganglia (Cohen, Levi-Montalcini,
& Hamburger, 1954). The factor was subsequently detected and isolated from
snake venom (Cohen & Levi-Montalcini, 1956) and from mouse submaxillary
salivary glands (Cohen, 1960).
An antiserum against this protein factor inhibits its action in tissue culture
(Cohen, 1960). Daily subcutaneous injections of antiserum into a variety of
mammals causes severe atrophy of the sympathetic chains (Levi-Montalcini &
Booker, 1960) suggesting a normal role of NGF in the growth and maintenance
of sympathetic neurons. Bueker, Schenkein & Bane (1960) have detected NGF
in serum and in a variety of organs, especially in the axial region, of mouse and
1
Author's address: Department of Zoology and Physiology, Rutgers University, Newark,
New Jersey, U.S.A.
122
j . s. W E I S
chick embryos. Winick & Greenberg (1965) have detected moderate amounts in
the axial region of young tadpoles, the sympathetic chain of newborn mice, and
the axial region of the goldfish.
The nerve growth factor has therefore been detected in representatives of five
main classes of vertebrates and has been shown to enhance the growth and
development of spinal and sympathetic ganglia in birds and mammals. Preliminary experiments in our laboratory with a variety of teleosts have indicated
that NGF is widespread in the spinal axes of these fishes. The experiments to be
presented here were undertaken to examine the effects of NGF and its specific
antiserum on the development of the spinal ganglia of the zebrafish, Brachydanio
rerio. The effect of NGF on sympathetic ganglia was also investigated. The
normal morphology and development of the nervous system in this species have
been described in the previous paper (Weis, 1968).
MATERIALS AND METHODS
In each experiment young fish from a single spawning of one male and one
female were raised on infusoria and microworms. When they were 2-3 weeks of
age the experimental work was started. They were anaesthetized, one at a time,
in a dilute solution of MS-222 (Tricaine methanesulfonate) and injected by means
of a micropipette attached to a micromanipulator. The operation was carried
out under a dissecting microscope at x 20 magnification.
Micropipettes were made by pulling capillary tubing of 0-7-1-0 mm diameter
in a microburner. A 45 ° bend was made in the shaft, and the pipette fitted into
the holder, with the point downward. The needle-holder was connected to a
glass syringe by means of a flexible plastic tube. The syringe, tube and needleholder were filled with distilled water before inserting the pipette into the holder.
Then the injection fluid was drawn up the shaft to the desired point by applying
negative pressure to the plunger of the syringe, thus leaving an air space between
the distilled water and the injection fluid. The anaesthetized fish was placed on
its side on a depression slide whose depression had been filled with paraffin.
The needle was then moved downward until it was immediately above the posterior part of the body cavity. By applying a steady pressure downward, the
pipette could puncture the body wall of the animal without injuring the internal
organs. By applying slow pressure on the piston of the syringe, the fluid was
injected into the body cavity of the animal. The fish was then placed in a fingerbowl of fresh water and would recover from the anaesthetic in a few minutes.
Agitation of the water could accelerate this process by increasing the flow of
water over the gills.
The effect of antiserum was tested on two broods of fish, one for each of two
experiments, designated Exps. 1 and 2. The effect of the nerve growth factor was
tested on three broods of fish. These investigations are designated Exps. 3-5.
In all experiments, approximately twelve fish were used as experimentals and
Zebrafish nervous system. II
123
the same number as controls. There was, however, a certain mortality of all
groups during the experimental period, and only those which survived the entire
time are considered.
In Exp. 1 ten fish from a single brood served as experimental fish, and ten
fish from the same brood served as untreated controls. In Exp. 2, a repeat of
Exp. 1, nine fish served as experimental fish, and seven from the same brood
served as untreated controls. At the time these experiments were done, normal
bovine serum was not available, and therefore it could not be used as an additional control. Experimental fish were injected twice weekly for 4 weeks with
0-1 fi\ of bovine anti-NGF serum (courtesy Abbott Laboratories, North Chicago,
Illinois) with a titer of 52000 anti-units/ml. (An anti-unit is the amount of
antiserum which inactivates one biological unit of NGF.) At the end of this
period they were fixed in formalin, dehydrated through graded ethyl alcohols
to xylene, embedded in paraffin, sectioned at 10/* from the operculum to the
post-anal region, and stained with Delafield's hematoxylin and counterstained
lightly with eosin. Some fish were fixed in de Castro's fixative and stained in the
block by de Castro's modification of the Cajal silver stain. Although this
technique stained the nerve fibers, the ganglia themselves were much clearer with
the hematoxylin stain. This silver impregnation technique was, in general,
unsatisfactory on fish material.
