Results 2. Hatching period 48 hpf stage

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 The distribution of VIP-like immunoreactive cells and At this stage of development, similarly to 24 hpf stage, a fibers in the brain, olfactory pit, and retina of the zebrafish, high number of VIP-like immunoreactive cells, exhibiting Danio rerio, was investigated in animals whose age varied a bright immunofluorescence, was found in the olfactory from the pharyngula period up to the juvenile period. pit Fig. 8, level c in Fig. 1. A few VIP-immunoreactive Incubation of sections with the VIP antiserum revealed the cells were occasionally found in the retina. In the brain, a presence of immunopositive cell bodies in all stages moderate number of bright fluorescent VIP-like immuno- investigated. By contrast, VIP-like positive nerve fibers reactive cells was found within the telencephalon Fig. 8, first appeared in the larval stages. No differences were level c in Fig. 1. In the diencephalon, during this stage, found between Bouin-fixed brains and paraformaldehyde- VIP-like immunopositive cell bodies were more concen- fixed brains. The anatomical distribution of VIP-like trated in the ventral region of the caudal hypothalamus immunoreactive perikarya and fibers in the brain of D . Fig. 9, level d in Fig. 1. In the mesencephalon, a larger rerio during the different stages of development is number of VIP-like immunoreactive cell bodies was schematically illustrated in Figs. 1 and 2. Incubations of present within the dorsal and ventral parts of the tegmen- brain sections with the antiserum agains PACAP38 showed tum Fig. 10, level d in Fig. 1. In the rhombencephalon, a Fig. 3. Frontal section of 24 hpf embryo of D . rerio showing the presence of VIP-like immunostained cells within the olfactory pit heads of arrow. Level a in Fig. 1. Paraformaldehyde-fixed tissue. op, olfactory pit; tel, telencephalon. Scale bar 100 mm. Fig. 4. Frontal section of 24 hpf embryo of D . rerio showing the occurrence of scattered immunopositive cells heads of arrow within the retina. Level a in Fig. 1. Paraformaldehyde-fixed tissue. Scale bar 200 mm. Fig. 5. Frontal section of a paraformaldehyde-fixed tissue through the brain of 24 hpf embryo of D . rerio, showing the presence of VIP-like positive cell bodies heads of arrow in the dorsal part of the rostral telencephalon. Level a in Fig. 1. tel, telencephalon. Scale bar 100 mm. Fig. 6. 24 hpf stage brain of D . rerio showing the presence of strong immunolabeled cells head of arrow within the ventral and dorsal regions of the caudal diencephalon. Level a in Fig. 1. Transverse section of a paraformaldehyde-fixed tissue. di, diencephalon; op, olfactory pit; tel, telencephalon. Scale bar 100 mm. Fig. 7. Transverse section through the 24 hpf embryo of D . rerio showing the presence of strong immunostained cells heads of arrow within the anterior pituitary. PAP immunoreaction. Level b in Fig. 1. Paraformaldehyde-fixed tissue. p, pituitary. Scale bar 50 mm. 240 M M . Mathieu et al. Brain Research 888 2001 235 –247 241 moderate concentration of brightly fluorescent VIP-like bulbs Fig. 15, level d in Fig. 2 was similar to that immunoreactive perikarya was detected within the medial described in 7-day-old larvae. By contrast, during this region. Similarly to 24 hpf stage, several VIP-like im- stage of development, several positive cells were detected munoreactive cells were found in the pituitary. in the retina at level of the inner layer Fig. 16. In the proper telencephalon, a small number of VIP-like positive 3.3. Larval period stages perikarya, exhibiting a weak immunofluorescence, was detected in the dorsal telencephalon Fig. 17, level e in 3.3.1. Day 7 Fig. 2. In the diencephalon, a small cluster of VIP-like During this stage, the distribution of VIP-like immuno- immunoreactive cells appeared in the periventricular pre- reactivity showed some changes as compared to that optic nucleus Fig. 17, level f in Fig. 2. A moderate described during the hatching period. A high density of number of positive cells was found in the ventral part of VIP-like immunoreactive fibers first appeared in the olfac- the caudal hypothalamus Fig. 17, level g in Fig. 2. In the tory bulbs, within the primary olfactory fiber layer. These mesencephalon, similarly to the previous stage, positive fibers, showing a bright immunostaining, were preferen- cell bodies were located in the tegmentum Fig. 17, level g tially distributed through the dorsal and medial region of in Fig. 2. More caudally, a