A study on preganglionic connections and possible viscerofugal projections from urinary bladder intramural ganglia to the caudal mesenteric ganglion in the pig

Abstract

The present study was designed to (1) ascertain the distribution and immunohistochemical characteristics of sympathetic preganglionic neurons supplying the caudal mesenteric ganglion (CaMG) and (2) verify the existence of viscerofugal projections from the urinary bladder trigone intramural ganglia (UBT‐IG) to the CaMG in female pigs (n = 6). Combined retrograde tracing and immunofluorescence methods were used. Injections of the neuronal tracer Fast Blue (FB) into the right CaMG revealed no retrogradely labelled (FB‐positive; FB ⁺) nerve cells in the intramural ganglia; however, many FB ⁺ neurons were found in the spinal cord sympathetic nuclei. Double‐labelling immunohistochemistry revealed that nearly all (99.4 ± 0.6%) retrogradely labelled neurons were cholinergic (choline acetyltransferase‐positive; ChAT ⁺) in nature. Many FB ⁺/ChAT ⁺ perikarya stained positive for vesicular acetylcholine transporter (63.11 ± 5.34%), neuronal nitric oxide synthase (53.48 ± 9.62%) or cocaine‐ and amphetamine‐regulated transcript peptide (41.13 ± 4.77%). A small number of the retrogradely labelled cells revealed immunoreactivity for calcitonin gene‐related peptide (7.60 ± 1.34%) or pituitary adenylate cyclase‐activating polypeptide (4.57 ± 1.43%). The present study provides the first detailed information on the arrangement and chemical features of preganglionic neurons projecting to the porcine CaMG and, importantly, strong evidence suggesting the absence of viscerofugal projections from the UBT‐IG.

Introduction

The caudal mesenteric ganglion (CaMG) is one of the prevertebral autonomic ganglia and is a source of postganglionic innervation to a number of abdominal and pelvic organs (e.g. Miolan & Niel, 1996; Pidsudko et al. 2001; Ragionieri et al. 2013; Pidsudko, 2014; Barbe et al. 2018). The ganglia themselves are targeted by nerve fibres (NF) arising from different sources. Some NF terminating in the CaMG represent collaterals of peripheral processes of primary afferent neurons found in dorsal root ganglia (DRG; Bossowska, 2002). Many axons that synapse with CaMG neurons are preganglionic nerve terminals with perikarya in spinal cord nuclei (sympathetic preganglionic neurons; SPN; Krier et al. 1982; Anderson et al. 1995). Although the morphology and chemical coding of CaMG neurons have been described in detail both in humans and in many animal species (e.g. Furness, 2015; Sienkiewicz et al. 2015; Lepiarczyk et al. 2017), the literature in the field contains only fragmentary data on the localization and chemical characteristics of SPN projecting to this ganglion. Detailed knowledge concerning the arrangement of autonomic pathways to different organs is mostly limited to the postganglionic connections and is thus incomplete because it does not include the preganglionic component. In addition to the two above‐mentioned sources of NF projecting to CaMG, another prominent one has been found, represented by a group of intestinal (intramural) neurons, and the pathway to prevertebral ganglia they provide is called viscerofugal or centripetal. The existence of viscerofugal projections has been reported in some mammalian species, such as the rat (Luckensmeyer & Keast, 1996), guinea pig (Messenger & Furness, 1992) and pig (Barbiers et al. 1994), and they are considered an important component contributing to reflex circuits controlling gastrointestinal tract function. Until now, no attempts have been made to investigate the existence of possible viscerofugal projections from intramural (local) ganglia associated with the other organs to prevertebral ganglia. One possible source of such connections to the CaMG could be the intramural ganglia of the urinary bladder (UB‐IG). It should be emphasized that the literature in the field suggests the existence of profound interspecies differences in the number and distribution of UB‐IG, especially between laboratory rodents and larger mammals. For instance, few or no nerve cells have been found in the urinary bladder wall of the rat (Uvelius & Gabella, 1995; Zvarova & Vizzard, 2005) or mouse (Grozdanovic et al. 1992). However, UB‐IG are very numerous in humans (Dixon et al. 1983, 2000; Gilpin et al. 1983), guinea pigs (Crowe et al. 1986; Gabella, 1990) and pigs (Pidsudko, 2004, 2013). Despite this, no attempts have been made to investigate the existence of possible viscerofugal projections from these ganglia. This could be partly because rats and mice have been the most frequently used animals thus far in studies concerning innervation of urogenital organs, but they possess no UB‐IG. The domestic pig, on the other hand, seems to be a perfect animal model for such investigations because of its conspicuous resemblance to humans with respect to the presence and arrangement of UB‐IG (Dalmose et al. 2000; Kuzmuk & Schook, 2011; Swindle et al. 2012; Bassols et al. 2014).