In Exp. 3 six fish from a single brood were injected three times weekly for 3
weeks with 0-1 jul of the CM-1 fraction of NGF (prepared from the salivary
glands of mice according to Cohen, 1960) with a titer of 10 biological units (B.U.)
per/d. Six other fish from the same brood were injected twice weekly for 4 weeks
with 0-1 fi\ of normal bovine serum, and nine fish from the same brood served
as untreated controls. The fish injected with normal bovine serum were treated
in this manner, paralleling the procedures in Exps. 1 and 2, as a belated control.
In analysis of data, however, these fish were contrasted with untreated controls
of the same brood. All were fixed in Bouin's fluid, then processed as above.
In Exp. 4 ten fish served as experimentals, seven fish from the same brood
served as serum-injected controls, and five from the same brood served as untreated controls. In Exp. 5 ten fish served as experimentals, and eleven fish from
the same brood served as untreated controls. In these two experiments, fish
were injected three times weekly for 3 weeks with 0-1 [A of the CM-3 fraction of
NGF (Cohen, 1960) which had been assayed at 100B.u.//d. Serum-injected
controls when used were injected twice weekly for 4 weeks, as in the previous
experiments.
All were fixed in Bouin's fluid, and most were processed as above. Some were
stained with the tribasic nerve stain described by Spoerri (1948). This combination of cresyl violet, toluidine blue and thionin has a special affinity for nerve cells
with their abundant basophilic Nissl substance. This staining technique further
delineated the structure of the nerve cells.
Ganglia of all fish were measured by means of paper reconstructions, as
124
J. S. WEIS
Table 1. Weights, in hundredths of a gram, of paper reconstructions of ganglia
1,2,3 and cloacal ganglion (cl), from fish injected with antiserum and untreated
controls
Specimen number!
C T
*
8
10
12
3
4
5
6
17
15
12
—
—
—
—
—
Eel
21
—
—
—
—
—
Cl||
C2
C3
27
32
27
15
26
20
Ccl
El
E2
C3
30
30
34
33
34
21
17
20
28
—
—
—
25
22
25
33
—
—
—
25
29
33
46
47
63
29
66
25
28
32
47
115
84
—
85
33
49
63
64
23
15
17
34
50
48
46
79
63
50
52
39
—
—
—
—
58
—
—
85
—
—
—
40
28
35
63
—
—
—
—
33
—
—
62
102
130
83
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ccl
47
Cl
C2
C3
Ccl
El
E2
43
43
32
48
10
39
E3
32
Eel
43
Cl
53
73
72
Ccl
El
E2
E3
Eel
Cl
C2
C3
18
2
E2
E3
C3
16
1
El§
C2
14
Ponied
A
(mm) Gang.
Ccl
El
E2
E3
Eel
Cl
C2
C3
Ccl
El
E2
E3
Eel
Cl
C2
C3
Ccl
—
—
—
32
37
45
45
99
94
94
16
24
22
39
—
—
—
—
—
—
—
—
—
110
31
52
64
64
94
94
139
107
—
39
49
70
103
56
77
57
70
101
129
108
—
—
—
—
80
77
—
—
—
—
111
156
93
—
—
—
—
87
80
—
—
88
99
100
126
—
—
—
—
—
—
—
—
123
—
—
—
180
—
—
—
—
—
—
—
—
70
97
66
64
159
Mean
S.E.
170
— )
150
12-0
21-0
22-3
26-6
240
32-3
25-8
24-5
26-6
42-8
46-6
51-3
35-6
64-3
32-6
410
40-2
47-2
890
83-6
830
99-3
44-5
59-3
61-3
701
820
1
mean
S.E.t
16-2
1-9
ify J
i &
ID
300
2-3
4Q-5
*+y J
39
-3-8]J
2-9 1
2-11
1-2J
3-5]
3 01
31
4-7J
2-n
6-21.
5-3
9-lJ
9-1]
31 ,
50 r
3-9J
18-4]
6-0' >
110
7-5J
7-4]
8-6
2-2
5-lJ
A(\.r)
tU L
^ y
RQ"?
5-3
CO.O
^•R
oy J
Jo 0
J O
120\
—
—
—
—
—
—
—
98-2
119-2
93-7
1-9
—
39
49
70
—
-1
—
—
—
—
—
—
—
—
—
—
88-5
22-5
1100
900
—
101-5
—
—
880
139-5
1000
142-5
460
100-8
13-5
8-2 J
52-6
90
y\j \J
1?Q
IL. y
101-5
21-5
—]
3-Oj
— '
21-5
•
—
— .
