Identification of neuron types in the submucosal ganglia of the mouse ileum
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- Mongardi Fantaguzzi, C., Thacker, M., Chiocchetti, R. et al. Cell Tissue Res (2009) 336: 179. doi:10.1007/s00441-009-0773-2
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The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found.
KeywordsEnteric nervous systemChemical codingSubmucosal gangliaSmall intestineNeuropeptidesMouse (C57black6)
Definition of the enteric neuron types in the mouse is important because of the advantages of this species for the investigation of the roles of specific molecules in these enteric neurons and their effectors; mouse mutants can be created that, for example, abolish or augment the functions of the targeted molecules (Mashimo et al. 2000; Abdu et al. 2002; Bian et al. 2003; Ren et al. 2003; Clarke et al. 2007). The need to define the neurons in mouse arises because the chemistries of orthologous enteric neurons differ between species and the extrapolation of data from other species to mice is not necessarily reliable (Timmermans et al. 1997, 2001; Chiocchetti et al. 2004; Brehmer 2006, 2007). For this reason, the neurons with cell bodies in the myenteric plexus of the mouse small intestine have recently been re-investigated (Qu et al. 2008). This re-investigation has defined all the major myenteric neuron types by their chemistries, morphologies and projections to targets and, overall, more than 90% of myenteric neurons have been accounted for. The study of myenteric neurons has built upon earlier investigations of the chemistries and projections of these neurons in the mouse small intestine (Sang and Young 1996, 1998; Sang et al. 1997).
Comparable studies have not been carried out for the submucosa, and there is a relative paucity of information, particularly a lack of quantitative information, about the neuron types present. The studies that have been performed indicate that calretinin immunoreactivity occurs in a high proportion of submucosal neurons in the mouse (Van Nassauw et al. 2000), that somatostatin-immunoreactive neurons are common and that choline acetyltransferase (ChAT) also occurs in the somatostatin neurons (De Jonge et al. 2003b). One study reports neurons immunoreactive for calcitonin gene-related peptide (CGRP; Pham et al. 1991), whereas a later study indicates that they are not present (De Jonge et al. 2003a). A small proportion of submucosal neurons is nitric oxide synthase (NOS)-immunoreactive (Young and Ciampoli 1998). Tyrosine hydroxylase (TH), rather surprisingly, is a marker of submucosal neurons in the mouse (Li et al. 2004) but where these neurons project or how they relate to the other populations is not known. This is important to determine, especially as there is evidence that not all nerve cells with TH synthesise catecholamines and that some neurons with TH in their cell bodies do not have TH in their terminals (Kummer et al. 1990; Weihe et al. 2005; Hoard et al. 2008). Only rarely is quantitative information available on the numbers of submucosal nerve cells that are defined by particular neurochemical markers in the mouse (Young and Ciampoli 1998; Li et al. 2004; Van Op Den Bosch et al. 2008). Thus, significant deficiencies exist in our knowledge of which neurons are present, of how populations that have been described relate to each other, and of the possible functional identities of the different classes of submucosal neurons.
Materials and methods
All animal procedures conformed to National Health and Medical Research Council of Australia guidelines and were approved by the University of Melbourne Animal Experimentation Ethics Committee. The subjects of study were C57black6 mice of either sex (6–10 weeks old) that were killed by cervical dislocation. All observations were repeated on specimens from at least three mice.
For tissue that was to be examined in whole-mount, segments of ileum were removed and fixed for immunohistochemistry. The ileum was cleaned of its contents, opened along the mesenteric attachment and pinned, mucosa down, to balsa-wood sheets for fixation in 2% formaldehyde plus 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.2, at 4°C. Fixation was continued overnight at 4°C. Preparations were cleared of fixative by washes (3 × 10 min) in dimethyl sulphoxide (DMSO) followed by washes (3 × 10 min) in phosphate-buffered saline (PBS: 0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.2). Fixed tissue was stored at 4°C in PBS containing sodium azide (0.1%).
