236 M
demonstrated in the brain of several species of teleosts, represents a simple model characterized by well defined
including tilapia [38], sea bream [58], green molly [5], developmental stages and it is one of the major models for
salmon, trout and carp [12]. To our knowledge, there are the genetic study of early brain development.
no studies on the ontogeny of VIP in the brain of fishes, in spite of developmental models notably different with
respect to: i mother embryo relationships; ii presence
2. Materials and methods
of separate developmental phases embryonic and larval phases. The study of different developmental models may
2.1. Animals improve our information to better understand the roles of
VIP during the various phases of the development. Specimens of zebrafish, Danio rerio, in different stages
In the present study we have investigated the immuno- of development during the pharyngula period [24-h post
histochemical distribution of VIP in the brain of the teleost fertilization hpf], the hatching period 48 hpf, the larval
fish Danio rerio during development. To confirm the period day 7, day 15, and the juvenile period 1 month, 2
presence of brain VIP-like molecules, an immunoblot months, 3 months, were sampled from different aquaria, at
analysis was performed on larvae of 7 days. Danio rerio 25–288C. At least 5 animals were used for each stage.The
Fig. 1. Schematic sections through the brain, olfactory pit and retina of embryos of D . rerio during 24 hpf stage, a and b, frontal sections and 48 hpf stage
c and d, sagittal sections. Black stars indicate VIP immunoreactive perikarya. ce, cerebellum; di, diencephalon; div, diencephalic ventricle; hyp, hypothalamus; l, lens; me, mesencephalon; op, olfactory pit; p, pituitary; re, retina; rh, rhombencephalon; tg, tegmentum of the mesencephalon; te, tectum
of the mesencephalon; tel, telencephalon.
M . Mathieu et al. Brain Research 888 2001 235 –247
237
27
development stages were classified according to Haffter et contrast, preincubation of VIP antiserum with 10
M al. [30]. The fishes were anesthetized with tricaine
synthetic pituitary adenylate cyclase activating peptide methane–sulfonate MS 222, Sigma Chemical Co., MO,
PACAP Bachem did not change the intensity of the fixed in freshly prepared Bouin’s fluid at room temperature
immunostaining. No immunoreaction was observed when under vacuum for 10 min, followed by immersion in fresh
the VIP antiserum was replaced by nonimmune rabbit fixative for 4 h, or in 4 paraformaldehyde in cold
serum or PBS. Rat intestine sections incubated with VIP phosphate buffered saline PBS 0.2 M, pH 7.4. Paraffin-
antiserum exhibited several immunostained nerve fibers. embedded, 4 mm thick, serial sagittal, frontal or coronal
sections were mounted on chrome alum gelatin-coated glass slides.
Animal manipulations and experimental protocols were performed according to the recommendations of the Ethi-
cal Committee at our institution and under the supervision of authorized investigators.
2.2. Immunofluorescence and peroxidase–antiperoxidase procedures
The sections were rehydrated and processed for indirect immunofluorescence microscopy. Briefly, the sections
were rinsed in cold phosphate-buffered saline, preincu- bated with normal swine serum 1:50 for 20 min to reduce
non specific staining, and incubated in a dark moist chamber for 18 h at 48C with a polyclonal antiserum raised
against porcine VIP Biomeda, CA or with a polyclonal antiserum raised against pituitary adenylate cyclase activat-
ing peptide PACAP38 Peninsula, Belmont, CA. The antisera were diluted 1:200 in PBS, containing 1 BSA
and 0.3 Triton X-100. Rat intestine sections were incubated with the VIP antiserum as positive controls.
Then, the sections were rinsed several times in PBS and incubated for 1 h at room temperature with fluorescein
isothiocyanate-conjugated
swine anti-rabbit
gamma globulin
SAR Nordic
Immunology, Tilburg,
The Netherlands, diluted 1:100 in PBS. Finally, the sections
were rinsed twice in PBS, mounted in glycerol–PBS 1:1, and examined under a Zeiss epifluorescence microscope
Oberkochen, Germany. Several sections were processed according to the peroxidase–antiperoxidase PAP pro-
cedure. For this method, the sections were incubated with the primary antiserum as described above. The slices were
washed in PBS and incubated with SAR diluted 1:500 for 1 h at room temperature and then with the PAP complex
Dakopatts, Glostrup, Denmark diluted 1:300 in PBS for another hour. The enzymatic activity was revealed by
using 3,39-diaminobenzidine tetrahydro-chloride DAB Sigma containing 0.01 H O , in PBS solution. Nomen-
2 2
clature of brain areas was based on the work of Wullimann et al. [69].
