4.1. The NPY Precursor is Processed to Amidated NPY and CPON

As shown in Figure 2, gel filtration elution profiles of NPY immunoreactivity from the pineal gland as measured both by antiserum 337 and 8999 demonstrated a single peak with an apparent molecular size corresponding to that of synthetic porcine NPY. Filtration, collections and assays of pineal extracts resulted in similar elution positions of desamidoNPY- (identified with antiserum 337) and NPYamide-immunoreactivity (identified with antiserum 8999) indicating that the vast majority, if not all, of NPY-

Figure 2. Concentration of NPY- (upper panel) and NPYamide-immunoreactivity (lower panel) in fractions extracted from rat superficial pineals using Sephadex G-50 gel filtration. The two profiles are comparable and show that NPY is present only in an amidated form.

immunoreactivity is amidated NPY. No molecular species corresponding to proNPY was identified. Furthermore, CPON is found in intrapineal nerve fibres (Figure 3), which suggests that proNPY is cleaved to the two peptide fragments, and the relative concentration of the two fragments is only dependent on their degradation rate and not synthesis or processing.

NPY/CPON-immunoreactive nerve fibres were found in the meningeal tissue, in fibres surrounding pial arteries, in the choroid plexus adjacent to the deep pineal gland, and in the connective tissue connected to the superficial pineal gland (Figures 3 and 4). Positive nerve fibres were found close to the intrapineal vessels and intermingled between groups ofpinealocytes.

The rostral part of the pineal complex, comprising the body of the deep pineal gland together with the pinealocytes attached to the pineal stalk contained several NPY/CPON-immunoreactive fibres.

4.3. NPY in Ganglionectomised Rats

Radioimmunoassays revealed that the content of NPY was 1181.2 ± 83.2fmol/gland in normal pineals, and in the pineal of ganglionectomised rats 27.4 ± 3.5fmol/gland (Figure 5). This was in accordance with the results of Zhang et al. (1991) showing that the number of NPY-containing fibres in the superficial pineal was considerably lower in bilateral ganglionectomised animals than in normal animals.

In the deep pineal and the pineal stalk, the number ofNPY-immunoreactive fibres was only moderately decreased after superior cervical ganglionectomy (6). These fibres are likely to originate from the brain. Dual-labelling showed that NPY in the deep pineal gland of ganglionectomised rats were not co-stored with tyrosine hydroxylase (Figure 6) indicating that these fibres originate from those central NPY cells that are not catecholaminergic.

Figures 3-4. Photomicrographs at different magnifications showing sections of the rat superficial pineal gland immunostained for CPON (Figure 3) and NPY (Figure 4).

Figures 3-4. Photomicrographs at different magnifications showing sections of the rat superficial pineal gland immunostained for CPON (Figure 3) and NPY (Figure 4).

Normal SCGX

Figure 5. The content of NPY in extracts of the superficial pineal gland measure with radioimmunoassay. Effects of superior cervical ganglionectomy on content of NPY-immunoreactivity in the pineal gland.

4.4. NPY Receptors in the Pineal

Binding of sections with iodinated peptide YY (PYY) revealed the presence of a low concentration of binding sites in the rat superficial pineal gland (Figure 7). High levels of binding was observed in the hippocampus and the cerebral cortex.

RT-PCR analysis of pineal total RNA revealed expression of Y1 mRNA, but virtually no expression of Y2,Y4 or Y5 mRNA (Figure 8). In the anterior pituitary, expres-




Figure 6. Section of the deep pineal gland from a superior cervical ganglionectomised rat immunoreacted for both tyrosine hydroxylase (TH, A) and CPON (B). A positive CPON-fibre not co-storing noradrenaline is present in the pinealopetal projection originating from the brain.

Figure 7. Receptor autoradiograms (A) showing the distribution of 125I-PYY binding in the rat pineal gland (arrows).The content of binding in the pineal gland is relatively low compared to the brain. The lower figure (B) shows an adjacent section incubated with an excess of NPY.

sion of Y2 mRNA was dominant and in the hippocampus all cloned receptors apart from Y4 were expressed (Figure 8). This shows that the Y1 subtype is the only postsynaptic receptor amongst the NPY receptors subtypes known today that is expressed in the pineal gland.

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