Primary antisera and labels and their respective dilutions
Tissue antigen or label
Dilution (for whole-mounts)
Sourcea or reference
Hu (RNA-regulating protein)
Fairman et al. 1995
CGRP (calcitonin gene-related peptide)
NPY (neuropeptide Y)
Maccarrone and Jarrott 1985
Furness et al. 1985
VIP (vasoactive intestinal peptide)
Furness and Costa 1979
Accili et al. 1995
TH (tyrosine hydroxylase)
Chemicon - AB1542
ChAT (choline acetyltransferase)
VAChT (vesicular acetylcholine transporter)
Weihe et al. 1996
NFM (neurofilament medium)
NF (neurofilament) 200 kDa
NOS (nitric oxide synthase)
Williamson et al. 1996
Secondary antisera and labels
Donkey α human TR
Donkey α rabbit 594
Donkey α rabbit 488
Donkey α rabbit 647
Donkey α sheep 594
Donkey α sheep 488
Donkey α mouse 594
Donkey α mouse 488
Donkey α chicken 488
Intestine to be examined in sections was opened and pinned to balsa wood, mucosa side up, without stretching. It was fixed in the same formaldehyde/picric acid mixture, cleared in DMSO as described above, placed in PBS-sucrose-azide (PBS containing 0.1% sodium azide and 30% sucrose as a cryoprotectant) and stored at 4°C overnight. The following day, tissue was transferred to a mixture of PBS-sucrose-azide and OCT compound (Tissue Tek, Elkhart, Ind., USA) at a ratio of 1:1 for a further 24 h before being embedded in 100% OCT. Sections of 12 μm thickness were cut and collected on 1% gelatinised slides and left to dry for 1 h at room temperature. Sections were then incubated in primary and secondary antibodies, as described above.
To clarify the origins of particular nerve fibres, the extrinsic nerves were cut in six mice. Mice were anaesthetised (via intraperitoneal injection) with a mixture of xylazine (20 mg.kg−1) and ketamine hydrochloride (100 mg.kg−1), both from Troy Laboratories (Sydney, Australia). The abdominal cavity was opened and part of the ileum was exteriorised. The nerves supplying 6–8 cm of ileum were stripped away from about 5 mm of the vessels supplying the intestine and this region of the vessels was swabbed with 5% phenol to kill any remaining nerve fibres. The animals were allowed to recover; the denervated region and non-denervated regions were taken 6 days later.
Preparations were analysed by confocal microscopy on a Zeiss Meta or Zeiss Pascal confocal laser scanning system. Fluorophores were visualised by using a 488-nm excitation filter and 505/530-nm emission filter for Alexa 488 or fluorescein isothiocyanate, a 568-nm excitation and 575/615-nm emission filter for Alexa 594 and a 633-nm excitation and 650-nm emission long-pass filter for Alexa 647. Single 1024 × 1024 pixel images (optical sections of 0.5 μm nominal thickness) were captured.
The proportions of neurons in which immunoreactivity was colocalised were determined by examining fluorescently labelled, double-stained, preparations. Neurons were first located by the presence of a fluorophore that labelled one antigen and then the illumination was switched to determine whether the neuron was labelled for a second antigen located with a fluorophore of a different colour. In this way, the proportions of neurons labelled for pairs of antigens were determined. In general, the cohort size was 200 neurons and data were collected from preparations obtained from at least three animals. In some cases, neuron numbers were low and cohorts of fewer than 200 were counted. Data are given as mean±SEM.
Nerve cell bodies
The synthesising enzyme for acetylcholine, ChAT, was present in 42.0 ± 1.5% of neurons. The immunoreactivity was cytoplasmic in these neurons. When ChAT and VIP immunoreactivity were localised in the same preparations, ChAT was never localised with VIP (Fig. 1b, b′). Triple-staining (ChAT, VIP, Hu) confirmed the counts made in double-stained preparations for three populations: VIP neurons, ChAT neurons, and 6.1 ± 1.1% of neurons in which only Hu immunoreactivity was detected (Fig. 1b).
