Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia

Autonomic Neuroscience: Basic and Clinical 132 (2007) 52 – 62 www.elsevier.com/locate/autneu

Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia Yongling Li ⁎, Zhanyou Wang, Annica Dahlström Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Göteborg, Box 420, SE-405 30 Göteborg, Sweden Received 30 November 2005; received in revised form 19 October 2006; accepted 19 October 2006

Abstract Neuroendocrine secretory protein 55 (NESP55) is a soluble, acidic and heat-stable protein, belonging to the class of chromogranins. It is expressed specifically in endocrine cells and the nervous system, and is probably involved in both constitutive and regulated secretion. In the present study, we investigated the distribution of NESP55 in various rat sympathetic ganglia by immunohistochemistry. The expression of NESP55-IR was detected in a subpopulation of principal neurons in the rat SCG, which was also TH positive, and, thus, adrenergic. In the rat stellate ganglion, more than two thirds of NESP55 positive neurons were adrenergic. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP) in cholinergic neurons was also observed. In the rat thoracic chain, however, the majority of NESP55 positive neurons appeared to lack TH. No detectable NESP55-IR was found in the mouse SCG. Furthermore, in the sexually dimorphic SCG, it was demonstrated that, 80% of the NESP55 positive principal neurons were also NPY positive in the male rat, while a slightly higher, but statistically significant proportion, 87%, was found in the female. Whether or not this small difference is physiologically significant is unknown. The present data provide basic knowledge about the expression of NESP55 in the sympathetic autonomic nervous system of rat, which may further our understanding of the functional significance of NESP55. © 2006 Elsevier B.V. All rights reserved. Keywords: Chromogranins; Autonomic nervous system; Sex difference; Immunohistochemistry; Confocal laser scanning microscopy

1. Introduction The chromogranin family is a unique group of acidic, soluble and heat-stable secretory proteins. It consists of three classic granins, chromogranin A (CgA) (Blaschko et al., 1967; Schneider et al., 1967), chromogranin B (CgB) (Lee and Huttner, 1983), and Secrotogranin II (SgII, sometimes called CgC) (Rosa and Zanini, 1983), all of which have been intensively studied during the past decades (for review, see Winkler and Fischer-Colbrie, 1992; Taupenot et al., 2003). In addition, several other proteins, secretogranin III (1B1075) (Ottiger et al., 1990), HISL-19 (Krisch et al., 1986), 7B2 (Marcinkiewicz et al., 1985), and in particular, secretogranin VI, namely neuroendocrine secretory protein 55 (NESP55) (Ischia et al., 1997), are also considered ⁎ Corresponding author. Tel.: +46 31 773 3366; fax: +46 31 82 96 90. E-mail address: [email protected] (Y. Li). 1566-0702/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.10.010

members of the chromogranin family. It was demonstrated that chromogranins are widely distributed in various neuronal, endocrine and neuroendocrine tissues where they are present in large dense-cored vesicles (LDVs)/hormone storage granules together with various neuropeptides/peptide hormones (Fischer-Colbrie et al., 1985; Rosa et al., 1985; Cozzi et al., 1989; Marksteiner et al., 1993; Klimaschewski et al., 1996; Kato et al., 2000). The functional significance of these proteins is not yet completely established. However, several suggestions have been presented: 1. Chromogranins may play an important role in the sorting and aggregation of secretory products in the trans-Golgi network, as well as in the subsequent formation of secretory granules (Chanat et al., 1991; Ozawa and Takata, 1995). 2. Chromogranins in different tissues may serve extracellularly as precursors that are proteolytically processed to small-sized, biologically active peptides (Li et al., 1998, 1999; Leitner et al., 1999; Lovisetti-Scamihorn et al., 1999b), and most of them appear

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Fig. 1. Confocal images of adult rat SCGs (male) processed for immunofluorescence. (a) The primary NESP55 antiserum was omitted. (b) This section was incubated with preabsorbed NESP55 antiserum. (c) Section incubated with NESP55 antiserum. No specific fluorescence staining is seen in neither A nor B (Scale bar = 50 μm).

