Peripheral projections of NESP55 containing neurons in the rat sympathetic ganglia

Autonomic Neuroscience: Basic and Clinical 141 (2008) 1 – 9 www.elsevier.com/locate/autneu

Peripheral projections of NESP55 containing neurons in the rat sympathetic ganglia Yongling Li ⁎, Annica Dahlström Department of Anatomy and Cell Biology, Institute of Biomedicine, Göteborg University, Box 420, SE-405 30 Göteborg, Sweden Received 4 December 2007; received in revised form 7 March 2008; accepted 20 March 2008

Abstract The peripheral projections of neurons expressing neuroendocrine secretory protein 55 (NESP55), a novel member of the chromogranin family, were studied by retrograde tracing technique. It was found that NESP55 positive neurons in the rat superior cervical ganglion projected to a number of targets including the submandibular gland, the cervical lymph nodes, the forehead skin, the iris, but not to the thyroid. Among these NESP55 positive target-projecting neurons, a subpopulation contained neuropeptide Y (NPY), a vasoconstrictor. Forepaw pad projecting neurons were found exclusively in the stellate ganglion, almost all of which (approximately 90%) were immunoreactive to NESP55. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP), a peptide involved in sudomotor effects, was observed in a subpopulation of these paw pad projecting neurons, as was colocalization of NESP55 and NPY. The data suggest that NESP55 may have a functional role in some populations of sympathetic neurons. © 2008 Elsevier B.V. All rights reserved. Keywords: Retrograde tracing; Chromogranins; Peptides; ANS; Chemical coding

1. Introduction Postganglionic sympathetic neurons show two main phenotypes characterized by their contents of the classical neurotransmitters: noradrenaline and acetylcholine. The majority, expressing noradrenline, are noradrenergic neurons responsible for autonomic functions including vasoconstrictor, pilomotor, secretomotor, and visceromotor activities, etc. (Gibbins, 1995). A much smaller population, for instance, those projecting to the paw sweat glands and the rib periosteum is cholinergic, utilizing acetylcholine as the transmitter (Anderson et al., 2006). In addition, a wide variety of neuropeptides has been observed in the sympathetic postganglionic neurons (Gibbins, 1992; Forehand, 1995; Gibbins, 1995; Grkovic and Anderson, 1997; Bergner et al., 2000). Distinctive combinations of these neurochemicals in the

⁎ Corresponding author. Department of Anatomy and Cell Biology, Box 420, SE-405 30 Göteborg, Sweden. Tel.: +46 31 786 3366; fax: +46 31 82 96 90. E-mail address: [email protected] (Y. Li). 1566-0702/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.03.008

postganglionic neurons, together with the preganglionic inputs, chemically code target-specific pathways (Landis and Fredieu, 1986; Anderson et al., 1995; Grkovic and Anderson, 1997; Chanthaphavong et al., 2003; Murphy et al., 2003; Richardson et al., 2006). The submandibular salivary gland of the rat was found to be innervated by neuropeptide Y (NPY) immunonegative postganglionic neurons, which, however, received calretinin positive preganglionic innervation (Grkovic and Anderson, 1995); Cholinergic sudomotor neurons contained immunoreactivities (IR) for vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) but were always devoid of calbindin-IR. However, cholinergic neurons projecting to the periosteum contained VIP-IR and sometimes calbindin-IR, but always lacked CGRP-IR (Hohmann et al., 1986; Anderson et al., 2006). Chemical coding of the postganglionic neurons differs among species. Opioid peptides were found in the noradrenergic NPY positive vasoconstrictor neurons of guinea pigs, rats, and cattle, but not in mice, nor in humans (Gibbins, 1995). Cholinergic vasodilator neurons were observed in cats (Anderson et al., 1995), and guinea pigs