Therefore, the present study had two main purposes: (1) to verify the possible existence of viscerofugal projections from the urinary bladder trigone intramural ganglia (UBT‐IG) to the CaMG and (2) to ascertain the distribution and immunohistochemical properties of preganglionic neurons projecting to the CaMG in the female pig.

Methods

Laboratory animals

The study was performed on six juvenile (8–12 weeks old, 15–20 kg bodyweight, b.w.) female pigs of the Large White Polish breed. The animals were kept under standard laboratory conditions. They were fed standard fodder (Grower Plus, Wipasz, Wadąg, Poland) and had free access to water.

Before any surgical procedures were performed, all the pigs were pretreated with atropine (Polfa, Poland, 0.05 mg kg⁻¹ b.w., s.c.) and azaperone (Stresnil, Janssen Pharmaceutica, Belgium, 2.5 mg kg⁻¹ b.w., i.m.); after 30 min, the main anaesthetic drug, propofol (Provive, 10 mg kg⁻¹ b.w.) and the main analgesic drug, ketamine (Bioketan 10 mg kg⁻¹ b.w.), were given intravenously in a slow, fractionated infusion. The depth of anaesthesia was monitored by testing the corneal reflex. The animals were housed and treated according to the guidelines of the local Ethics Committee for Animal Experimentation in Olsztyn (affiliated with the National Ethics Committee for Animal Experimentation, Polish Ministry of Science and Higher Education; decision No. 44/2017 from 25 July 2017).

Surgical procedures

A midline laparotomy was performed in all the pigs and the right CaMG was gently exposed to a total volume of 3 μL of 5% aqueous solution of the fluorescent retrograde tracer Fast Blue (FB; Dr K. Illing, KG & Co. GmbH, Gross Umstadt, Germany) in a few injections. To avoid leakage, the needle was left in each injection site for ˜ 1 min. After the injections, the ganglion was rinsed with physiological saline and gently wiped with gauze.

Three weeks later (which is the minimum time period needed for the retrograde tracer to be transported to the urinary bladder and the spinal cord, to label neurons supplying the CaMG; Juranek & Wojtkiewicz, 2015; Lepiarczyk et al. 2015), all the pigs were deeply anaesthetized with sodium pentobarbital and transcardially perfused with 4% buffered paraformaldehyde (pH 7.4). Immediately after perfusion, all the urinary bladders and all remaining CaMG were collected. The thoracic (Th), lumbar (L) and sacral (S) segments of the spinal cord were then exposed by laminectomy and dissected out. The tissues collected were postfixed in the same fixative (spinal cord segments for 20 min and urinary bladders and CaMG for 10 min, all at room temperature), washed several times in 0.1 m phosphate buffer and stored in 18% buffered sucrose at 4 °C until sectioning.

Sectioning and immunohistochemical staining of the urinary bladder wall trigones

The bladder trigones were cut from the collected organs. To facilitate freezing, tissue cutting and analysis of cryostat sections, the trigones were first divided with scalpels into three parts: the lower part and two (left and right) upper parts. All of these samples were cut with an HM525 Zeiss cryostat into 10‐μm‐thick sections. To ensure that all the intramural ganglia neurons located in the urinary bladder wall trigone region were analysed, the cryostat sections were processed for single‐labelling immunofluorescence, performed according to a previously described method (Bossowska & Majewski, 2012), using an antibody against mouse anti‐human protein gene product (PGP 9.5; 7863‐2004; 1 : 2000; Bio‐Rad, Kidlington, UK), which is considered to be a general neuronal marker. All immunostained sections were evaluated under an Olympus BX61 microscope equipped with an epifluorescence filter and an appropriate filter set for CY3 and fluorescein isothiocyanate (FITC).