Immunoreactivity for TH was found in a minority of neurons and double-staining with VIP showed that all of these were VIP-positive (Fig. 3c, c′). Counts in these double-stained preparations indicated that 42.5 ± 1.8% of VIP neurons had TH immunoreactivity, i.e. 22% of all neurons.
Calretinin immunoreactivity occurred in 91.7 ± 1.7% of neurons. These could be divided into strongly immunoreactive calretinin neurons and weakly immunoreactive calretinin neurons. In preparations that were stained for VIP and calretinin, 88.8 ± 0.5% of VIP neurons had weak calretinin immunoreactivity. None of the neurons that were strongly immunoreactive for calretinin had VIP immunoreactivity (Fig. 3d, d′).
We have confirmed earlier observations that there is a small proportion of neurons (less than 3%) that has immunoreactivity for NOS (Young and Ciampoli 1998). Our preliminary investigations indicate that these are not calretinin-immunoreactive.
Nerve fibre populations
Within the ganglia, numerous CGRP-, NPY- and TH-immunoreactive nerve fibre varicosities surrounded most nerve cells (Fig. 4). Despite the observation of a high degree of co-localisation of VIP and NPY in nerve cells, the NPY-positive fibres around nerve cells were not VIP-immunoreactive (Fig. 4b, b′). Indeed, few VIP-immunoreactive fibres were found in the ganglia and those that were present crossed the ganglia without providing terminals around individual nerve cells; many of the VIP-positive axons that traversed the ganglia were NPY-immunoreactive. Strongly immunoreactive TH fibre varicosities were common in the ganglia; these were almost all NPY-immunoreactive (Fig. 4c, c′) but not VIP-immunoreactive. The peri-neuronal varicose NPY/TH fibres in the ganglia were not seen after extrinsic denervation, although some non-varicose fibres ran through the ganglia, without forming pericellular baskets (Fig. 4d, d′). Rare calretinin-immunoreactive terminals were observed in the ganglia and the majority of nerve cells had no calretinin fibres in close association with them (Fig. 3d). Most of the calretinin fibres that were present entered the ganglia in interconnecting nerve fibre bundles, ran through the ganglia and exited in an interconnecting bundle. In a few cases, a single calretinin fibre ran around the surface of a nerve cell. Fibres with NOS immunoreactivity were numerous in the ganglia but the sources of these fibres have not yet been determined.
No changes in expression of the neurochemical markers were observed in the submucosal neurons following extrinsic denervation.
This work has defined five groups of nerve cells in submucosal ganglia: cell bodies of VIP/NPY/calretinin neurons (about 30%), VIP/NPY/calretinin/TH neurons (about 20%), ChAT/CGRP/SOM neurons (about 30%; including a small sub-group of ChAT/CGRP/SOM/NPY neurons), neurons with ChAT and no other marker (about 10%) and neurons with neither ChAT nor VIP (about 8%). The proportion of neurons showing somatostatin immunoreactivity (30%) is similar to that determined previously (32%; Van Op Den Bosch et al. 2008). The proportion of submucosal neurons with TH immunoreactivity (20%) is slightly larger than the 13% found in a previous study in which the CD-1 mouse strain was used (Li et al. 2004). Indeed, it is probably prudent to consider that the quantitative results of the present study may not be applicable to other strains of mice. Three targets of submucosal neurons, viz., the submucosal ganglia themselves, submucosal arterioles and the mucosa, have been investigated. These targets also receive innervation from extrinsic sources that can lead to difficulties of interpretation, because the sympathetic neurons contain TH, some contain NPY and many dorsal root ganglion neurons contain CGRP. Therefore, to help clarify targets of submucosal neurons, we have studied tissues from mice in which the extrinsic nerves had been cut and time allowed for their terminals to degenerate.