2

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

(Anderson et al., 1996), but never in rats (Guidry and Landis, 2000). The chromogranin family is a group of acidic, soluble, and heat-stable proteins widespread in various neuronal, neuroendocrine and endocrine tissues, where they were suggested to have both intracellular and extracellular roles, acting as inducers in the formation of the secretory granules or as small peptide precursors, respectively (Taupenot et al., 2003). Neuroendocrine secretory protein 55 (NESP55) is the most recently identified member of this family (Ischia et al., 1997). It, like its siblings, is subcellularly located in the secretory granules or the large dense core vesicles of the adrenal medulla and the splenic nerves (Ischia et al., 1997; Bauer et al., 1999b). However, NESP55 was also observed in vesicles with a density slightly lighter than the large dense core vesicles and constitutively secreted into the exterior in the mouse AtT-20 cells (Eder et al., 2004). In the rat spinal cord, NESP55-IR was found to be concentrated in the prenuclear region in the sympathetic neurons. In contrast, in the spinal motoneurons, NESP55-IR was diffusely distributed throughout the whole cytoplasm (Li et al., 2008). Thus, NESP55 may be involved in both regulated and constitutive pathways. NESP55 is proteolytically processed to smaller peptide, like GAIPIPRRH, in various bovine tissues to a varying degree (Lovisetti-Scamihorn et al., 1999). Moreover, NESP55 is genomically imprinted and expressed exclusively from the maternal allele (Hayward et al., 1998; Plagge et al., 2005). Previously, we demonstrated that NESP55 was expressed in both noradenergic and cholinergic postganglionic neurons of the rat (Li et al., 2007). In the present study, we attempted to analyze the peripheral projections of these NESP55 positive sympathetic neurons by retrograde tracing technique. Our results showed that the majority of the forepaw pad projecting neurons contained NESP55-IR. Also, NESP55 positive neurons in the rat superior cervical ganglion (SCG) were shown to innervate a number of target tissues. 2. Materials and methods 2.1. Animals Adult male Sprague–Dawley rats ((9–10 weeks old) purchased from B & K Universal (Aldbrough, England) 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 Gothenburg University and all efforts were made to minimize animal suffering and the number of animal used. 2.2. Retrograde tracer injections and tissue preparation Fluoro-Gold (fluorochrome, Englewood, USA), dissolved as 4% in distilled water, was used as tracer to examine the peripheral projections of NESP55 positive neurons in different sympathetic ganglia (Schmued and

Fallon, 1986). Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, 4% Fluoro-Gold was injected unilaterally (right side) into five different targets including the submandibular gland, the thyroid, the cervical lymph nodes, the forepaw pad and the anterior chamber of the eye (iris). The forehead skin (around midline area) was also applied as a target. For each investigated target, three animals were used. The submandibular salivary gland, the thyroid and the lymph nodes were exposed via a midline neck incision. Injections of FluoroGold (1–1.5 μl each) were made into the above organs at 2– 5 sites and the skin incisions were sutured. Tracer injections, 3–4, were also done for the forehead skin. In the forepaw 1– 2 injections of tracer were made into each paw pad tubercle, totally up to 10 sites. A total volume of 4 μl of tracer was injected into the anterior chamber of the eye after insertion of the needle into the lateral corner of the anterior chamber. In all cases, the injecting device, Microliter Syringe (Hamilton Bonaduz, Schweiz), was left in the place for 1–2 min after injection to minimize dye leakage. Any leakage after this time was soaked up with a cotton pad. After a survival period of 4–5 days, all animals were perfused transcardially with 4% paraformaldehyde (pH 7.4). The SCG, the stellate ganglion (SG), as well as the sympathetic chain ganglia from both sides, were then removed and post-fixed overnight in the same fixative and stored at 4 °C in a PBS solution containing 0.1% sodium azide and 20% sucrose. The ganglia were frozen with compressed CO2, sectioned longitudinally in a cryostat at 10–12 µm, and mounted on gelatinized glass slides for immunohistochemistry. 2.3. Immunofluorescence procedures 2.3.1. Primary antibodies Polyclonal Guinea pig anti-NESP55, kindly donated by Dr. Reiner Fischer-Colbrie, University of Innsbruck, Austria. The antiserum was prepared against a synthetic octapeptide representing the C-terminus (GAIPIRRH, amino acids 234– 241) of NESP55 and staining a single band of Mr 55,000 on western blots (Ischia et al., 1997), dilution 1:4000. In brains of NESP55 knock-out mice (Plagge et al., 2005) no positive staining was seen with this antiserum (Ischia et al., 1997; Eder et al., 2004). Polyclonal rabbit anti-calcitonin gene-related peptide (CGRP), produced against a synthetic peptide corresponding to amino acids 1–37 of rat CGRP (Cambridge Research Biochemicals, Cleveland, UK; cat# CA-08-220), dilution 1:400. Polyclonal rabbit anti-neuropeptide Y (NPY), produced against synthetic porcine NPY-KLH (Sigma, St. Louis, MO, USA; cat# N9528), dilution 1:4000. Polyclonal rabbit anti-Tyrosine Hydroxylase (TH), produced against tyrosine hydroxylase purified from pheochromocytoma (Sigma-Aldrich, Sweden; cat# T8700), dilution 1:800 Polyclonal goat anti-neuropeptide Y (NPY), produced against synthetic porcine neuropeptide tyrosine (Affiniti