Sectioning of the left caudal mesenteric ganglia and estimation of the total number of Fast Blue‐positive neurons

Left CaMG were cut with a cryostat into 10‐μm‐thick serial sections and analysed under an Olympus BX61 microscope. To calculate the number of FB‐positive (FB⁺) perikarya, they were counted in every fourth CaMG section to avoid double counting of the same neuron (most neurons were ~ 40 μm in diameter). Only neurons with clearly visible nuclei were considered. The total numbers of nerve cells counted in the left CaMG are presented as the means ± standard deviation (SD).

Sectioning of the spinal cord, estimation of the total number and immunohistochemical staining of the caudal mesenteric ganglion‐projecting preganglionic neurons

The dissected portions of the spinal cords were divided into segments, which were sectioned longitudinally with an HM525 Zeiss cryostat into 10‐μm‐thick serial sections. To calculate the number of FB⁺ perikarya, they were counted in every fourth section to avoid double‐counting of the same neuron; the calculated neurons were 38.13 ± 4.94 μm in diameter (~ 40 μm). Only neurons with clearly visible nuclei were considered. The total numbers of nerve cells counted in each spinal cord section and relative frequencies of the perikarya in sections from either side at each segmental level are presented as the means ± SD.

Immunohistochemistry involved double‐staining, which was performed according to a previously described method (Bossowska & Majewski, 2012). The stainings were applied to cryostat sections from the second and third L spinal cord sections because these sections were found to contain the largest number of FB⁺ neurons. Immunohistochemical characteristics of FB⁺ neurons were investigated using primary antibodies against choline acetyltransferase (ChAT; marker of cholinergic neurons), cocaine and amphetamine‐regulated transcript peptide (CART), calbindin (CB), calcitonin gene‐related peptide (CGRP), galanin (GAL), Leu5‐enkephalin (L‐ENK), neuronal nitric oxide synthase (NOS), neuropeptide Y (NPY), pituitary adenylate cyclase‐activating polypeptide (PACAP), phoenixin (PNX), serotonin (5‐HT), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and vasoactive intestinal polypeptide (VIP); details concerning all the primary and secondary antibodies used are listed in Table 1. ChAT antiserum was applied in a mixture with antisera against CART, CB, CGRP, GAL, L‐ENK, NPY, NOS, PACAP, PNX, 5‐HT, SOM, SP, TH, VAChT or VIP.

Table 1.

List of primary antisera and secondary reagents used for immunohistochemical staining of the CaMG preganglionic neurons

Antigen	Code	Dilution	Species	Supplier
Primary antibodies
CART	H‐003‐61	1 : 6000	Rabbit	Phoenix Pharmaceuticals Inc, Burlingame, CA, USA
CB	CB‐38a	1 : 1500	Rabbit	Swant, Marly, Switzerland
ChAT	AB144P	1 : 200	Goat	Millipore, Temecula, CA, USA
CGRP	AB15360	1 : 2500	Rabbit	Merck, Darmstadt, Germany
GAL	AB 5909	1 : 5000	Rabbit	Millipore, Temecula, CA, USA
L‐ENK	EA1149‐0025	1 : 6000	Rabbit	Enzo Life Sciences, Farmingdale, NY, USA
NOS	AB5380	1 : 6000	Rabbit	Merck, Darmstadt, Germany
NPY	Ab30914	1 : 4500	Rabbit	Abcam, Cambridge, UK
PACAP	T‐4465	1 : 2500	Rabbit	Peninsula, San Carlos, CA, USA
PNX	H‐079‐01	1 : 2500	Rabbit	Phoenix Pharmaceuticals Inc, Burlingame, CA, USA
5‐HT	S5545	1 : 7000	Rabbit	Peninsula, San Carlos, CA, USA
SOM	T‐4103	1 : 4000	Rabbit	Peninsula, San Carlos, CA, USA
SP	8450‐0004	1 : 2500	Rabbit	Bio‐Rad, Kidlington, UK
TH	Ab112	1 : 600	Rabbit	Abcam, Cambridge, UK
VAChT	H‐V007	1 : 6000	Rabbit	Phoenix Pharmaceuticals Inc, Burlingame, CA, USA
VIP	VA 1285‐0025	1 : 5000	Rabbit	Enzo Life Sciences, Farmingdale, NY, USA
Secondary reagents
Biotinylated anti‐rabbit immunoglobulins	711‐1622	1 : 1100	Goat	Rockland, Limerick, PA, USA
CY3‐conjugated streptavidin	711‐165‐152	1 : 9000	–	Jackson I.R., West Grove, PA, USA
Alexa Fluor 488 anti‐goat IgG	A‐11055	1 : 1300	Donkey	Thermo Fisher Scientific, Waltham, MA, USA