to exhibit inhibitory effects (Natori and Huttner, 1994; Taupenot et al., 2003). 3. From a clinical point of view, chromogranins are valuable indicators of sympathoadrenal activity and useful markers of secretion from normal and neoplastic neuroendocrine cells (Jakobsen et al., 2003; Taupenot et al., 2003). NESP55 shares similar features with other chromogranins. It is highly conserved among mammalian species and was first cloned from bovine chromaffin cells. The primary amino acid sequence comprises 241 amino acids and contains 6 pairs of dibasic amino-acid residues where NESP55 can be cleaved by endopeptidases (Ischia et al., 1997). The cellular localization and molecular forms of NESP55 have been studied in different species, for instance, in cow, rat, and pig (Bauer et al., 1999a,b; LovisettiScamihorn et al., 1999a; Li et al., 2002). NESP55 is present in the adrenal medulla, pituitary, various brain regions, spinal cord, splenic and sciatic nerves, pancreas, etc. In these tissues NESP55 partly overlaps with neurotransmitters and other neuropeptides. The processing of NESP55 is comparatively limited, except in the posterior pituitary and the vas deferens where the free peptide GAIPIRRH, a proteolytic product of NESP55 at the C terminal end, is the predominant molecular form (Lovisetti-Scamihorn et al., 1999a; Li et al., 2002). Within the brain, NESP55 is expressed mainly in ontogenetically old parts, such as the brain stem and the hypothalamus, but has not been found in cortical areas, the hippocampus or the cerebellum (Bauer et al., 1999a). Interestingly, NESP55 was found in vesicles of slightly lighter density, in addition to its presence in LDVs. The authors, therefore, suggested that NESP55 was probably involved in both regulated and constitutive secretory pathways (Fischer-Colbrie et al., 2002; Eder et al., 2004). It is worth mentioning that NESP55 was previously speculated to

be the precursor of Leu–Ser–Ala–Leu (LSAL), a peptide with 5-HT1B receptor antagonist activities. This peptide is flanked within bovine NESP55 by lysine residues that can serve as proteolytic cleavage signals (Ischia et al., 1997). The subsequent report from the same laboratory, however, demonstrated that LSAL was mutated to Leu–His–Ala– Leu (LHAL) in rat and man (Fischer-Colbrie et al., 2002). NESP55 is genomically imprinted and expressed exclusively from the maternal allele (Hayward et al., 1998). A recent study reported that NESP55-deficient mice developed without obvious phenotypic signs and were fertile. However, these mice showed increased curiosity/excitement toward a novel environment, but spent less time exploring the new surrounding. Thus, NESP55 may play a role in the control of specific types of behavior in adult mice (Plagge et al., 2005). We previously demonstrated that NESP55 was expressed in the rat superior cervical ganglion (SCG) (Li et al., 2002). Now we extend the study by comparing the distribution of NESP55 in various rat sympathetic ganglia, as well as in the Table 1 Prevalence of NESP55 positive neurons in various rat sympathetic ganglia Ganglia

NESP55 + neurons in rat sympathetic ganglia Population size

SCG

17 ± 3% (13~19%) Stellate 8 ± 1 % (6∼9%) Thoracic 5 ± 2 % (3∼7%)

Proportion of NESP55 + neurons containing TH

NESP55 + neurons scored (in total)

Scattered

Almost 100%

2674

Scattered

82 ± 6% (75∼90%) 22 ± 7% (15∼30%)

1379

Distribution pattern

Grouped

323

The percentages above are based on cell counts in individual sympathetic ganglion from a total of 4 rats (two males and two females). Values are expressed as mean ± SD and ranges.