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

Research Products Limited, Devon, UK; cat# NA1236), dilution: 1:100. 2.3.2. Immunofluorescence The sections were preincubated with normal donkey serum for 1 h, followed by incubation overnight with antiNESP55 and one of the polyclonal rabbit primary antibodies listed above. The sections were then incubated with a mixture of biotin-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch, dilution 1:200) and Texas Redconjugated AffiniPure (TxR) donkey anti-rabbit IgG (Jackson ImmunoResearch, dilution 1:50) for 2 h, followed by incubation with Fluorescein (DTAF)-conjugated streptavidin (Jackson ImmunoResearch), diluted to 1:800, for 1.5 h. Double labeling with goat anti-CGRP and rabbit anti-NPY was also carried out on sections of the SG after tracer injection into the forepaw pad. In this case, biotin donkey anti-goat and TxR donkey anti-rabbit IgG were applied as the secondary antibodies. All experiments were carried out at room temperature. 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 X-100 to allow penetration intracellularly. Finally, the sections were mounted with anti-fading medium (DakoCytomation, Carpinteria, CA, USA) and examined in a CLSM (Bio-Rad MRC 1024). Images were captured using a digital camera (Nikon D70) mounted to a fluorescence microscope (MICROPHOT-FXA, Nikon), and processed using Adobe Photoshop (Version 5.5).

3

Control experiments where the primary antibodies were omitted, were carried out in every incubation series. No specific fluorescence staining was observed. 2.4. Cell count The SCG and SG were sectioned in their entirety. Every tenth section (at least at 100 μm interval) was subjected to examination to avoid double counting of cells and totally 4–8 sections were selected for each ganglion. All retrogradely labeled cells, clearly distinguishable from background levels and displaying a nucleus, were counted. The longest and shortest diameters of cells were measured with a calibrated eyepiece graticule. The average of these two values, referred to by Gibbins (1991), was taken as a measure of cell diameter. The presence of NESP55-, NPY- and CGRP-IR in the labeled cells was recorded. Cells positive to both NESP55 and CGRP in the SG sections were counted, as was the clear labeling of tracer in these cells. All counts were present as uncorrected numbers. 3. Results The SCG, SG, as well as the rostral thoracic chain ganglia were subject to investigation in the present study. Injections of tracer into the submandibular gland, the cervical lymph nodes, the thyroid, and the iris resulted in retrogradely labeled neurons in the ipsilateral SCG. In the case of the forehead skin, labeled neurons were seen in the SCG bilaterally. Most, if not all, of the tracer labeled neurons were positive for TH, thus

Fig. 1. SCG sections, after injection of Fluoro-Gold into a number of targets, were incubated with anti-TH, showing these neurons are noradrenergic in nature. Scale bar = 30 μm (applies to all images).

4

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

Fig. 2. SCG sections, after injection of Fluoro-Gold into a number of targets, immunostained with anti-NESP55 and -NPY, showing the different subpopulations of these target-projecting neurons. Thin arrows indicate NESP55+/NPY+ target-projecting neurons; small arrows indicate NESP55+/NPY− target-projecting neurons; arrowheads indicate NESP55−/NPY+ target-projecting neurons; ⁎ indicates NESP55−/NPY− target-projecting neurons. Scale bar = 30 μm.

noradrenergic (Fig. 1). The majority of these labeled neurons had an average diameter between 20–35 μm. However, some cells had a smaller average diameter (less than 20 μm). Cells with a diameter larger than 35 μm were also seen, especially noted for the submandibular gland projecting neurons. Thus, we classified these cells, by their size, into three types as small (b 25 μm), medium (25–30 μm), or large (≥30 μm). No labeled neurons were observed in the SG and the thoracic chain ganglia. Injection of tracer into the forepaw pad resulted

in labeled neurons, noradrenergic (TH positive) or nonnoradrenergic (TH negative), present in the ipsilateral SG, but not in the SCG nor in the thoracic chain ganglia. The size criterion for the SCG was also applied for the SG. NESP55-IR was present in some of these tracer labeled neurons, both in the SCG and the SG, showing a perinuclear (trans-Golgi complex region) expression pattern, typical for autonomic neurons (Li et al., 2008). 3.1. NESP55 positive neurons in the SCG projecting to a number of targets

Table 1 NESP55 and NPY immunoreactivity in retrogradely labeled SCG neurons after injection of tracer into various targets (n = 3) NESP55+ NPY+ SMG Lymph node Forehead skin Eye chamber Thyroid