40-5
•
—
125-6
36-6
16-5J
* Standard length—snout to caudal peduncle
t Specimens in vertical columns 1-3 above from a single brood (Exp. 1). Fish in columns 4-6 from
another brood (Exp. 2).
J In 8 mmfishthe t value between experimentals and controls was 40, significant to 001. In 10 mm
fish the t between experimental and control was 4-3, in 12 mm fish the t between experimentals and
controls was 8-1, and in 14 mm fish the / between experimentals and controls was 6-4, all of which
§ E=experimental.
|| C=control.
are significant to beyond the 0-005 level.
Zebrafish nervous system. II
125
described in the previous paper (Weis, 1968). The first three typical spinal
ganglia and the one at the level of the cloaca were measured. Cell counts and
measurements of nuclear diameters of cells in ganglion 2 were performed as in
the previous paper. Counts were corrected for the spread of nuclei between
sections as in the previous paper. Also, for a limited number of fish the greatest
diameter of the caudal sympathetic ganglia just posterior to the cloaca was
measured.
140 r
120
100
ft 60
ctf
ft
40
5
20
18
16
10
12
14
Length of fish (mm)
Fig. 1. Relative size of spinal ganglia of antiserum-treated and control fish, x, Antiserum;
O, control.
RESULTS
Since observations on the development of the ganglia indicated a relationship
between the size of fish and the size of ganglia, and fish grew at different rates,
each experimental fish was compared with controls of the same size, as measured
by standard length (snout to caudal peduncle).
The effect of antiserum on sensory ganglia
The weights of paper reconstructions of ganglia numbers 1-3 and the
ganglion in the cloacal region ('cl') from Exps. 1 and 2 (antiserum injections)
are presented in Table 1. The cloacal ganglion can be number 10-13 and is most
frequently number 11. As can be observed, the control values from Exp. 1 (fish
numbers 1-3) and Exp. 2 (fish numbers 4-6) are very similar. Likewise, the values
9
JEEM 19
126
J. s. WEIS
for experimental fish are very similar in these two experiments. Consequently,
the data from the two experiments were pooled. Statistical analysis of data
indicated no significant differences between weights of ganglia 1-3 and 'cl' in
any group offish. The cloacal ganglion was frequently larger than the other three
ganglia, but when the largest and smallest ganglia were compared by a t test
the differences were not significant. In 8 mm controls the t between ganglia 1
and 'cl' was 2-5, in 10 mm controls the t between ganglia 3 and 'cl' was 2-7, for
12 mm controls the t between ganglia 3 and 'cl' was 1-2, and for 14 mm controls
the t between ganglia 1 and 3 was 2-0, none of which are significant to the 001
level. Therefore, values for all four ganglia were pooled and are shown in the
table and in graphic form in Fig. 1. These data indicate highly significant
differences between experimental and control values at each size level. For 8 mm
fish, the mean value (in hundredths of a gram) of control ganglia was 26-3 + 1-6,
( ± S . E . ) , whereas the mean value for experimental ganglia was 16-2 ±1-9, a
difference significant to the 0-01 level. In 10, 12 and 14 mm fish, control values
were 49-5 ±3-9, 89-3 ±5-3, and 100-8 ±5-3, respectively, as compared with experimental values which were 30-0 + 2-3, 40-2 ±2-9 and 58-8 ±3-8, respectively,
Table 2. Corrected cell counts and nuclear diameters in ganglion 2
of antiserum-treated and control fish
(mm)
Mean cell
countf
No.
specimens
8 Exp.
Cont.
10 Exp.
Cont.
12 Exp.
Cont.
14 Exp.
Cont.
16 Exp.
Cont.
18 Exp.
Cont.
51-1
54-9
50-4
761
52-8
96-6
761
1150
78-3
141-3
94-8
142-9
1
3
6
3
4
3
3
4
1
1
2
2
S.L.*
S.E.
11
4-9
2-2
2-4
12-3
1-7
7-9
—
201
14-3
Nuclear
diameters (ji)%
4-8
4-8
4-8-6-0
6-0-7-2
6-0-7-5
7-5
6-0-7-5
6-0-7-5
7-5
7-5
7-2-10-0
100
* Standard length.
t These data based on all neurons whose nuclei were present, counted in all sections (10 (i)
in which the ganglion appeared. Counts for left and right ganglia combined, then corrected.