5‐HT, serotonin; CART, cocaine‐ and amphetamine‐regulated transcript peptide; CB, calbindin; CGRP, calcitonin gene‐related peptide; ChAT, choline acetyltransferase; GAL, galanin; L‐ENK, Leu5 – enkephalin; NOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; PACAP, pituitary adenylate cyclase‐activating polypeptide; PNX, phoenixin; SOM, somatostatin; SP, substance P; TH, tyrosine hydroxylase; VAChT, vesicular acetylcholine transporter; VIP, vasoactive intestinal polypeptide.

The application of antisera raised in different species enabled us to investigate the coexpression of ChAT with other substances. Retrogradely labelled and immunohistochemically stained perikarya were evaluated under an Olympus BX61 microscope equipped with an epifluorescence filter and an appropriate filter set for CY3 and FITC.

To determine the percentages of particular neuronal subpopulations, at least 200 FB⁺ neuronal profiles were investigated with one combination of the primary antisera for the coexpression of biologically active substances in each pig studied. The percentages of the retrogradely labelled neurons immunopositive for particular biologically active substances or their markers were pooled in the individual animals and presented as the means ± SD. Micrographs were taken using an Olympus XM10 digital camera. The microscope was equipped with cellsens dimension image processing software.

Control of specificity of the tracer staining and immunohistochemical procedures

Thorough macroscopic examinations of FB injection sites and tissues adjacent to the right CaMG were performed before sample collection. The injection sites were easily identified by the yellow‐labelled deposition left by the tracer within the right CaMG. Moreover, the sites were observed under an UV lamp in the darkroom. The tissues adjacent to the right CaMG were not found to be contaminated with the tracer. In none of these procedures was there any leakage of the tracer, validatinf the specificity of the tracing protocol.

Standard controls, i.e. preabsorption for the neuropeptide antisera (20 μg of appropriate antigen per 1 mL of corresponding antibody at working dilution; all antigens purchased from Abcam, Sigma or Phoenix), as well as omission and replacement of the respective primary antiserum with the corresponding non‐immune serum, completely abolished immunofluorescence and eliminated specific staining.

Results

Investigation of the presence of viscerofugal projections from the urinary bladder trigone intramural ganglia to the caudal mesenteric ganglion in the female pig

The comprehensive analysis of cryostat sections of the urinary bladder trigone (UBT), taken from all the animals studied, unquestionably excluded the existence of viscerofugal projections from the UBT‐IG to the CaMG. To visualize UBT‐IG neurons, the sections were stained with PGP (Fig. 1A). However, after injection of fluorescent tracer FB into the right CaMG, no retrogradely labelled (FB+) nerve cell bodies were found in the intramural ganglia in any of the animals studied (Fig. 1B).

Figure 1.

Representative images of neurons in the urinary bladder trigone intramural ganglia (UBT‐IG) (A,B) and Fast Blue (FB)‐positive neurons found in the left caudal mesenteric ganglion (CaMG) (C) after injection of the tracer to the right CaMG. The images were taken separately in green (A) and blue (B,C) fluorescent channels. White brackets indicate the UBT‐IG neurons that stained positive for mouse anti‐human protein gene product (PGP; image A) and were simultaneously FB‐negative (image B). Scale bars: (A,B) 20 μm; (C) 200 μm.

Sectioning of the left caudal mesenteric ganglia and estimation of the total number of Fast Blue‐positive neurons in these ganglia

After injection of FB into the right CaMG, 1017 ± 76.18 FB⁺ neurons were found in the left CaMG (Fig. 1C).

Distribution of sympathetic preganglionic neurons supplying the right caudal mesenteric ganglion in the pig

After injection of FB into the right CaMG, FB⁺ neurons were found in all the pigs; these neurons were distributed from the last two Th segments to the fifth L segment of the spinal cord. Distinct left–right differences were observed, as after injection of FB into the right CaMG, the vast majority of FB⁺ neurons (1509 ± 276; 93.79 ± 2.96%) were found on the ipsilateral (in this case, right) side of the spinal cord and 107.8 ± 67.51 FB⁺ neurons (6.21 ± 2.98%) on the left side of the spinal cord. Moreover, the right L2‐L3 segments appeared to contain distinctly more FB⁺ neurons than any of the other segments. The relative frequency of FB⁺ SPN in particular segments of the spinal cord is shown in Fig. 2.