NESP55NPY−

NPY+

NPY−

Labeled cells

19 (2.1%) 11 (1.2%) 4 (0.4%) 858 (96.2%) 892 16 (10.9%) 5 (3.4%) 18 (12.2%) 108 (73.5%) 147 21 (35.6%) 0 3 (7.7%) 0

0 0

16 (27.1%) 22 (37.3%) 31 (79.5%) 5 (12.8%) 60 (65.9%) 31 (34.1%)

3.1.1. Projecting to the submandibular gland Following injection of tracer into the submandibular gland, retrogradely labeled neurons were found in the Table 2 NESP55 and NPY immunoreactivity in retrogradely labeled neurons in the stellate ganglion after injection of tracer into the forepaw pad

59 39 91

NPY+ NPY−

NESP55+

NESP55−

17 (19%) 62 (70%)

0 10 (11%)

A total of 89 cells was counted in 3 rats.

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9 Table 3 NESP55 and CGRP immunoreactivity in retrogradely labeled neurons in the stellate ganglion after injection of tracer into the forepaw pad

CGRP+ CGRP−

NESP55+

NESP55−

26 (30%) 54 (62%)

0 7 (8%)

5

3.1.5. No NESP55 positive neurons in the SCG projected to the thyroid Most of the labeled neurons after injection of tracer into the thyroid were of small size. Very few, if any, of these contained NESP55-IR. However, the majority of the labeled neurons (60 of 91) were NPY positive (Table 1, Fig. 2).

A total of 87 cells was counted in 3 rats.

3.2. NESP55 positive neurons in the SG projecting to the forepaw pad ipsilateral SCG. The majority of these neurons were of the large type. One third (70 of 243) was classified as the medium type, and a few neurons were of the small size. After incubation with anti-NESP55, only 3% (30 of 858) of these submandibular gland projecting neurons were NESP55 positive. Double labeling experiments revealed that some of these NESP55 positive submandibular gland projecting neurons were also NPY positive (Fig. 2). In general, only a minor proportion of submandibular gland projecting neurons was positive to both NESP55 and NPY (2%), or positive to either NESP55 (1%) or NPY (less than1%), which were always of the small or medium size. The majority, thus, were devoid of immunostaining for NESP55 or NPY (Table 1). 3.1.2. Projecting to the cervical lymph nodes Neurons which were retrogradely labeled from the cervical lymph nodes were scattered in the middle or caudal part of the ipsilateral SCG. A high proportion was small or medium type neurons. NESP55-IR was present in the labeled neurons with medium to strong intensity (Fig. 2). Of 147 labeled cells, 21 (14%) were NESP55 positive. These lymph node projecting neurons appeared to form four subgroups after double staining with anti-NESP55 and anti-NPY: NESP55+/NPY+, NESP55+/NPY−, NESP55−/NPY+, and NESP55−/NPY− (Table 1). 3.1.3. Projecting to the forehead skin Labeled neurons were found in the SCG bilaterally after injection of tracer into the forehead skin. The neurons were located in the rostral part of the SCG, and most of them were of small size. Some medium and large type neurons were also seen. NESP55-IR was present in 21 of a total of 59 labeled neurons. Interestingly, all of these NESP55 positive forehead skin projecting neurons also displayed NPY-IR. NESP55−/NPY+ and NESP55−/NPY− neurons were also observed, accounting for 27% and 37% of the total tracer labeling, respectively (Table 1, Fig. 2). 3.1.4. Projecting to the iris Relatively few neurons (39) were observed to project to the iris in the present study. The neurons were scattered in the rostral half of the ipsilateral SCG. Three of the 39 tracer labeled neurons were immunoreactive to NESP55, and these 3 neurons were also NPY positive (Fig. 2). The majority of the tracer labeled neurons were NESP55 negative, but instead NPY positive (Table 1).