% These data based on maximum-sized nuclei in sections through the middle of the ganglion
of all fish.
indicating a significant reduction in ganglion size (to 40-60 %) after antiserum
treatment. All values are significant to the 0-005 level. Fish larger than 14 mm
were so few in number that the results are not significant, but the same trends can
t>e observed.
Zebrafish nervous system. II
127
Cell counts and nuclear diameters for ganglion 2 are presented in Table 2.
The nuclear diameters of the largest neurons in the ganglia were quite similar
within any group of fish, with a maximum difference of 2-S/i observed. The
nuclear diameters are seen to increase with the size of the fish, but are not very
different in experimental and control fish of any given size. Cell number is also
seen to increase with the size of the fish, and there is, furthermore, a significant
difference between experimental and control fish. In 10, 12 and 14 mm fish,
for example, control ganglia averaged 76-1 ±2-2, 96-6 ±12-3 and 115-0 ±7-9
cells, respectively, whereas experimental ganglia averaged 50-4 ± 4-9, 52-8 ± 2-4
and 76-1 ±1-7 cells, respectively, indicating a significant reduction in cell number
in the ganglia of fish treated with antiserum. These differences are significant to
the 0-005 level.
The effect ofNGF on sensory ganglia
In Exp. 3 injections of the partially purified CM-1 fraction of NGF proved to
be quite toxic, and there was a great mortality of these fish. Furthermore, the
survivors appeared to be stunted. The largest experimental fish was less than
8 mm (standard length), whereas this is usually near the lower limit of size
reached by the end of an experiment. The growth and development of the
untreated and serum-injected controls of this brood were also retarded, specimens
reaching a maximum size of 10 mm. This retardation of the entire brood allowed
the effects of NGF injection to be seen quite strikingly. For example, in the 6 mm
controls the ganglia were still primitive in condition, appearing like very young
ganglia in which new cells are being added to pre-existing Rohon-Beard cells.
Cells for the most part were immature neuroblasts. However, in the NGFtreated fish the ganglia were more mature, approaching the adult condition in
tapering down ventrally from the broadest dorsal part. Rohon-Beard cells were
not present, and ganglion cells were more differentiated, appearing like bipolar
neurons with a large amount of cytoplasm. Ganglia from serum-injected fish
were similar to untreated controls in degree of development. This is further
borne out by a similarity of weights of reconstructions of serum-injected and
untreated controls. Therefore, the two are considered together as a combined
control.
Injections of the more purified CM-3 fraction did not produce the undesirable
side effects produced by the CM-1 injections. In addition, the general growth and
development offish in these two litters (Exps. 4, 5) were much better than in the
previous experiment, experimental fish reaching a maximum size of 12 mm and
controls reaching 16 mm.
The weights (in hundredths of a gram) of paper reconstructions of ganglia from
Exps. 3-5 are shown in Table 3. The values for control fish (both serum-injected
and untreated) from Exp. 3 (fish numbers 1-5), Exp. 4 (fish numbers 6-11) and
Exp. 5 (fish numbers 12-17) were very similar. Likewise, values for experimental
fish from all three experiments were similar. Therefore, results from the three
9-2
128
J. s. WEIS
experiments were pooled. When the values for the four ganglia were examined,
and the largest and smallest compared by a t test, the differences were not
statistically significant. In 4 mm controls between ganglia 2 and 3, t = 0-18;
Table 3. Weights in hundredths of a gram of paper reconstructions of ganglia
1, 2, 3 and cloacal (cl) from NGF-treated and control fish (bold figures indicate
controls injected with normal bovine serum)
Specimen numbert
S.L.*
(mm.)