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mouse SCG. A special focus was the expression of NESP55 in the rat SCG in relation to neuropeptide Y (NPY), a peptide, which is widespread in vasoconstrictor neurons in sympathetic ganglia. A possible difference concerning the expression of NESP55 and NPY in the rat SCG between adult male and female is presented. 2. Material and methods 2.1. Animals Adult (10–11w) Sprague-Dawley rats (10 females and 7 males purchased from B and K Universal) and 6 male BALB/ c mice (20–25 g, purchased from Mollegaard Breeding Center, Denmark) were used in this study. The animals were housed on a 12 h light/dark cycle with food and water available ad libitum. All experimental procedures were approved by the Animal Ethical Committee of University of Gothenburg (for rat experiments) and University of Aarhus (for mouse experiments). All efforts were made to minimize animal suffering and the number of animals used. 2.2. Tissue preparation Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, the superior cervical ganglia in three mice were surgically exposed and injected with 2 μl colchicine (1% in distilled water) in order to facilitate the detection of NESP55 in the ganglia by blocking export. After operation, the animals were allowed to

survive for 24 h before sacrifice. Three un-operated mice were used to study the normal level of NESP55 in the superior cervical ganglion. The rats and mice, both operated and unoperated, were anaesthetized and transcardially perfused with 4% paraformaldehyde (pH 7.4). The SCG in the mice and the SCG, stellate ganglia, thoracic sympathetic chain (thoracic ganglia), and celiac ganglia of the rats were dissected and postfixed in the same fixative overnight and then stored at 4 °C in a PBS solution containing 0.1% sodium azide and 20% sucrose. The ganglia were frozen with compressed CO2, sectioned in a cryostat at 10–12 μm, and mounted on gelatinized glass slides for immunohistochemistry. 2.3. Immunofluorescence procedures Indirect immunofluorescene incubation was carried out using the following primary antibodies: Guinea pig anti-NESP55, recognizing the free terminal end (GAIPIRRH) of NESP55, dilution 1:4000. The antiserum reacts not only with the free peptide GAIPIRRH, but also with intermediate- and large-sized peptides including the precursor NESP55, containing GAIPIRRH at their C-terminal end (Ischia et al., 1997; Eder et al., 2004). Sheep anti-TH, produced against the native TH from rat pheochromocytoma (Chemicon), dilution 1:800. Rabbit anti-TH, isolated from human pheochromocytoma (donated by Dr M Goldstein), dilution 1:800.

Fig. 2. Confocal images of adult rat SCGs. The distribution of NESP55-IR is showed in both male (a) and female (b) rats. NESP55 positive principal neurons are scattered throughout the sections. (a′ and b′) High magnification images from the areas in (a) and (b) indicated by arrows. NESP55-IR in the principal neurons is predominantly distributed in the perinuclear regions. Nerve fiber structures containing NESP55-IR are also evident. Scale bars = 10 μm (a′, b′), 100 μm (a, and b).

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Rabbit anti-NPY, produced against synthetic NPY (Sigma N9528), dilution 1:4000. Rabbit anti-choline acetyltransferase (ChAT), produced against ChAT (Chemicon), dilution 1:400. Rabbit anti-calcitonin gene related peptide (anti-CGRP), produced against synthetic CGRP (Genosys CA-08-220), dilution 1:400. Rabbit anti-secretoneurin (anti-SN), produced against rat secretogranin II, sequence 154–186, dilution 1:800 (Kirchmair et al., 1993). Mouse anti-transGolgi network 38 (anti-TGN38), recognizing the trans-Golgi network (Oncogene), dilution 1:800. The secondary antibodies used in the study were: Biotin-conjugated AffiniPure donkey anti-guinea pig IgG, dilution 1:200. Texas Red-conjugated AffiniPure (TxR) donkey antirabbit IgG, dilution 1:50. Texas Red-conjugated AffiniPure (TxR) donkey antimouse IgG, dilution 1:50. FITC-conjugated AffiniPure (FITC) donkey anti-rabbit IgG, dilution 1:50. Cy™5-conjugated AffiniPure (Cy5) donkey anti-sheep IgG, dilution 1:50.