Following injection of Fluoro-Gold into the forepaw tubercles, retrogradely labeled neurons were found in the ipsilateral SG, but very few were found in the SCG and the chain ganglia. The labeled neurons were scattered but tended to be more numerous in the middle third of the ganglion. The neurons were of varying size, from small to large. The majority were positive to NESP55, a subset of which, also contained NPY or CGRP (Tables 2, 3). However, colocalization of NPY and CGRP was rarely observed (Fig. 3). 3.2.1. NESP55-IR was colocalized with NPY-IR in the forepaw pad projecting neurons. Of a total of 89 tracer labeled neurons in the SG, 89% were NESP55 positive. NPY-IR in the tracer labeled neurons was exclusively present in cells also positive to NESP55, representing 19% of the total number of tracer labeled neurons (Table 2). These neurons usually were of small or medium size (Fig. 3). 3.2.2. NESP55-IR was colocalized with CGRP-IR in the forepaw pad projecting neurons Three subgroups of the forepaw pad projecting neurons were characterized after double immunostaining of the SG with anti-NESP55 and anti-CGRP (Table 3, Fig. 3). a. NESP55+/CGRP+ neurons: Of a total of 87 tracer labeled neurons, 26 (30%) were immunoreactive to both NESP55 and CGRP. Colocalization of NESP55-IR and CGRP-IR was frequently observed in large neurons. Some neurons had an average diameter more than 40 μm. The intensity of NESP55-IR was comparatively weaker in larger neurons than in smaller NESP55+ pad projecting neurons. NESP55-IR and CGRP-IR were also colocalized in neurons with medium-sized cell bodies. b. NESP55+/CGRP− neurons: The majority (54 of 87, accounting for 62%) of the forepaw pad projecting neurons were NESP55 positive, but CGRP negative. In contrast to the neurons in the first group, these neurons were medium or small in size. c. NESP55−/CGRP− neurons: A few (7 cells) paw pad projecting neurons were found to contain neither NESP55-IR nor CGRP-IR. This group of neurons had medium-sized cell bodies. They were unlikely to be NPY positive since NPY-IR was only present in the NESP55 positive paw pad projecting neurons, as described above.

6

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

Fig. 3. SG sections, after injection of Fluoro-Gold into the forepaw pad, were double stained with different antibody combinations of anti-NESP55, -NPY and -CGRP, showing different subpopulations of the forepaw pad projecting neurons. (1a, 1b) NPYpositive neurons are devoid of CGRP-IR, and vice versa. (2a–2c) A forepaw pad projecting neuron immunoreactive to both NESP55 and NPY. (3a–3c) Two retrogradely labeled neurons contain NESP55-IR but no NPY-IR. (4a–4c) A retrogradely labeled neuron contains neither NESP55-IR, nor NPY-IR. (5a–5c) Two retrogradely labeled neurons contain both NESP55-IR and CGRP-IR (arrows), and two NESP55+/CGRP+ neurons were not retrogradely labeled (arrowheads). (6a–6c) A retrogradely labeled neuron is devoid of NESP55-IR and CGRPIR. (7a–7b) Three retrogradely labeled neurons contain NESP55-IR but no CGRP-IR. Scale bar = 50 μm in 1a (also applies to 1b); 30 μm in 7a (applies to 2a–7c).

3.2.3. Not all NESP55+/CGRP+ neurons in the SG, detected by immunofluorescence, were labeled by tracer A total of 69 cells in the SG (3 animals) was observed to be immunoreactive to both NESP55 and CGRP, but only 30 cells (44%) were retrogradely labeled by tracer.

3.2.4. NESP55-IR was absent from the CGRP-IR terminals around the sweat gland acini The sections of the forepaw pad were incubated with antiNESP55 and anti-CGRP. CGRP-IR was found to surround the sweat gland acini. However, NESP55-IR could not be

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

7

Fig. 4. Sections of forepaw pad processed by double incubation with anti-NESP55 and anti-CGRP. CGRP-IR is present in nerve terminals in the sweat glands but NESP55-IR appears to be absent from the terminal processes. Scale bar = 50 μm.

observed in these CGRP+ nerve fibers despite the presence of both peptides in the postganglionic neurons projecting to the sweat glands (Fig. 4). 4. Discussion Previously NESP55-IR was reported to be present in the rat sympathetic ganglia (Li et al., 2007). In this study we investigated the peripheral projections of the NESP55 containing neurons by retrograde tracing technique. Several organs, including the submandibular gland, the thyroid, the hairy forehead skin, the iris, and the cervical lymph nodes, were selected as possible targets of NESP55-IR neurons in the SCG. In the rat SG 10–25% of NESP55 positive neurons appeared to be non-noradrenergic (TH negative) (Li et al., 2007). We speculated that these neurons may be cholinergic, projecting to the sweat glands in the forepaw pad since numerous studies have shown that the sweat glands were the target of cholinergic postganglionic neurons (Landis and Fredieu, 1986; Anderson et al., 1995; Anderson et al., 2006). In the SCG NESP55 positive neurons were found to project to the forehead skin, the submandibular gland, the cervical lymph nodes, as well as to the iris. Thyroid projecting neurons appeared to lack NESP55-IR. Because only three rats were recruited in every experimental group, the number of labeled cells from different targets, especially in the iris group, where only 39 neurons were retrogradely labeled, was relatively low. It should be kept in mind that the number of retrogradely labeled cells observed here probably represents only a fraction of all neurons projecting to a certain tissue. Presumably, the larger the target, the more incomplete the diffusion of the injected tracer, and the less likelihood that all projecting neurons will be labeled. Thus, the raw figures we report in the present study provide qualitative, not quantitative information. To further characterize these NESP55 positive neurons, we performed double staining experiments with antiNESP55 and anti-NPY. All NESP55 positive neurons, projecting to the iris and the forehead skin, also contained NPY, suggesting NESP55 is probably involved in the vascular regulation like NPY. However, among the NESP55 positive neurons projecting to the submandibular