4
6
8
10
12
14
1 0
,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
,
17 Mean
El
E2
E3
16
10
14
9 13
17 18
2115
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12-6
150
16-6
21
2-5
21
C2
C3
(2C\
El
E2
E3
Eel
Cl
C2
C3
Ccl
6
5
10
7
7
10
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
7-6
7-3
1-7
1-5
2118
35 19
36 17
22
9
10
9
7 11
8
8
—
9
—
—
—
—
9
6
7
6
—
—
—
—
13
6
10
9
—
—
—
—
6
9
7
6
13
12
13
12
—
—
—
—
18
17
23
23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
17-5
20-2
22-2
16-5
9-4
7-2
80
7-5
1-7
5-1
50
El
E2
E3
Eel
Cl
C2
C3
Ccl
26
34
37
31
16
14
10
12
—
—
—
—
17
19
20
22
—
—
—
—
14
19
14
25
—
—
—
—
—
—
—
—
26
23
22
33
16
17
17
15
—
—
—
—
16
16
16
16
—
—
—
—
18
19
18
17
—
—
—
—
13
20
15
17
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
26
34
26
25
17
19
19
15
24
28
28
26
12
25
17
26
27
37
38
28
11
12
16
22
26
37
28
42
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
25-8
32-2
29-8
30-8
15-3
17-8
16-6
191
0-4
2-3
2-6
2-5
0-7
10
09
1-4
El
E2
E3
Eel
Cl
C2
C3
Ccl
— — — —
— — — —
_ _ _ _ _
_ _ _ _ _
16 28 23 —
38 40 38 —
38 37 34 —
37 49 41 —
—
—
28
50
33
53
23
30
25
25
44
39
32
38
20
19
21
21
57
67
99
57
20
21
26
32
70
52
58
53
19
25
26
21
63
55
51
55
33
28
35
46
—
—
—
—
24
25
30
34
47
38
52
80
27
25
31
34
60
73
93
77
20
19
24
23
36
50
36
58
11
21
19
16
41
42
49
63
19
24
23
24
46
53
57
61
20
16
27
27
54
58
67
60
29
39
31
35
49-6
52-5
59-5
59-5
22-3
27-2
28-5
310
3-6
3-1
6-4
3-3
1-7 "k
201
1-5 j
^
El
E2
E3
Eel
_
_
_
_
_
_
—
—
52
_
_
_
_
9
8
_ _ _ _ 1 O 3
_
_
_
_
8
2
83
69
89
86
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
67-5
83-5
960
84-0
CNone
—
—
_
ENone
Cl
C2
C3
Ccl
—
_
_
E*
~~*
Gang.
^(OnC
Cl
C2
C3
Ccl
—
—
—
—
18
16
21
23
—
—
—
—
—
—
—
_
_
_
_
_
_
_
—
—
—
—
—
—
—
—
—
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
——
——
——
——
—
_ _ _ _ _ 9 0
_ _ _ _ _ 6 0
— — — — — 86
_ _ _ _ _
107
•
•
• •
80
77
92
89
—
—
—
—
—
—
—
—
—
—
—
—
——
——
_
—
_
6
5
_
_
—
56
4
7
60
_
_
—
—
—
63
—
—
5
5
5
6
54 —
—
i i
•"• ••
•
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
S.E.
in
11
no
0-9 I
0-6
0-9
15-5
14-5
70
20
_
_
—
59-5
59-5
56-5
570
—
3-5
4-5
0-5
30.
"
""•
•^^
^~~
~ ^
• " •
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
850
68-5
890
980
50
8-5
30
90
Pooled
mean s.E.J
...
7-5
1-6
19-3
1-
8-2
0-4
29-7
11
17-2
05
i4-/
27 2
'
I 0
82'8
5-7
581
1-3
* Standard length—snout to caudal peduncle.
t Fish in vertical columns 1-5 from one brood (Exp. 3), fish in columns 6-11 from another brood (Exp. 4), and fish in
columns 12-17 from a third brood (Exp. 5).
t In 4mm fish the t value between experimentals and controls was 3-5, significant to 001. In 6mm fish the / between
experimentals and controls was 6-0, in 8 mm fish the t between experimentals and controls was 10-4, and in 10 mm fish the
t between experimentals and controls was 11-4, all of which are significant beyond the 0-005 level.
Zebrafish nervous system. II
129
in 6 mm controls, between ganglia 1 and 2, t = 1-5; in 8 mm controls, between
ganglia 1 and 'cl', t =2-4; in 10 mm controls, between ganglia 1 and 'cl',
/ =2-5; in 14 mm controls, between ganglia 1 and 'cl', t =0-7; and in 16 mm
controls, between ganglia 2 and 'cl', t = 2-4; none of which are significant to
the 0-01 level. Therefore, the values for the four ganglia were pooled, and are
summarized in Table 3, and shown in graphic form in Fig. 2. These data show
a significant increase in ganglion size as a result of the NGF treatment. In 4 mm
controls the ganglia averaged 7-5 ±1-6 (S.E.), whereas experimental ganglia
averaged 14-8 + 1-3, a difference significant to the 0-01 level. In 6, 8 and 10 mm
controls, ganglia averaged 8-2 ±0-4, 17-2 + 0-5 and 27-2 ±1-0, whereas corresponding values for experimental ganglia were 19-3 ± 1-8, 29-7 ±1-1 and 54-7 ±
2-2, respectively, indicating, in many cases, a doubling in the size of the ganglia.