Figure 2.

Bar diagram showing relative frequencies of Fast Blue (FB)‐positive preganglionic neurons supplying the right caudal mesenteric ganglion (CaMG) in the female pig (n = 6). Each bar represents the mean ± SD of pooled data from the right (R – solid bars) or left (L – open bars) side of each (separate) spinal cord segment. L, lumbar segment of the spinal cord; Th, thoracic segment.

FB⁺ neurons were predominantly distributed in the nucleus intermediolateralis pars principalis (NILpp; 1603 ± 337.8 cells; 99.11 ± 0.34%). Solitary perikarya were also found in the nucleus intermediolateralis pars funicularis (NIpf; 2.83 ± 1.47 cells; 0.19 ± 0.13%) and the nucleus intercalatus spinalis (NIS; 11 ± 2.37 cells; 0.7 ± 0.22%).

Immunohistochemical characteristics of Fast Blue‐positive spinal preganglionic neurons supplying the right caudal mesenteric ganglion

Double‐labelling immunohistochemistry revealed that nearly all (99.4 ± 0.6%) retrogradely labelled preganglionic nerve cells were ChAT positive (ChAT⁺; Figs 3B,E,H,K,N,R and 4B,E,H,K,N,R).

Figure 3.

Representative images of sympathetic preganglionic neurons (SPN) supplying the right caudal mesenteric ganglion (CaMG) in the female pig (n = 6), taken from the nucleus intermediolateralis pars principalis region of the spinal cord. All images were taken separately in blue (A,D,G,J,M,P), green (B,E,H,K,N,R) and red (C,F,I,L,O,S) fluorescent channels. Single arrows indicate Fast Blue‐positive (FB ⁺) neurons (A,D,G,J,M,P) that were simultaneously ChAT ⁺ (B,E,H,K,N,R) and immunonegative for VAChT (C), NOS (F), CART (I), CGRP (L), PACAP (O) or CB (S). Double arrows indicate FB ⁺ neurons (A,D,G,J,M) that were simultaneously ChAT ⁺ (B,E,H,K,N) and immunopositive for VAChT (C), NOS (F), CART (I), CGRP (L) or PACAP (O). Scale bar: (A‐L,P‐S) 100 μm; (M‐O) 50 μm.

Figure 4.

Representative images of sympathetic preganglionic neurons (SPN) supplying the right caudal mesenteric ganglion (CaMG) in the female pig (n = 6), taken from the nucleus intermediolateralis pars principalis region of the spinal cord. All images were taken separately in blue (A,D,G,J,M,P), green (B,E,H,K,N,R) and red (C,F,I,L,O,S) fluorescent channels. Single arrows indicate Fast Blue‐positive (FB ⁺) neurons (A,D,G,J,M,P) that were simultaneously ChAT ⁺ (B,E,H,K,N,R) and immunonegative for L‐ENK (C), NPY (F), 5‐HT (I), SOM (L), SP (O) or TH (S). Scale bar: (G‐I) 100 μm; (A‐F, J‐S) 50 μm.

Among FB⁺/ChAT⁺ neurons, many perikarya also stained for VAChT (63.11 ± 5.34%; Fig. 3C), NOS (53.48 ± 9.62%; Fig. 3F) or CART (41.13 ± 4.77%; Fig. 3I). Small numbers of retrogradely labelled perikarya stained for CGRP (7.60 ± 1.34%; Fig. 3L) or PACAP (4.57 ± 1.43%; Fig. 3O). No FB⁺/ChAT⁺ nerve cells were immunoreactive for CB (Fig. 3S), GAL, L‐ENK (Fig. 4C), NPY (Fig. 4F), PNX, 5‐HT (Fig. 4I), SOM (Fig. 4L), SP (Fig. 4O), TH (Fig. 4S) or VIP. Retrogradely labelled SPN supplying the CaMG were surrounded by moderately dense networks of TH⁺ (Fig. 4S), 5‐HT⁺ (Fig. 4I) or VACHT⁺ (Fig. 3C) NF and very dense networks of axons immunopositive for CART (Fig. 3I), L‐ENK (Fig. 4C), PACAP (Fig. 3O) or SOM (Fig. 4L), sometimes forming basket‐like structures around the FB⁺ perikarya (upper double arrow, Fig. 3L; double arrow, Fig. 3O; second and third single arrows from the top, Fig. 4L).