gland and the cervical lymph nodes, there is also a small subpopulation lacking NPY-IR. Therefore, NESP55 may also participate in other autonomic activities, probably secretomotor functions, since a subpopulation of NPY negative noradrenergic neurons in the rat SCG, were thought to be secretomotor neurons (Gibbins, 1995; Bergner et al., 2000). In the present study, the forepaw pad projecting neurons were restricted to the ipsilateral SG. Almost all (approximately 90%) of them appeared to contain NESP55-IR. Of all the forepaw pad projecting neurons 30% contained both CGRP- and NESP55-IR, suggesting that NESP55, or modified fragments from this molecule, may have a role in sudomotor functions like CGRP (Landis and Fredieu, 1986; Anderson et al., 2006). In addition, approximately 62% of the forepaw pad projecting neurons were NESP55 positive, but CGRP negative, and 8% contained neither NESP55-IR nor CGRP-IR. We also found that a subpopulation of the forepaw pad projecting neurons contained NPY-IR, in agreement with the previous report by Dehal et al. (1992) of NPY-IR being present in SG neurons projecting to blood vessels within the forepaw pad. After double staining of SG sections with anti-CGRP and anti-NPY, we found that CGRP+ neurons appeared devoid of NPY-IR and vice versa. In addition, NPY-IR and CGRP-IR were observed exclusively in the NESP55 positive neurons in the present study. Thus, we classify the forepaw pad projecting neurons into four different subpopulations: 1. NESP55+/CGRP+/NPY–, possibly sudomotor; 2. NESP55+/CGRP–/NPY+, possibly vasoconstrictor; 3. NESP55+/CGRP–/NPY–; 4. NESP55–/CGRP–/NPY–. The functions of the last two classes are unknown. Another target tissue of cholinergic neurons in the SG and the thoracic chain ganglia is the rib periosteum (Hohmann et al., 1986; Anderson et al., 2006). Our previous study demonstrated a group of neurons in the thoracic chain ganglia resembling the periosteum projecting neurons described by Anderson et al. (2006). This group consisted of small neurons forming clusters and were devoid of CGRP-IR (Li et al., 2007). Therefore, we suspect that NESP55 may be present in neurons projecting to the periosteum, participating in the regulation of metabolism and maturation of the bone. However, this hypothesis needs to be verified by further experiments.

8

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9

Despite careful study, we could not observe any NESP55 positive nerve terminals in the forepaw pad sections although approximately 90% of the projecting neurons contained NESP55-IR, nor in the sections of the rib (unpublished observation). Generally, there are high levels of peptides and other transmitters in the nerve terminals. However, our experiments and those of others', using immunohistochemistry, showed that NESP55-IR appeared totally absent from the varicose terminals in neuronal tissues (Li et al., 2007), as well as in the peripheral target tissues as demonstrated in the present study and also by (Li et al., 2002), despite its strong presence in nerve cell bodies in both CNS (Bauer et al., 1999a) and PNS (Li et al., 2007). The reason for the distinct phenomenon of NESP55 remains unexplained, but may be related to a proteolysis or modification of the peptide during axonal transport, rendering the product unrecognizable by the antiserum used. The sympathetic innervations of sweat glands and periosteum undergo a switch of neurotransmitter phenotype from noradrenergic to cholinergic in the late embryonic stage or postnatally (Leblanc and Landis, 1986; Landis et al., 1988; Asmus et al., 2000). This change is considered to be induced by target derived differentiation activity (Schotzinger and Landis, 1988; 1990). Cytokines can also induce the same phenotype changes in sympathetic neurons in vitro (Rao and Landis, 1990; Rao et al., 1992). A number of peptides, including NPY, VIP, galanin, and somatostatin, were reported to undergo a dynamic change in the sympathetic ganglia during development (Tyrrell and Landis, 1994; Masliukov and Timmermans, 2004). The NESP55 gene is an imprinted gene exclusively expressed from maternal allele. Imprinted genes are of importance in the regulation of placental development, fetal growth, and neurodevelopment (Davies et al., 2005; Fowden et al., 2006). The phenomenon of wide expression of NESP55 in the sweat gland projecting neurons, possibly also in the periosteum projecting neurons, raises a question whether NESP55 has influence in the differentiation towards a cholinergic phenotype of sweat gland or periosteum projecting neurons. Taking this into account, it would be interesting to investigate the appearance/expression of NESP55 in the SG/thoracic ganglia during different developmental stages. Acknowledgments This study was supported by the Swedish Medical Research Council (14X-2207), the Göteborg Medical Society, the Medical Faculty, Göteborg University and the Royal Society of Arts and Science in Göteborg. We wish to thank Dr. Reiner Fischer-Colbrie (Dept. of Pharmacology, University of Innsbruck, Innsbruck, Austria) for a generous supply of antiserum against NESP55, Dr. Cecilia Falkenberg (Dept. of Pedicatric Cardiology, the Queen Silvia Children's Hospital, Sahlgrenska/Ostra University Hospital) for the loan of her digital camera (Nikon D70), and Dr. Jiabin Sun (Dept.