All differences were significant beyond the 0-005 level. There were too few
fish larger than 10 mm to provide significant results, but the same trends can be
observed.
100 r
80
60
40
43
20
4
6
8
10
12
14
16
Length of fish (mm)
Fig. 2. Relative size of spinal ganglia of NGF-treated and control fish, x, NGF; O, control.
Cell counts and nuclear diameters of cells in ganglion 2 are shown in Table 4.
Again nuclear diameters are seen to increase with the size of the fish, but in this
case there seems to be a consistent effect of NGF, resulting in larger cells in the
experimental fish than in controls at comparable sizes. Cell number can also be
seen to increase with fish size, and there is a striking increase in cell number in
NGF-treated fish over control fish at comparable sizes. In 4 mm fish control
ganglia averaged 14-1 ±0-8, whereas experimentals averaged 26-9 ± 1-8 cells. In
6, 8 and 10 mm fish controls averaged 24-9 ± 2-8, 45-2 + 3-8 and 61-8 + 2-6 cells,
respectively, whereas corresponding experimentals averaged 37-6 ± 1-7,63-8 ± 4-2
and 82-0 + 4-4, indicating substantial hyperplasia, as well as hypertrophy of
individual neurons. Differences were significant to the 0-005 level.
130
J. S. WEIS
The effect ofNGF on sympathetic ganglia
Sympathetic ganglia were also markedly enlarged by the NGF treatment. For
the 10 mm fish in which diameters of caudal sympathetic ganglia were measured
by ocular micrometer, the values for ten control fish are 12-0, 9-6, 10-8, 13-2,
9-6, 15-6,13-2,15-6, 12-5 and 13-2/*, averaging 12-5 ±0-6/*. The values for eleven
experimental fish are 15-6, 14-4, 12-0, 12-5, 15-0, 180, 19-2, 15-6, 22-8, 16-8 and
19-2/i, averaging 16-5 ±0-9/*. The difference is significant beyond the 0-005 level.
Table 4. Corrected cell counts and nuclear diameters in
ganglion 2 of NGF-injected and control fish
S.L.*
(mm)
4
6
8
10
12
14
16
Exp.
Cont.
Exp.
Cont.
Exp.
Cont.
Exp.
Cont.
Exp.
Cont.
Exp.
Cont.
Exp.
Cont.
Mean cell
count f
No.
specimens
26-9
141
37-6
24-9
63-8
45-2
820
61-8
109-3
3
3
4
5
6
10
11
15
2
0
0
2
0
2
900
90-8
S.E.
Nuclear
diameters (/*)
2-8
3-6^-2
2-4-3-6
4-8-6-0
3-6^-8
6-0-7-2
4-8-6-0
7-2
6-0-7-2
7-2-8-4
150
7-2-8-4
110
7-2-8-4
1-8
0-8
1-7
2-8
4-2
3-8
4-4
2-6
* Standard length.
t These data based on all neurons whose nuclei were present, counted in all sections (10 /i)
in which the ganglion appeared. Counts for left and right ganglia combined, then corrected.
% These data based on maximum-sized nuclei in sections through the middle of the ganglion
of all fish.
DISCUSSION
The spinal ganglia of B. rerio have been shown to be reduced in size as a
result of antiserum treatments. A response to antiserum has previously been
obtained only from sympathetic but not from spinal ganglia, although it has been
demonstrated that NGF is necessary for survival of dissociated spinal ganglion
cells in culture (Levi-Montalcini & Angeletti, 1963). In antiserum experiments
done by other investigators (Levi-Montalcini & Booker, 1960) the times of
treatment have been mostly on neonatal to adult stages, at periods when the
spinal ganglia were essentially mature, while the sympathetics were still developing. The mature spinal ganglion cells are therefore refractory to the antiserum.
In the zebrafish, with its late-developing spinal ganglia, it is possible to reduce
Zebrafish nervous system. II
131
ganglion growth by antiserum treatments. The spinal ganglia normally show a
continuing increase in cell number during their growth period, and this number
of cells can be reduced by antiserum treatments.
The cytological effects of antiserum on mouse sympathetic ganglion cells
were not seen in the fish spinal ganglia. In treated mammalian ganglia after
12 h of incubation with antiserum the nerve cells appear as a uniform undifferentiated population of young neuroblasts, with many pyknotic cells. Controls,
on the other hand, show all developmental stages from immature to differentiated
neurons. In treated ganglia degenerative processes increase with time, so that
the neuroblasts gradually disappear, and the ganglia consist mainly of satellite
cells (Levi-Montalcini, 1964).