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of NESP55 antiserum, sections were incubated with the antiserum preabsorbed with the peptide fragment of NESP55 used for immunization (GPIPIRRH). No specific immunofluorescence was observed (Fig. 1). 2.4. Cell count and statistical analysis Eight ganglia from four rats of either sex were used for investigating the distribution of NESP55-IR (Immunoreactivity) in relation to NPY-expression in the rat SCG. Three to five sections were randomly selected from each ganglion at 100 μm intervals. Cells co-labeled with anti-NPY and anti-NESP55, as well as cells labeled with only one of the two antibodies, were counted throughout the sections. The ratio of co-expression of NPY-IR in the NESP55 positive cells, as well as of NESP55IR in the NPY positive cells, was examined. The data were expressed as mean ± SD and compared between male and female using ANOVA (single factor). A p-value b0.05 was considered statistically significant (Fig. 6). Sections from the SCG, stellate ganglia, as well as midthoracic ganglia of the rats were incubated with anti-NESP55 and anti-TH. The NESP55 positive neurons, TH positive neurons, NESP55+/TH+ neurons, and the whole cell population in the ganglia were counted as described above (Table 1). 3. Results

All the secondary antibodies were purchased from Jackson ImmunoResearch. Single staining with anti-NESP55, double staining with anti-NESP55 together with a variety of rabbit/mouse primary antibodies listed above, as well as triple staining with antisera against NESP55/NPY/TH, were carried out as described previously (Li et al., 2005). All the procedures were performed in room temperature. Briefly, the sections were pre-incubated with normal donkey serum for 1 h. The primary antibodies were then applied overnight. After washing, the secondary antibodies, Biotin-anti-guinea pig for single staining, Biotin-antiguinea pig/TxR-anti-rabbit (or-mouse) or FITC-anti-rabbit/ TxR-anti-mouse for double staining, and Biotin-anti-guinea pig/TxR-anti-rabbit/Cy5-anti-sheep for triple staining, were added to the sections for 2 h. This step was followed by incubation with Fluorescein (DTAF)-conjugated streptavidin (Jackson ImmunoResearch), diluted to 1:800, for 1.5 h. Between each incubation step the sections were rinsed in 0.01 M PBS (3× 15 min.). All antibody dilutions contained 1% bovine serum albumin, 0.1% sodium azide, and 0.2% Triton X100 to allow penetration intracellularly. Finally, the sections were mounted with anti-fading medium (Mowiol/DABCO) and examined in a CLSM (Bio-Rad MRC 1024). Images were processed using Adobe Photoshop (Version 5.5). Control experiments were carried out in every incubation series. The sections were processed in the same way except that primary antibodies were omitted from the initial incubation. No specific fluorescence staining was observed in the control sections (Fig. 1). In addition, to further demonstrate the specificity of the immunostaining pattern

3.1. Localization of NESP55-IR in the rat SCG and its colocalization with other peptides and neurotransmitters NESP55-IR was observed mainly in medium-sized principal neurons scattered throughout the ganglia, distributed in the perinuclear region with a granular appearance. In some of these cells NESP55-IR was also present in the peripheral cytoplasm in small granules. The intensity of NESP55-IR varied between neurons. Some nerve fiber structures were also reactive for NESP55. No NESP55-IR was observed in nerve terminals or varicosities (Fig. 2). NESP55 positive neurons, as well as NESP55 positive fibers, were also TH positive, although the intensity of TH-IR was variable. Compared to the distribution pattern of NESP55-IR, which was restricted to the perinuclear area, TH-IR was evenly distributed in the cytoplasm. NESP55 positive neurons, apparently lacking TH-IR, were rarely seen (Fig. 3). A subpopulation of principal neurons was observed to contain both NESP55-IR and NPY-IR. NPY-IR, with a granular appearance like that of NESP55-IR, was highly concentrated in the perinuclear regions, and also present in the peripheral cytoplasm. All NPY positive cells were TH positive (Fig. 3). Small intensively fluorescent (SIF) cells, as identified by their size, and their distribution mainly in clusters, were strongly positive for NESP55, but NPY negative (Fig. 3). Dense ChAT-IR network of nerve terminals was seen throughout the SCG sections. These, probably preganglionic, ChAT positive terminals, formed basket-like structures surrounding both NESP55 positive and negative cells. The