population of positive the olfactory bulbs Fig. 11, level a in Fig. 2. Similarly to perikarya was found in the dorsal region of the rostral the previous stages, a high number of strongly immuno- rhombencephalon Fig. 17, level h in Fig. 2. As reported fluorescent VIP-like positive cells was found in the in the previous stages, a small group of immunoreactive olfactory organ. In particular, a high accumulation of cells was found in the pituitary, pars distalis. VIP-like immunoreactivity was observed in the external layer of the organ Fig. 12. As reported in the previous 3.4. Juvenile period stages stage of development, a few positive cells were found in the retina. In the telencephalon, very few VIP-like im- 3.4.1. 1 month 2 months munoreactive cells were found in the dorsal region. In In the brain of 1 month and 2 months old larvae, the contrast, the diencephalon contained a moderate concen- anatomical distribution and relative frequency of VIP-like tration of VIP-like immunoreactive cells at level of the immunoreactive elements was substantially similar. A ventral region of the caudal hypothalamus Fig. 13, level c dense plexus of VIP-immunoreactive fibers, showing a in Fig. 2. In the mesencephalon, VIP-like positive cells bright fluorescence, was found in the olfactory bulbs. Also exhibiting a moderate fluorescence were present within the the olfactory organ showed a high concentration of VIP- tegmentum Fig. 14, level b in Fig. 2. Occasional positive like immunoreactive cells. During these stages, VIP-like cell bodies were found in the ventral rhombencephalon. positive beaded nerve fibers first appeared through the Few immunoreactive cell bodies were present in the ventral telencephalon Fig. 18, level i in Fig. 2, whereas pituitary. During this stage of development, no VIP-like no VIP-like immunoreactive perikarya were found in the immunoreactive cell bodies and fibers were found in the telencephalon. In the diencephalon, similarly to the previ- other regions of the brain. ous stage, VIP-like immunoreactive cell bodies were observed within the periventricular preoptic nucleus. In 1 3.3.2. Day 15 month 2 months old larvae, the number of these cells was During this larval stage, the distribution of VIP-like higher if compared to that of 15-day-old larvae Fig. 19, immunoreactivity in the olfactory pit and the olfactory level l in Fig. 2. On the contrary, no VIP-like positive Fig. 8. Sagittal section through the 48 hpf embryo of D . rerio showing the presence of brightly immunofluorescent cells within the olfactory pit head of arrow. VIP-immunoreactive perikarya are present in the rostral telencephalon arrow. Level c in Fig. 1. Bouin-fixed tissue. div, diencephalic ventricle; hyp, hypothalamus; op, olfactory pit; te, tectum of the mesencephalon; tel, telencephalon. Scale bar 200 mm. Fig. 9. Sagittal section through the 48 hpf brain of D . rerio showing the occurrence of VIP-like immunopositive cells head of arrow in the diencephalon, at level of the ventral region of the caudal hypothalamus. Level d in Fig. 1. Bouin-fixed tissue. hyp, hypothalamus. Scale bar 200 mm. Fig. 10. Sagittal section of a Bouin-fixed tissue through the 48 hpf embryo showing the presence of immunoreactive perikarya in the tegmentum of the mesencephalon head of arrows. Level d in Fig. 1. tg, tegmentum of the mesencephalon. Scale bar 200 mm. Fig. 11. Sagittal section through the brain of a 7-day-old larva of D . rerio showing the presence of high densities of bright fluorescent VIP-like positive nerve fibers entering the olfactory bulb at level of the glomerular layer. Level a in Fig. 2. Bouin-fixed tissue. bo, olfactory bulb. Scale bar 200 mm. Fig. 12. Sagittal section of a 7-day-old larva of D . rerio showing a high accumulation of VIP-like immunoreactivity in the olfactory organ. Bouin-fixed tissue. Scale bar 100 mm. Fig. 13. Sagittal section of a Bouin-fixed tissue through the brain of a 7-day-old larva of D . rerio showing the occurrence of VIP-like immunoreactive cell bodies within the ventral region of the caudal hypothalamus head of arrow. Level c in Fig. 2. hyp, hypothalamus. Scale bar 200 mm. Fig. 14. This figure shows the presence of a group of VIP-like positive cells within the tegmentum of the mesencephalon head of arrow. Sagittal section of a Bouin-fixed tissue through the brain of a 7-day-old larva of D . rerio. Level b in Fig. 2. tg, tegmentum of the mesencephalon. Scale bar 200 mm. 242 M M . Mathieu et al. Brain Research 888 2001 235 –247 243 Fig. 20. Sagittal section through the brain of 