Discussion

It is well known that the functions of the urinary bladder, i.e. the collection and storage of urine and its periodic emptying, are regulated by complex neuronal pathways (de Groat, 2006). One of the sources of autonomic innervation of this organ are ganglia distributed in its wall (Gabella, 1990; Dixon et al. 2000; Pidsudko, 2013). Intramural ganglia are associated with many visceral organs, but the most numerous are those of the gastrointestinal tract (Furness et al. 2004). The results of many studies confirm that the role of intramural ganglia is not limited to a simple transfer of autonomic activity to the smooth muscles or glands in their host tissues. It is well known that these neurons have many complex interconnections with adjacent ganglia or collaterals of primary sensory fibres, forming local neural networks capable of influencing and regulating the intrinsic motor/sensory system. In the intestines, some intramural nerve cells, called viscerofugal neurons, send their axons to prevertebral sympathetic ganglia, thereby actively participating in extrinsic reflex control of intestinal peristaltic activity and secretion (Furness et al. 2004; Hibberd et al. 2012). To some extent, the urinary bladder wall has a comparable micro‐anatomical arrangement to that of the gastrointestinal tract. Moreover, it has been suggested that intramural neurons participate in the control of urinary bladder function (Gillespie et al. 2006). Therefore, we decided to investigate whether, similar to the gastrointestinal tract, the urinary bladder has intramural neurons that send their axons outside the organ to the neighbouring prevertebral ganglion, CaMG. However, a thorough analysis of urinary bladder trigone sections, after injections of the tracer FB into the CaMG, clearly excluded the existence of the viscerofugal projections from the UBT‐IGN to this ganglion, at least in the female pig. Therefore, for the first time, we can exclude the possibility of retrograde control of prevertebral ganglion neuronal activity by UBT‐IG neurons.

The present study revealed that after administration of FB in the right CaMG, ~ 1000 FB⁺ neurons were found in the left one. Interestingly, Ragionieri et al. (2012) found that after injection of FB tracer into the first sacral sympathetic chain ganglion in the pig, FB⁺ nerve cells were observed not only in the intermediolateral nucleus of the spinal cord segments but also in adjacent sympathetic chain ganglia (L3‐Co1) or even in the CaMG and pelvic ganglia. On the one hand, the present findings and the results obtained by Ragionieri et al. (2012) could indicate the existence of very intriguing visceral neuronal circuits between different autonomic ganglia. A positive verification of this theory would invalidate the traditionally understood arrangement of the autonomic motor pathways, which assumes a simple transmission of nerve signals from preganglionic to postganglionic neurons and then to the innervated tissue. However, in our opinion, the most probable explanation of this phenomenon is that FB administered through injections to a certain ganglion is absorbed not only by the nerve endings that synapse in this ganglion but also by nerve fibres that are simply passing through (or in the close vicinity of) the injected tissue. Aside from the possibility that the tracer may be taken up by uninterrupted nerve projections, many fibres passing through the ganglion are certainly disrupted by the inserted needle, which also facilitates FB absorption. From that point of view, the present data suggest only that the nerve cells in each (left and right) CaMG send postganglionic axons to targets on both the ipsi‐ and contralateral sides of the body. It must be mentioned that the existence of cross‐innervation from different autonomic ganglia has been proven before (Lepiarczyk et al. 2015) and the level of the CaMG has been previously suggested to be such a crossing point (Kaleczyc et al. 2002).

The present results provide further evidence of the irrefutable significance of preganglionic projections in the CaMG nerve supply. SPN are more than the source of the entire sympathetic outflow to the sympathetic ganglia and adrenal medulla (Cabot, 1990), they should also be considered a final station where autonomic activity can be integrated and modulated within the central nervous system. Interestingly, projections of preganglionic neurons exhibit considerable divergence, as thousands of postganglionic autonomic pelvic and intramural urinary bladder neurons are supplied by a distinctly smaller number of SPNs (for review see Keast, 1999).