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Fig. 3. Confocal images of sections from adult rat SCGs. Colocalization of NESP55-IR and NPY-IR (yellow) is seen in a subpopulation of the principal neurons in both male (a) and female (b) SCG. NESP55-IR (green) and NPY-IR (red), mutually exclusive in some neurons, are also seen. SIF cells displayed strong NESP55-IR, but were NPY negative (arrow in a). (c–c″) High magnification images (from a male SCG) show three subpopulations of the principal neurons immunoreactive for NESP55 (green) and/or NPY (red). (d–d‴) Section from a female SCG, triple incubated with anti-NESP55 (green), anti-NPY (red) and anti-TH (blue). NESP55 and/or NPYpositive neurons were also immunoreactive for TH. A fraction of TH positive fibers contained NESP55-IR, but were NPY negative. Scale bar=10 μm (c–c″, d–d‴), 50 μm (a, b).

Fig. 4. Confocal images of sections from rat SCGs. The sections were double-labeled with anti-NESP55 (green) and anti-ChAT, anti-SN or anti-CGRP (red). Preganglionic ChAT immunoreactive terminals surround both NESP55 positive and negative neurons (a–c, a′–c′, from male rat). NESP55 positive nerve fibers appear to lack ChAT-IR (arrows in a–c). SN and CGRP positive varicosities are seen in close apposition to the postganglionic neurons with or without NESP55IR (d–f and g–i, from females). (d′–f′) SIF cells are immunoreactive to both anti-SN and-NESP55. Scale bar = 10 μm (a′–c′), 20 μm (a–i, and d′–f′).

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Fig. 5. Confocal images of sections from adult rat SCGs (female), double-labeled with anti-NESP55 or -NPY and anti-TGN38, marker for the trans-Golgi network. Granular NESP55-IR and NPY-IR are concentrated in the trans-Golgi network area. Note that both NESP55-IR and NPY-IR are also seen in pre-, and post-TGN regions. (c–e) High magnification images of three neurons in (a″) and (b″). Scale bar = 5 μm (c–e), 10 μm (a–a″, b–b″).

principal neurons rarely exhibited ChAT-IR. NESP55 positive fibers appeared ChAT negative, and were therefore probably postganglionic (Fig. 4a–c, a′–c′). SN positive varicosities were seen in close apposition to some postganglionic neurons with or without NESP55-IR (Fig. 4d–f). Colocalization of NESP55-IR and SN-IR was evident only in the SIF cells (Fig. 4d′–f′). A small fraction of CGRP positive nerve fibers and varicosities was preferentially distributed in the superficial part of the SCG. A few CGRP positive fibers/varicosities were present around a subset of the principal neurons, which were either NESP55 positive or negative. CGRP-IR appeared, however, absent from the perikarya of the neurons (Fig. 4g–i).

regions. Thus, the whole Golgi complex appeared to contain NESP55-IR. NPY-IR had a distribution pattern similar to that of NESP55-IR (Fig. 5).

