Table 1.
Antisera and antibodies used in the study.
Table 2.
VGF assay characterization.
Fig 1.
VGF molecular characterization.
Upper panels: immunoreactivity deteced by ELISA after HPLC. Note that in the cortex, TLQP immunoreactivity was mainly found in fractions having the same elution times of synthetic TLQP-21 and TLQP-62. In the hypothalamus, TLQP-21 and TLQP-62 were also detected but a major further uncharacterized form eluted at 19 min. Immunoreactivity for NERP-1 was found exclusively in one fraction having the same elution time as synthetic NERP-1. TPGH immunoreactivty was found in one MW form eluting at the same time as the synthetic peptide but also in some uncharacterized fractions. Lower panels: gel chromatography revealed a large form of 60kDa that was recognised by both the antisera raised against the two extremes of the proVGF in plasma, but not in the cortex. In the cortex, two further MW forms were revealed by the C-terminus antiserum corresponding to TLQP-62 and peptide V.
Fig 2.
VGF peptides abundance as detected by ELISA.
Values are group mean ± SEM. The short day (SD) produced a reduction in the VGF peptide levels that regards almost all the peptides in the cortex, NERP-1 and TPGH in the hypothalamus, and TPGH in the pituitary. [*p < 0.05, **p < 0.0005 vs long day (LD)]. N-terminus: p** = 0.0003; NERP-1:**p = 0.0003 and *p = 0.04; TPGH: **p = 0.0004, *p = 0.02; TLQP: *p = 0.008; C-terminus: *p = 0.01 in the cortex and 0.04 in the hypothalamus.
Fig 3.
VGF C-/N- terminus peptide localization in the hamster brain.
According to Morin and Wood, 2001 [36] coronal sections were stained at the level of (top to bottom) OVLT including area (a, b, c), preoptic area (d, e, f), supraoptic region (g, h, i, j, k), median eminence (ME, l, m, n) and arcuate nucleus (o, p, q, insert). VGF peptides are found in small axons and a number of nerve terminals of OVLT (a, C-terminus), and in the brain cortex within single punctuate cell bodies (b, N- terminus, frontal; c, C-terminus, parietal; arrows indicate small perikarya) as well as axons labelled by the VGF C-terminus only (c, C-terminus, parietal). Within the preoptic area, labelling was seen in the entire MPN (d, low magnification; C-terminus), within widely distributed axons and nerve terminals (e, f: C- and N- terminus, respectively), and small scattered perikarya (f, N- terminus arrows indicate the small perikarya). In the supraoptic region, labelling was abundant in many nuclei (g, low magnification, C- terminus) including Pa (h, C-terminus), SON (i, C- terminus) and SCN (j, N-terminus). In the same section, hippocampus CA3 (k, C- terminus) was rich in positive perikarya. ME (l, C- terminus) was labelled more intensively in the internal than in external layer, with Herring bodies brightly labelled. Perikarya were also visible in DM nucleus (m, C- terminus) and in an area just underneath the SON hence may compatible with LH (n, C- terminus). Immunopositive axons and nerve terminals (o, p; C- and N- terminus, respectively) were also found through the entire actuate nucleus and in amygdaloidei nuclei (q, C-terminus), in which scattered cell bodies were also visible (q, insert). Scale bars: a, c, f, m, p:100 μm; e, j, o, q: 100 μm; d, g, l, n: 200 μm; b, h, i, k, insert:100μm. SON: supraoptic nucleus, LH: lateral hypothalamic area.
Fig 4.
Localization of NERP-1, TPGH, and TLQP peptides in the hamster brain.
Coronal sections are at the levels of (top to bottom) the preoptic area (a, b, c), supraoptic region (d, e, f, g), median eminence (ME, h, i, j) and arcuate nucleus (k, m, l, n). Cell bodies of the cortex were immunoreactive for NERP-1 peptides (a), this was also present in axons and nerve terminals of the MPN (b) while the TPGH peptides were present in small axons of MNP (c). In the supraoptic region, TLQP peptides were found in perikarya of the SCN (d, e: low and high magnitude, respectively), and NERP-1 peptides in a few cell bodies of the Pa (f) and SON (g). In the ME: the internal layer was more intensively stained than the external with NERP-1 (h) and TPGH (i) antisera, while TLQP peptides were more intensively labelled in the external layer (j). In the arcuate nucleus containing section, small perikarya, axons and nerve terminals were stained by the TPGH (k) and TLQP antisera (l: axons only), while TPGH antiserum labelled a few cell bodies and axons within the LH (m) as well as in amygdaloid nuclei (n). Scale bars: a, e, m: 100 μm; b, k, l: 100 μm; c, d, g, h, i, j, n: 100 μm.
Fig 5.
Colocalization profiles of the VGF peptides in hamster brain and pituitary.
In the cortex, VGF C-terminus antiserum stained axons (a, red labelling) also positive for orexin antibody (b, green labelling), as well as perikarya (c, red labelling) containing ChAT (d, green labelling). In hypothalamic nyclei, NERP-1 (e, red labelling) and TLQP (g, red labelling) peptides, respectively in the Pa and SCN, were present in vasopressin cell bodies (f and h, respectively; green labelling, arrows identify colocalized cells). In the ME, NERP-1 (i, red labelling) and TLQP (k, red labelling) antisera stained vasopressin (j, green labelling) and somatostatin (l, green labelling) containing axons, respectively. In the anterior pituitary, VGF C-terminus antiserum labelled more cells in the LD than SD state (m versus n, respectively; red labelling). VGF C-terminus positive cells were found to contain LH (o versus p; red and green labelling, respectively) or ACTH (q versus r; red and green labelling, respectively). In the posterior pituitary, VGF C-terminus antiserum (s, red labelling) stained almost all vasopressin axons (t, blue labelling). Red, green and blue labelling reflects Cy3, Cy2 and AMCA, respectively. Scale bars: a, b, e, f: 100 μm; c, d: 50 μm; g–l: 100 μm m–t: 50 μm.