The present study has revealed that in the female pig, the SPN projecting to the right CaMG are found from the last two Th segments to the L5 segment of the spinal cord, but most of them are localized in the L2‐L3 neuromeres, predominantly in the NILpp. The injection of FB only into the right ganglion allowed us to identify distinct left–right differences, as the vast majority (~ 94%) of FB⁺ neurons were found on the right (ipsilateral) side of the spinal cord. Therefore, ~ 6% of SPN supplying the right CaMG reach the innervated ganglion from the contralateral (left) NILpp. The present results are consistent with findings obtained in other species. Anderson et al. (1995) found that in the guinea pig, after injection of tracer FB into the CaMG, most of the retrogradely labelled nerve cells were distributed in Th12‐L2 segments of the spinal cord. As in the present experiment, the majority of the neurons projecting to the CaMG were located in the intermediolateral column (i.e. in the NILpp). In contrast to the present findings, in both the guinea pig (Anderson et al. 1995) and the rat (Strack et al. 1988), the CaMG receives most of its preganglionic inputs from the L1 segment of the spinal cord; however, it must be mentioned that it is always difficult to compare spinal cord segments between different species because they have different vertebral formulae.

Immunohistochemistry revealed the complex neurochemical composition of CaMG‐projecting SPN. As mentioned in the introductory section, CaMG nerve cells send their axons to multiple targets in the abdominal and pelvic cavities (e.g. Miolan & Niel, 1996; Pidsudko et al. 2001; Ragionieri et al. 2013; Pidsudko, 2014; Barbe et al. 2018). Therefore, differentiated chemical coding of the SPN may suggest that different populations of these perikarya supply discrete groups of CaMG neurons that project to specific targets.

As expected, virtually all FB⁺ nerve cells in the spinal cord were immunoreactive for ChAT, implying their cholinergic nature. Among FB⁺/ChAT⁺ neurons, ~ 60% of perikarya were VAChT⁺. This finding corresponds well with the results of Całka et al. (2008b), who found that in the pig, the distribution and morphology of ChAT⁺ perikarya in the intermediolateral cell column of Th1‐L2 spinal cord segments were similar to that of VAChT⁺ nerve cells. Both ChAT and VAChT are considered markers of cholinergic nerve cells, but their expression in the same perikarya may vary according to the different roles they play in neurons. ChAT is an enzyme responsible for acetylcholine (ACh) synthesis, whereas VAChT ensures the loading of this neurotransmitter into synaptic vesicles (Parsons, 2000; Prado et al. 2002). Therefore, all cholinergic nerve cells should express ChAT but not necessarily VAChT. A large number of FB⁺ cholinergic SPNs projecting to porcine CaMG expressing VAChT may indirectly indicate prominent ongoing release of ACh from these nerve cells. This assumption may be confirmed by experiments using vesamicol and vesamicol analogues (Parsons, 2000), which have demonstrated that inhibition of ACh transport into synaptic vesicles decreases ACh release from the nerve terminals. Interestingly, the present results stand in contrast to findings of Juranek & Wojtkiewicz (2015), who found a surprisingly low number (only 7%) of VAChT‐expressing nerve cells in the population of ChAT⁺ preganglionic neurons supplying the superior cervical ganglion in the pig.

More than half of the FB⁺/ChAT⁺ neurons stained for NOS, which corresponds well with the results of Całka et al. (2008a), who investigated the coexpression of ChAT and NOS in neurons of the porcine thoracic and sacral spinal cord. Those authors found that ~ 50–60% of cholinergic perikarya in the intermediolateral nucleus (depending on the investigated segment of the spinal cord) also stained for NOS. Similar results were obtained in rats (Wetts & Vaughn, 1994), in which ~ 62% of autonomic motor neurons in the spinal cord were double‐labelled for ChAT and NOS. Because NOS⁺ perikarya constitute a large population of all the SPN, it seems likely that most NOS⁺ NF in sympathetic ganglia represent axons of these nerve cells. It has been found, for instance, that sectioning of the sympathetic trunk abolishes immunostaining for NOS in the superior cervical ganglion (Ceccatelli et al. 1994). Moreover, the large number of neurons co‐reactive for both ChAT and NOS strongly suggests that nitric oxide (NO) should be considered a significant factor modulating the cholinergic activity of SPN. Indeed, it has been revealed that in the central nervous system, NO alters cholinergic transmission (Garthwaite, 1991) and is capable of controlling both ACh release and metabolism (Morot Gaudry‐Talarmain et al. 1997). Although endogenous NO enhances the release of ACh from neurons in the basal forebrain (Prast & Philippu, 1992), in the spinal cord, this neurotransmitter seems to affect both excitatory and inhibitory transmission in SPN (Wu & Dun, 1995, 1996; Wu et al. 1997).