3.2. Colocalization studies of anti-NESP55 or -NPY with anti-TGN38 in the rat SCG The Trans-Golgi network (TGN) is the main compartment for sorting, aggregation and packing of secretory proteins. It has been agreed that Chromogranins are involved in these processes. Colocalization studies of antiNESP55 and -TGN38, a marker for trans-Golgi network, were carried out in the rat SCG. NESP55-IR was found to partly overlap with TGN38-IR, but NESP55-IR was also observed in pre- as well as post-trans Golgi network

Fig. 6. The frequencies of NPY immunoreactivity in NESP55 positive cells (NESP55 + NPY / NESP55) and NESP55 immunoreactivity in NPY positive cells (NESP55 + NPY / NPY) in the rat SCG. Values represent mean ± SD (n = 4). Cell numbers scored in the study were 1268 ± 200 per male rat and 1196 ± 196 per female rat. The relative frequencies between male and female are compared using ANOVA. ⁎p b 0.05.

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3.3. More NESP55 positive neurons contained NPY-IR in the female rat SCG than in the male Sex differences in the rat SCG were demonstrated early in 1980's (Wright and Smolen, 1987). Adult male rats contain more principal neurons in SCGs than do females. In the present study, the intracellular distribution of NESP55-IR in the male and female rat SCG was compared. No differences were observed (Fig. 2). We also counted the neurons labeled either for anti-NESP55 or anti-NPY, as well as the neurons expressing both peptides. It was found that of the NPY positive neurons 23% contained NESP55IR in the male rat SCG, and a very similar ratio, 25%, was found in the female. Interestingly, of the NESP55 positive

neurons, 80% in the male rat also expressed NPY, while a somewhat higher proportion, 87%, was found in the female. This difference, although small, was statistically significant (p b 0.05, Fig. 6). 3.4. Expression of NESP55 in other sympathetic ganglia of the rat and in the mouse SCG 3.4.1. NESP55-IR in the sympathetic ganglia of the rat NESP55-IR was present in the stellate ganglion, thoracic chain ganglia as well as celiac ganglion of the rat (Fig. 7). NESP55 positive neurons were scattered in the SCG and the stellate ganglion, representing 13–19% and 6–9% of the

Fig. 7. Sections of different autonomic ganglia from female rats and male mice incubated with anti-NESP55 (green) and anti-TH (red). NESP55-IR is scattered in the rat SCG (a–a″) and celiac ganglion (d–d″), and almost completely colocalized with TH-IR. (b–b″) In the rat stellate ganglion, the majority of NESP55 containing neurons are TH positive. NESP55 positive non-adrenergic (TH negative) neurons are also seen (arrows). (c–c″) A higher proportion of NESP55 positive neurons appears to lack TH-IR in the thoracic sympathetic chain, while neurons co-stained with anti-NESP55 and anti-TH are also seen (arrows). (e–e″) NESP55-IR was not detectable in the mouse SCG. Scale bar = 50 μm.

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principal neurons, respectively. In the SCG, NESP55-IR was almost completely co-localized with TH-IR. In the stellate ganglion, 75–90% of the NESP55 positive neurons contained TH-IR (Table 1). Neurons expressing NESP55, but lacking TH-IR, appeared preferentially located in the middle part of the ganglion. In the thoracic ganglia NESP55 positive neurons represented approximately 3–7% of the whole population of principal neurons. The distribution of NESP55 expressing neurons in these ganglia was distinct from that in the stellate ganglion and the SCG. Rather than scattered, like in the stellate ganglion and the SCG, the NESP55 positive cells were grouped. In addition, the majority of these NESP55-IR containing neurons appeared to lack TH-IR (Table 1, Fig. 7). Double labeling studies showed that these groups of neurons also lacked ChAT-, CGRP-, and NPY-IR (data not shown). 3.4.2. NESP55-IR in the rat stellate ganglion in comparison with CGRP-IR and ChAT-IR A dense network of nerve terminals with ChAT-IR was seen throughout the stellate ganglion sections. A subset of the principal neurons expressed ChAT. The ChAT containing terminals surrounded both NESP55 positive and negative cells. NESP55-IR was found in some of the ChAT positive neuronal perikarya and these neurons appeared to be surround by varicose terminals with very strong ChAT-IR (Fig. 8). CGRP positive nerve fibers and varicosities were observed in the rat stellate ganglion. These CGRP positive varicosities were seen around a subset of the principal neurons with or without NESP55-IR. A small proportion of the principal neurons was CGRP positive, and some of them also contained NESP55-IR (Fig. 8). The intensity of NESP55-IR in these CGRP containing neurons varied among cells. NESP55-IR in large cell bodies was usually stronger in fluorescence intensity than in small cells. Consecutive sections (with 6 μm interval) showed that some CGRP+/NESP55+ neurons were also cholinergic in nature, since ChAT-IR was clearly present. However TH-IR was not observed in these CGRP+/NESP55+ cell bodies (Fig. 9). 3.4.3. NESP55-IR in the mouse SCG NESP55-IR in the normal mouse SCG could not be observed. If present, the level was too low to be detectable. No NESP55-IR could be seen even in ganglia treated with cochicine (Fig. 7). 4. Discussion Chromogranins are thought to play a crucial role in the sorting and packaging process of secretory proteins in the trans-Golgi network. In our present study NESP55-IR was concentrated in the area where TGN38-IR was located, and extended to pre- and post-trans Golgi network areas. This