of Microbiology and Immunology, Inst. of Biomedicine, Gothenburg University) for the help with image producing. References Anderson, C.R., McAllen, R.M., Edwards, S.L., 1995. Nitric oxide synthase and chemical coding in cat sympathetic postganglionic neurons. Neuroscience 68, 255–264. Anderson, R.L., Gibbins, I.L., Morris, J.L., 1996. Non-noradrenergic sympathetic neurons project to extramuscular feed arteries and proximal intramuscular arteries of skeletal muscles in guinea-pig hindlimbs. J. Auton. Nerv. Syst. 61, 51–60. Anderson, C.R., Bergner, A., Murphy, S.M., 2006. How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion? Neuroscience 140, 567–576. Asmus, S.E., Parsons, S., Landis, S.C., 2000. Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum. J. Neurosci. 20, 1495–1504. Bauer, R., Ischia, R., Marksteiner, J., Kapeller, I., Fischer-Colbrie, R., 1999a. Localization of neuroendocrine secretory protein 55 messenger RNA in the rat brain. Neuroscience 91, 685–694. Bauer, R., Weiss, C., Marksteiner, J., Doblinger, A., Fischer-Colbrie, R., Laslop, A., 1999b. The new chromogranin-like protein NESP55 is preferentially localized in adrenaline-synthesizing cells of the bovine and rat adrenal medulla. Neurosci. Lett. 263, 13–16. Bergner, A.J., Murphy, S.M., Anderson, C.R., 2000. After axotomy, substance P and vasoactive intestinal peptide expression occurs in pilomotor neurons in the rat superior cervical ganglion. Neuroscience 96, 611–618. Chanthaphavong, R.S., Murphy, S.M., Anderson, C.R., 2003. Chemical coding of sympathetic neurons controlling the tarsal muscle of the rat. Auton. Neurosci. 105, 77–89. Davies, W., Isles, A.R., Wilkinson, L.S., 2005. Imprinted gene expression in the brain. Neurosci. Biobehav. Rev. 29, 421–430. Dehal, N.S., Kartseva, A., Weaver, L.C., 1992. Comparison of locations and peptide content of postganglionic neurons innervating veins and arteries of the rat hindlimb. J. Auton. Nerv. Syst. 39, 61–72. Eder, S., Leierer, J., Klimaschewski, L., Wilhelm, A., Volknandt, W., Laslop, A., Fischer-Colbrie, R., 2004. Secretion and molecular forms of NESP55, a novel genomically imprinted neuroendocrine-specific protein from AtT-20 cells. Neurosignals 13, 298–307. Forehand, C.J., 1995. Peptides in Autonomic ganglion cells: control of expression and release. In: McLachlan, E.M. (Ed.), Autonomic Ganglia. Harwood academic publishers GmbH, Australia, pp. 123–151. Fowden, A.L., Sibley, C., Reik, W., Constancia, M., 2006. Imprinted genes, placental development and fetal growth. Horm. Res. 65 (Suppl 3), 50–58. Gibbins, I.L., 1991. Vasomotor, pilomotor and secretomotor neurons distinguished by size and neuropeptide content in superior cervical ganglia of mice. J. Auton. Nerv. Syst. 34, 171–183. Gibbins, I.L., 1992. Vasoconstrictor, vasodilator and pilomotor pathways in sympathetic ganglia of guinea-pigs. Neuroscience 47, 657–672. Gibbins, I., 1995. Chemical neuroanatomy of sympathetic ganglia. In: McLachlan, E.M. (Ed.), Autonomic Ganglia. Harwood academic publishers GmbH, Australia, pp. 73–121. Grkovic, I., Anderson, C.R., 1995. Calretinin-containing preganglionic nerve terminals in the rat superior cervical ganglion surround neurons projecting to the submandibular salivary gland. Brain Res. 684, 127–135. Grkovic, I., Anderson, C.R., 1997. Calbindin D28 K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat. J. Comp. Neurol. 386, 245–259. Guidry, G., Landis, S.C., 2000. Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice. Auton. Neurosci. 82, 97–108. Hayward, B.E., Kamiya, M., Strain, L., Moran, V., Campbell, R., Hayashizaki, Y., Bonthron, D.T., 1998. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 10038–10043.