3 months old larva of D . rerio showing the presence of a high number of bright fluorescent immunoreactive perikarya within the periventricular preoptic nucleus. Level m in Fig. 2. Bouin-fixed tissue. npo, preoptic nucleus. Scale bar 200 mm. Fig. 21. Sagittal section through the pituitary of 3 months old larva of D . rerio showing the location of bright fluorescent VIP-like immunopositive cells within the anterior lobe. Level n in Fig. 2. Bouin-fixed tissue. p, pituitary. Scale bar 200 mm. cells were found in the caudal hypothalamus, as well as in the mesencephalon and the rhombencephalon. These re- gions only contained few scattered positive nerve fibers. A moderate number of VIP-like immunoreactive cells, ex- hibiting a weak immunofluorescence, was observed in the pituitary, pars distalis. 3.4.2. 3 months During this stage of development, the distribution of VIP-like immunoreactivity in the olfactory organ and olfactory bulbs was similar to that described in 1 month 2 months stages. By contrast, a substantially larger number Fig. 22. Western blot analysis of VIP related protein of Danio rerio. Lane of VIP-like immunoreactive fibers was found in the ventral B: brain lysates of 7 days Danio rerio larvae; lane C: synthetic VIP telencephalon as compared with previous stages. Similarly, peptide; lane A: prestained molecular weight markers. a higher concentration of positive cell bodies was observed in the hypothalamus at level of the periventricular preoptic stages, a moderate number of immunoreactive cells, ex- nucleus Fig. 20, level m in Fig. 2. High densities of hibiting a bright fluorescence, was present in the pituitary, immunoreactive fibers were distributed in the ventral pars distalis Fig. 21, level n in Fig. 2. Outside these region of the caudal hypothalamus. As reported in previous Fig. 15. Sagittal section through a 15-day-old larva brain of D . rerio showing the presence of a high density of VIP-like immunoreactive fibers in the olfactory bulb. Level d in Fig. 2. Bouin-fixed tissue. bo, olfactory bulb. Scale bar 200 mm. Fig. 16. Sagittal section showing the presence of several VIP-like immunoreactive cells within the most internal layer of the retina head of arrow. Bouin-fixed tissue of a 15-day-old larva of D . rerio. Scale bar 200 mm. Fig. 17. Sagittal section through the brain of a 15-day-old larva of D . rerio showing the occurrence of scattered immunoreactive cell bodies head of arrows within the dorsal telencephalon level e in Fig. 2, the periventricular preoptic nucleus level f in Fig. 2, the ventral hypothalamus level g in Fig. 2, the mesencephalon level g in Fig. 2 and the dorsal part of the rostral rhombencephalon level h in Fig. 2. Bouin-fixed tissue. cc, crista cerebelli; CC , e corpus cerebelli; hyp, hypothalamus; npo, preoptic nucleus; te, tectum of the mesencephalon; tel, telencephalon; tg, tegmentum of the mesencephalon; rh, rhombencephalon. Scale bar 400 mm. Fig. 18. Sagittal section through the brain of 1 month old larva of D . rerio showing the occurrence of strong immunofluorescent nerve fibers within the ventral telencephalic area. Level i in Fig. 2. Bouin-fixed tissue. Scale bar 200 mm. Fig. 19. Sagittal section through the brain of 1 month old larva of D . rerio showing the presence of a group of brightly fluorescent VIP-like immunoreactive cells within the periventricular preoptic nucleus. Level l in Fig. 2. Bouin-fixed tissue. npo, preoptic nucleus. Scale bar 200 mm. 244 M regions, no VIP-like immunopositive material was found in and salmon [12] and VIP immunoreactive structures have the brain of zebrafish. been described in the olfactory bulbs of rat [21,24], hedgehog and sheep [2], brown bat [43] and chick [42]. In the retina of zebrafish, a transient expression of 3.5. Western immunoblot VIP-like immunoreactive cells occurred during pharyngula, hatching and larval stages. These cells ap- A western immunoblot analysis of VIP extracts from the peared early in the retina of 24 hpf embryos as scattered brain of embryos of zebrafish at 7 day of development elements and their number increased significantly in 15- revealed a protein band with a mobility corresponding to day-old larvae. By contrast, no VIP-like immunoreactive that of synthetic VIP Fig. 22. structures were found in the retina of juvenile fishes. Previously, VIP immunoreactivity has been demonstrated in amacrine cells of rabbit [15] and in the retina of snail [37] and highly selective VIP receptors have been de- 4. Discussion scribed in the retina of rat [64]. In rat, it has been