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suggests that, in addition to a role in trans-Golgi network, NESP55 may also be involved in the early sorting stage of secretory granules, e.g., in cis-Golgi network, as well as in the maturation process of the immature secretory granules after their budding off from the trans-Golgi network. Among various neuropeptides expressed in the rat SCG (Karhula, 1995; Kroesen et al., 1997; Bergner et al., 2000; Shafer et al., 2000), NPY is probably the most abundant one. As previously reported, 50–70% of principal neurons in the rat SCG contain NPY(Gibbins, 1995; Karhula, 1995). In the present study, a similar proportion was observed. In the sheep SCG, a small proportion of ganglionic neurons projecting to the submandibular gland, contained NPY, but apparently lacked TH (Arciszewski et al., 2004). This indicates that NPY containing neurons are not necessarily adrenergic in the sympathetic nervous system, at least not in sheep. Thus, non-adrenergic, NPY-ergic, neurons may exist in some species. Our present data, however, showed that NPY was completely colocalized with TH in the rat SCG. Therefore, principal neurons in SCG may differ between species regarding their different combinations of transmitters/peptides. Most of the NPY containing neurons in the sympathetic ganglia certainly are vasoconstrictor neurons, while a small proportion may also have other functions in different target organs (Lundberg et al., 1982; Gibbins, 1995). In the rat SCG, about 23–25% of all NPY-positive neurons contained NESP55-IR in our present study. The functional significance of NESP55 in the rat SCG is not known yet. Colocalization of NESP55-IR and NPY-IR in some neurons may represent a subpopulation of neurons with a specialized function, differing from neurons expressing NPY only. Interestingly, Seidah et al. (1987) first hypothesized that the granins might compete with the co-packaged peptide precursors for prohormone convertases. Like most neuropeptides, NPY is synthesized as a larger precursor molecule in the rough endoplasmic reticulum. ProNPY requires cleavage by prohormone convertase 2 plus the actions of carboxypeptidease E to generate mature NPY (Paquet et al., 1996; Marx et al., 1999). Prohormone convertase 2 was also demonstrated to liberate GAIPIRRH from its precursor NESP55 (Fischer-Colbrie et al., 2002). In the present study high intensities of both NPY-IR and NESP55-IR were found in the trans-Golgi network, as well as in the post-trans Golgi network region, probably localized to immature secretory granules. These two organelles are the main compartments where the proteolytic processing of peptide precursors has been shown to occur (Huttner and Natori, 1995). Moreover, the activity of prohormone convertases is rate-limiting in every secretory granule (Natori and Huttner, 1994). Thus, it is possible that NESP55 may indirectly inhibit the processing of NPY in the rat SCG by competing for prohormone convertase 2, and vice versa. SCGs of male and female rats have equal numbers of neurons at the time of birth. A difference arises during the first postnatal week, due to a reduction of 20–30% neurons by apoptosis in females. Testosterone has been shown to play