Y. Li, A. Dahlström / Autonomic Neuroscience: Basic and Clinical 141 (2008) 1–9 Hohmann, E.L., Elde, R.P., Rysavy, J.A., Einzig, S., Gebhard, R.L., 1986. Innervation of periosteum and bone by sympathetic vasoactive intestinal peptide-containing nerve fibers. Science 232, 868–871. Ischia, R., Lovisetti-Scamihorn, P., Hogue-Angeletti, R., Wolkersdorfer, M., Winkler, H., Fischer-Colbrie, R., 1997. Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J. Biol. Chem. 272, 11657–11662. Landis, S.C., Fredieu, J.R., 1986. Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands. Brain Res. 377, 177–181. Landis, S.C., Siegel, R.E., Schwab, M., 1988. Evidence for neurotransmitter plasticity in vivo. II. Immunocytochemical studies of rat sweat gland innervation during development. Dev. Biol. 126, 129–140. Leblanc, G., Landis, S., 1986. Development of choline acetyltransferase (CAT) in the sympathetic innervation of rat sweat glands. J. Neurosci. 6, 260–265. Li, J.Y., Lovisetti-Scamihorn, P., Fischer-Colbrie, R., Winkler, H., Dahlstrom, A., 2002. Distribution and intraneuronal trafficking of a novel member of the chromogranin family, NESP55, in the rat peripheral nervous system. Neuroscience 110, 731–745. Li, Y., Wang, Z., Dahlstrom, A., 2007. Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia. Auton. Neurosci. 132, 52–62. Li, Y., Fischer-Colbrie, R., Dahlstrom, A., 2008. Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: an immunocytochemical study. J. Comp. Neurol. 506, 733–744. Lovisetti-Scamihorn, P., Fischer-Colbrie, R., Leitner, B., Scherzer, G., Winkler, H., 1999. Relative amounts and molecular forms of NESP55 in various bovine tissues. Brain Res. 829, 99–106. Masliukov, P.M., Timmermans, J.P., 2004. Immunocytochemical properties of stellate ganglion neurons during early postnatal development. Histochem. Cell Biol. 122, 201–209.

9

Murphy, S.M., McAllen, R., Campbell, G.D., Howe, P.R., Anderson, C.R., 2003. Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets. Neuroscience 117, 347–360. Plagge, A., Isles, A.R., Gordon, E., Humby, T., Dean, W., Gritsch, S., Fischer-Colbrie, R., Wilkinson, L.S., Kelsey, G., 2005. Imprinted Nesp55 influences behavioral reactivity to novel environments. Mol. Cell Biol. 25, 3019–3026. Rao, M.S., Landis, S.C., 1990. Characterization of a target-derived neuronal cholinergic differentiation factor. Neuron 5, 899–910. Rao, M.S., Tyrrell, S., Landis, S.C., Patterson, P.H., 1992. Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons. Dev. Biol. 150, 281–293. Richardson, R.J., Grkovic, I., Allen, A.M., Anderson, C.R., 2006. Separate neurochemical classes of sympathetic postganglionic neurons project to the left ventricle of the rat heart. Cell Tissue Res. 324, 9–16. Schmued, L.C., Fallon, J.H., 1986. Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res. 377, 147–154. Schotzinger, R.J., Landis, S.C., 1988. Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo. Nature 335, 637–639. Schotzinger, R.J., Landis, S.C., 1990. Acquisition of cholinergic and peptidergic properties by sympathetic innervation of rat sweat glands requires interaction with normal target. Neuron 5, 91–100. Taupenot, L., Harper, K.L., O'Connor, D.T., 2003. The chromograninsecretogranin family. N. Engl. J. Med. 348, 1134–1149. Tyrrell, S., Landis, S.C., 1994. The appearance of NPY and VIP in sympathetic neuroblasts and subsequent alterations in their expression. J. Neurosci. 14, 4529–4547.