Fig. 2. Schematic sagittal sections illustrating the anatomical localization of VIP-like immunoreactive perikarya black stars and nerve fibers
black dots in the central nervous system of the zebrafish, Danio rerio,
2.3. Specificity of the immunoreaction
during development: 7 days stage; 15 days stage; 1 month stage; 3 months stage. bo, olfactory bulbs; cc, crista cerebelli; CC , corpus
27
e
Preincubation of VIP antiserum with 10 M synthetic
cerebelli; hyp, hypothalamus; npo, preoptic nucleus; rh, rhombence-
vasointestinal peptide Bachem, Bubendorf, Switzerland
phalon; te, tectum of the mesencephalon; tel, telencephalon; tg, tegmen-
resulted in complete loss of the immunoreaction. By
tum of the mesencephalon.
238 M
2.4. Western immunoblot analysis homogenized in a Teflon-glass homogenizer in 20 vol of
0.01 PBS, pH 7.4, containing proteases inhibitors: ap- 2.4.1. Tissue preparation for immunoblots
rotinin 20 mg ml; Sigma, 5 mM PMFS Sigma, 50 mM Fishes, during larval period stage 7 day, were deeply
NaF Sigma, 2 mM EDTA, pH 8.0 Sigma and 0.2 mM anesthetized with MS 222, decapitated and the brain
NaVaO Sigma. The tissue homogenates were centrifuged
3
removed and stored at 2708C. Frozen brains were at 13 0003g at 48C for 10 min. A 6 ml aliquot of the
M . Mathieu et al. Brain Research 888 2001 235 –247
239
supernatant was used for protein determination using a that the brain of zebrafish, during development and in
micro BCA protein assay reagent kit Pierce, IL. The juvenile animals, does not contain any PACAP 38-like
remainder of the supernatant was diluted with 1 vol of immunoreactivity.
sodium dodecyl sulfate SDS reducing buffer 2 ml 0.5 M Tris–HCl, 1.6 ml glycerol, 3.0 ml 10 SDS, 0.8 M
3.1. Pharyngula period 24 hpf stage mercaptoethanol, 0.4 ml 0.05 Bromophenol Blue boiled
for 3 min and stored at 2708C until immunoblot analysis. During this stage of development, VIP-like positive cells
were observed in several regions of the brain, in the 2.4.2. Immunoblot procedure
olfactory pit and retina of D . rerio. In particular, a high
For VIP analyses, 20 mg brain supernatant, pepared as density of VIP-like immunopositive cells, exhibiting a
described above, were separated electroforetically on a strong immunoreaction, appeared in the olfactory pit Fig.
20 SDS–polyacrylamide gel Tris–Tricine buffer. The 3, level a in Fig. 1. The retina contained only scattered
separated proteins were transferred to a nitro–cellulose VIP-like immunopositive cells. Fig. 4, level a in Fig. 1.
membrane Bio-Rad Laboratories, Hercules, CA at 350 In the brain, a moderate number of VIP-like immuno-
mA for 2 h at 48C. The VIP was detected by incubating the reactive cell bodies was found in the dorsal telencephalon
nitrocellulose filter at 48C overnight with polyclonal VIP and within the dorsal and ventral regions of the dien-
antiserum Biomeda diluted 1:1000 with 5 milk TBS–T. cephalon Figs. 5 and 6, level a in Fig. 1. In the
Bands were visualized by subsequent incubations with mesencephalon, a few cells, exhibiting their processes
peroxidase-conjugated anti-rabbit gamma immunoglobulin directed laterally to the ventricle, were present within the
Vector Laboratories at room temperature for 2 h and then tegmentum. Similarly, scattered VIP-like immunoreactive
developed in a 1-Step E NBT BCIP plus Suppressor-sys-
cell bodies were detected in the medial region of the tem Pierce.
caudal rhombencephalon. During this stage, VIP-like im- VIP synthetic peptide Bachem was used as positive
munoreactive cells, exhibiting a strong immunostaining, control. Prestained molecular weight markers were ob-
first appeared in the anterior part of the pituitary Fig. 7, tained by New England Biolabs MA, USA.
level b in Fig. 1. The other regions of the brain did not show any VIP-like immunoreactivity.
3. Results 3.2. Hatching period 48 hpf stage