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Fig. 8. Confocal images of rat stellate ganglia. The sections were double incubated with anti-NESP55 (green) and anti-ChAT or anti-CGRP (red). (a–c, from male) ChAT-IR is present in the nerve terminals as well as in a subset of the postganglionic neurons. NESP55 positive neurons are scattered. Colocalization of NESP55-IR and ChAT-IR is seen in two neurons (arrows), one of which is highlighted in a′–c′. (d–f, from male) CGRP-IR is seen in neurons as well as in nerve fibers. Arrows indicate neurons containing both CGRP- and NESP55-IR. (g–i, from female) High magnification images show that CGPR positive nerve terminals surround a NESP55 positive neuron. NESP55-IR in a CGRP positive cell is also shown. Scale bar = 10 μm (a′–c′); 20 μm (a–c, g–i); 50 μm (d–f ).

Fig. 9. Confocal images of the rat stellate ganglion. Consecutive sections with 6 μm interval show that a NESP55 positive neuron contains CGRP-IR and appears to be cholinergic (ChAT positive), but non-adrenergic (lacking TH-IR). Scale bar = 20 μm.

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a critical role to prevent this apoptotic cell death in males, possibly by elevating levels of nerve growth factor or other trophic factors (Wright, 1995). NESP55 containing neurons comprise a subpopulation of the principal neurons in the rat SCG. It was therefore of interest to investigate whether sex differences also existed in the NESP55 positive neurons with reference to NPY containing neurons, whose function in the rat SCG has already been established (Lundberg et al., 1982; Gibbins, 1995). In our present study about 23–25% of all NPY-positive principal neurons also contained NESP55-IR in the rat SCG in both sexes. Interestingly, from the other angle, of the NESP55 positive neurons 80% also contained NPY-IR in the male rat, while a slightly higher, but statistically different proportion of 87% was found in the female. Whether or not this small difference has physiological significance is unknown. In this study, we have observed different patterns regarding the expression and colocalization of NESP55 together with TH and other peptides in various sympathetic ganglia. In the rat autonomic sympathetic nervous system the distribution of NESP55-IR is marked. However, in the mouse SCG no detectable NESP55-IR could be observed. Even pretreatment with colchicine, which blocks export of peptide granules into the axon, thereby increasing peptide levels in the cell body, resulted in negative findings. One possibility is that this antibody does not recognize the mouse peptide, which may have a slightly different sequence than rat NESP55. However, this appears to be not the case, since NESP55-IR was clearly observed in the spinal cord of the mouse (unpublished observation). Therefore, it is possible that there is no NESP55 synthesized in the mouse SCG. Alternatively, the levels of NESP55 in the peripheral tissue may be too low to be detected immunohistochemically. Landis and Fredieu (1986) claimed that cholinergic CGRP containing neurons in the stellate ganglia give rise to the innervation of rat front paw sweat glands. In the present study, colocalization of NESP55 and CGRP in the cholinergic cells was evident (Fig. 9). Thus, NESP55 may have a role in the regulation of the activities of sweat glands. Interestingly, the number of non-adrenergic NESP55 positive neurons in the thoracic ganglia was pronounced, and unlike the distribution in SCGs and other ganglia, the neurons appeared in groups. Also, they appeared to lack the immunoreactivities of the other antibodies used in this study. Altogether, the present study demonstrated that NESP55 was differentially expressed in various sympathetic ganglia of the rat. The influence of NESP55 on the functions of the autonomic ganglia is still a matter of speculation. Studies regarding the projection of NESP55 expressing ganglion cells in the rat by retrograde tracing technique are planed, which would add information regarding this novel peptide. Acknowledgements This study was supported by the Swedish Medical Research Council (14X-2207), the Göteborg Medical Society,

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