The effect on fish spinal ganglia, however, was one of reduction of cell number,
though the cells appeared normal. Zaimis, Berk & Collingham (1965) have shown
a similar phenomenon in the coeliac ganglion of rats after antiserum treatment.
This prevertebral sympathetic ganglion was reduced in size due to a decrease in
cell density, though the nerve cells appeared normal. In the sympathetic chain of
mammals, however, the antiserum reduces the cell population to as little as
3-5 % of normal (Levi-Montalcini & Booker, 1960) and this atrophy is permanent, no regeneration taking place. The dosage used by these workers was 0-05
ml/g body wt per day. Assuming the potency of their antiserum was in the same
range as the Abbott preparation used in this study (52 000 anti-units/ml), one
can calculate 1500 anti-units were administered per gram mouse. In my experiments with fish weighing approximately 0-03 g, the dosage comes to only 150
anti-units/g, one-tenth as much per injection. It is possible that with greater
dosage more striking results could have been obtained.
Injections of NGF have been shown to increase cell size and cell number in
the spinal ganglia of the zebrafish. The response of the fish to NGF is due in a
large part to hyperplasia, whereas in mammals and chicks it is primarily due to
hypertrophy (Levi-Montalcini, 1964; Bueker & Schenkein, 1964). These
differences are compatible with the differences in the main method of growth of
ganglia in these different classes of vertebrates.
NGF injections into mammals and chicks have been at the level of 300-500
B.u./g body wt (Bueker & Schenkein, 1964). In my injections of NGF with a
titer of 100 B.u.//d into fish weighing about 0-03 g, the dosage is 300 B.u./g, the
same general level (Exps. 4, 5). In Exp. 3 the dosage was only one-tenth of this
level. However, the response seemed to be about the same as that in the succeeding experiments. This may indicate that there is a maximum response of which
the ganglia are capable, and at dosage levels above those which yield this
response no further hyperplasia or hypertrophy can be produced.
Although most of the quantitative data are from Exps. 4 and 5, perhaps the
most revealing results are seen in Exp. 3. It is interesting to note that the CM-1
fraction produced stunting and toxic effects in the fish, similar to those which had
been previously noted in mice (Bueker & Schenkein, 1964). The general retarda-
132
j . s. W E I S
tion of the entire brood including controls permitted the visualization of striking
qualitative differences in ganglion development in the NGF-treated and control
groups. At certain size levels controls still showed the primitive Rohon-Beard
system, whereas injected fish showed a development of ganglia approaching the
adult appearance. This acceleration of ganglion development and neuronal
differentiation cannot be seen so strikingly in Exps. 4 and 5. In these latter fish,
which had more typical growth patterns, the ganglia of controls have developed
normally. However, ganglia of experimental fish are much larger. The limited
data on size of the caudal sympathetic ganglia indicate that they also are
markedly enlarged by the NGF injections.
Goldberg (1963) compared the morphology ofNGF-treated and untreated
chick spinal ganglion cells, and found an increase in cell size, Nissl substance,
nucleolar size and nucleolar number. In the zebrafish nucleolar changes were
not noted, but the Nissl substance appeared to be denser in the treated cells as
seen in sections stained with the Spoerri stain.
It can be observed that the sizes of control ganglia are much larger in fish
from Exps. 1 and 2 than in fish from Exps. 3-5. This might be due to genetic
differences in the fish, but there are additional factors operative. Experiments
1 and 2 were done at the same time, in the spring of 1966, with two different
broods from the same breeding stock, and all fish were fixed in formalin,
dehydrated, etc., at the same time, and for the same length of time. Fish from
Exps. 3-5 were treated many months later, when a different breeding stock of
fish was used. There may be an effect of season as well as a genetic basis for these
differences. Also, fish from these latter experiments were fixed in Bouin's fluid
rather than formalin, and the mode of preservation could very likely influence
the sizes of the ganglia. Another source of variation might be due to the time
spent in the dehydrating solutions. Ganglion cells from all fish from Exps. 1 and
2 seemed to be less compact than those from subsequent experiments, indicating
that the formalin fixation may be responsible. This cell dispersal can partially
account for the larger sizes of the control ganglia from these broods as contrasted
with subsequent broods. Furthermore, cytological detail was not as well
preserved in these fish as in fish from subsequent experiments.