Approximately 41% of the FB⁺/ChAT⁺ nerve cells were simultaneously immunolabelled for CART. This finding is consistent with some results obtained in the rat. Dun et al. (2000) found that the vast majority of cholinergic SPNs in the thoracolumbar segments of the spinal cord were CART⁺. Moreover, Fenwick et al. (2006) revealed that ~ 35–40% of SPN projecting to the coeliac ganglion or to the major pelvic ganglion contain CART immunoreactivity. Interestingly, it has also been determined that CART is selectively expressed in a population of sympathetic preganglionic neurons but not in parasympathetic preganglionic neurons (Dun et al. 2000; Fenwick et al. 2006). CART is a neurotransmitter widely expressed in the central nervous system, especially in areas involved in the control of autonomic functions. It has been suggested that a significant number of CART⁺ SPNs may be involved in cardiovascular regulation. However, it has also been found that the occurrence of this neurotransmitter is not restricted to SPN controlling the heart and blood vessels (Fenwick et al. 2006), suggesting that CART may play a variety of roles in central autonomic control. The results of some experiments imply that this peptide may enhance sympathetic activity (Matsumura et al. 2001).

The present results indicate that ~ 8% of cholinergic SPNs projecting to the CaMG display immunoreactivity for CGRP. Colocalization of ChAT‐negative (ChAT^–) and CGRP has been previously found in the intermediolateral preganglionic neurons in the female pig (Całka et al. 2009), rat (Senba & Tohyama, 1988) and cat (Sámano et al. 2006). Moreover, Majewski & Heym (1992) revealed a dense network of CGPR⁺ NF which closely apposed noradrenergic neurons in the porcine CaMG. Considering the present findings, it can be assumed that at least some of them represent preganglionic sympathetic processes.

A small number (~ 4.5%) of retrogradely labelled cholinergic perikarya found in the present investigation were immunostained for PACAP. This finding correlates with the results of in situ hybridization studies that revealed the existence of preproPACAP mRNA in rat spinal cord intermediolateral neurons (Beaudet et al. 1998). Beaudet et al. (2000) suggested that in the rat, PACAP released from preganglionic sympathetic projections may not necessarily act as an ACh modulator but can play a role as a noncholinergic regulator of sympathetic function. Those authors, using electrophysiological and pharmacological approaches, have revealed that PACAP itself is able to depolarize most postganglionic sympathetic neurons in the superior cervical ganglion or sympathetic chain ganglia.

One of the interesting findings obtained in the present study was the lack of L‐ENK immunoreactivity in the FB⁺ SPN. This is quite surprising, as in many animal species, including the pig (Kaleczyc et al. 1995; Pidsudko et al. 2001; Ragionieri et al. 2013; Pidsudko, 2014), sheep (Sienkiewicz et al. 2015) and guinea pig (Dalsgaard & Elfvin, 1979), a dense meshwork of L‐ENK⁺ nerve terminals, often forming ‘basket‐like’ structures, were found to surround perikarya in CaMG. Therefore, it should be expected that these fibres originate from the SPN. Moreover, combined retrograde tracing and immunohistochemistry revealed the existence of L‐ENK⁺ SPN projecting to CaMG in the guinea pig (Dalsgaard et al. 1982). Nevertheless, the present data indicate that, at least in the pig, L‐ENK⁺ projections to CaMG must originate from another source. It is possible that L‐ENK⁺ NF supplying CaMG neurons may represent collaterals of peripheral processes of DRG neurons, because, in the pig, they have been found to stain for L‐ENK (Zacharko‐Siembida et al. 2014). The association of the L‐ENK⁺ axons with viscerofugal projections from the gastrointestinal tract (suggested previously by Kaleczyc et al. 2003) should also be taken into consideration.

Author contributions

Ewa Lepiarczyk and Mariusz Majewski conceived and designed the experiments; Ewa Lepiarczyk, Agnieszka Bossowska, Agnieszka Skowrońska and Mariusz Majewski performed the surgical procedures; Ewa Lepiarczyk performed the immunohistochemical procedures, analysed the data and wrote the paper.

Conflict of interests

The authors declare no conflict of interests.