As has been shown, NGF from the salivary glands of mice evokes a response
in the ganglia of zebrafish. It has been previously noted by other workers that
NGF exhibits a lack of species specificity, since chicks and a variety of young
mammals have responded to the factor from mouse submaxillary salivary glands.
NGF in extracts of sympathetic ganglia of mouse, rat, cat, cow and man were
all precipitated by the same antiserum (Levi-Montalcini & Angeletti, 1961).
Winick & Greenberg (1965) state that NGF from axial regions of chick, tadpole
and goldfish were all immunologically similar to mouse NGF, as tested by agar
diffusion bands and inhibition of activity by incubation with antiserum to mouse
NGF.
It is not unusual for biologically important proteins to retain similar activity
Zebrafish nervous system. II
133
across phylogenetic lines. For example, the various protein hormones of
vertebrates, such as the growth hormone, gonadotropins and melanophorestimulating hormone, have been shown to act similarly among the various
classes. It is therefore not surprising that NGF should be effective in fishes.
However, Cohen (1960) found only partial cross-reactivity in Ouchterlony plates
of anti-mouse serum with snake venom NGF, indicating some differences in the
molecules. Salivary gland NGF was not inactivated by anti-snake venom,
further indicating antigenic differences between the molecules. Burdman &
Goldstein (1964) found only partial cross-reactivity of goat anti-mouse NGF
with NGF from the sera of children with neuroblastoma, a tumor of neural
crest origin. In this light, the conclusion of Winick & Greenberg (1965) from
Ouchterlony plate studies of NGF that the goldfish, tadpole, human, chick and
mouse growth factors have identical molecular structures seems improbable.
SUMMARY
1. When a nerve growth factor isolated from mouse submaxillary salivary
glands is injected into young zebrafish, Brachydanio rerio, it causes enlargement
of spinal ganglia due to hypertrophy and hyperplasia.A large part of the response
is due to hyperplasia, as compared with that of chicks and mammals, in which
the response is primarily hypertrophy of cells. The nature of the response to
NGF corresponds to the chief method of normal growth of ganglia in this
species.
2. Sympathetic ganglia of B. rerio are also markedly enlarged by treatment
with the nerve growth factor.
3. Injections of a specific antiserum to the nerve growth factor cause a
reduction in size of the spinal ganglia of the zebrafish. This decrease in size is due
to a reduction in the cell population of the ganglia, while the cells in the reduced
ganglion appear normal. An effect of antiserum has previously been noted in
other animals, but only on sympathetic ganglia.
RESUME
Analyse du developpement du systeme nerveux du Poisson-zebre,
Brachydanio rerio. II. Action dufacteur de croissance nerveuse et
de son antiserum, sur le systeme nerveux
1. Quand on injecte a de jeunes poissons-zebres {Brachydanio rerio) un
facteur de croissance nerveuse (NGF) isole des glandes salivaires sous-maxillaires de la souris, il provoque un accroissement de taille des ganglions rachidiens, par hypertrophie et hyperplasie. Une grande partie de l'effet obtenu
provient de l'hyperplasie, par comparaison avec celui qu'on obtient chez les
oiseaux et les mammiferes, ou la reaction consiste principalement en une
134
j . s. W E I S
hypertrophie des cellules. La nature de la reaction au NGF correspond au
principal mode de croissance normale des ganglions chez cette espece.
2. La taille des ganglions sympathiques de B. rerio est aussi nettement accrue
par le traitement au facteur de croissance nerveuse.
3. Des injections d'un serum specifique anti-NGF provoquent une reduction
de la taille des ganglions rachidiens du poisson-zebre. Cette diminution de la
taille est due a une reduction de la population cellulaire de ganglion, alors que
l'aspect des cellules du ganglion reduit apparait normal. On a note auparavant
un effet de l'anti-serum chez d'autres animaux, mais seulement sur les ganglions
sympathiques.
I wish to express my appreciation to Dr Alfred Perlmutter for his interest and helpful
suggestions throughout the course of this study. I wish to thank Dr Elmer Bueker for his
advice and the use of his equipment. My appreciation is extended to my husband, Dr Peddrick
Weis, for the use of laboratory space and for his constant interest and encouragement
throughout the course of this study. This work was supported in part by U.S.P.H.S. grants
NB-05755 and NB-03979. This paper is part of a dissertation submitted in partial fulfilment
of the requirements for the Ph.D. degree at New York University.
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LEVI-MONTALCINI,
{Manuscript received 16 June 1967, revised 17 October 1967)