VIP/TH nerve fibres have only been identified in the mucosa and VIP fibres do not supply varicosities around submucosal nerve cells. These results are similar to observations in the guinea-pig (Furness et al. 2003) and suggest that the VIP/NPY/calretinin/TH neurons are secretomotor neurons. This accords with retrograde tracing studies that have shown that NPY-immunoreactive submucosal neurons project to the mucosa of the mouse small intestine (Van Nassauw et al. 2000) and is consistent with VIP being a primary transmitter of secretomotor neurons, as has been demonstrated in other species, including human, rat, guinea-pig and cat (Furness et al. 1984; Lundgren 2002; Banks et al. 2005; Furness 2006). VIP is also a stimulant of secretion in the mouse small intestine (Cox et al. 2001), although its role as a secretomotor neurotransmitter has not been directly investigated. VIP-immunoreactive fibres also innervate the submucosal arterioles, as they do in other species, where functional evidence indicates that they are vasodilator neurons (Vanner and MacNaughton 2004). Although, in other species, the VIP-immunoreactive vasodilator terminals are considered to be collaterals of secretomotor neurons (Furness et al. 1987), we have not detected TH immunoreactivity in the VIP endings around the blood vessels. In the mouse, the populations of VIP-immunoreactive secretomotor (coding VIP/NPY/calretinin/TH) and vasodilator neurons (coding VIP/NPY/calretinin) apear to be separate. In order to confirm this, it will probably be necessary to fill individual neurons by intracellular injection and to trace them to their targets.
In other species, in addition to the VIP-containing non-cholinergic secretomotor neurons, one or more classes of cholinergic secretomotor neurons occur. A likely candidate for these neurons are the CGRP/SOM/ChAT neurons that have previously been identified as SOM/ChAT neurons in mouse submucosal ganglia by De Jonge et al. (2003b). These neurons have a coding that is partly shared by cholinergic secretomotor neurons that have the coding CGRP/SOM/ChAT/GAL/NPY/CCK in the guinea-pg small intestine (Furness et al. 1987).
Curiously, in the mouse (this study), guinea-pig (Furness et al. 1987) and rat (Ekblad et al. 1984), the same secretomotor neurons express transmitter systems that activate secretion and transmitter systems that reduce secretion: cholinergic neurons with somatostatin in guinea-pig and mouse, and VIP neurons with NPY in rat and mouse. In rat, VIP and NPY are found in the same secretory vesicles of submucosal neurons, which strongly suggests that these peptides are released together (Cox et al. 1994). This may indicate that the extent to which secretion can be increased is regulated by the co-release of a transmitter that limits the amount or duration of secretion. A problem in investigating the significance of the co-localisation of peptides that enhance and inhibit secretion is the lack of good pharmacological tools, for example, blockers of VIP actions, that can be used to investigate their relative roles. Nevertheless, we might speculate that the inhibitory peptides have a role in limiting the life-threatening secretion that can occur in response to pathogens that activate secretomotor neurons (Lundgren 2002).
The complexity of the submucosal plexus changes remarkably with the size of animal (Brehmer 2006). In species with large body sizes, such as the pig, horse and dog, the submucosa has clearly defined outer and inner plexuses (Timmermans et al. 2001). Descriptions in human have described a third intermediate plexus (Hoyle and Burnstock 1989; Wedel et al. 1999). Studies in the pig, dog and human show that the outer plexus contains motor neurons that innervate the external musculature (Sanders and Smith 1986; Furness et al. 1990; Porter et al. 1999). Structural studies, including retrograde tracing experiments, indicate that type-II neurons are common in the submucosal ganglia of pig and human (Timmermans et al. 1992; Hens et al. 2000, 2001). In contrast to larger mammals, the submucosa of the guinea-pig small intestine contains only one layer of ganglia and, in this species, all nerve fibres that innervate the external muscle derive from myenteric ganglia (Wilson et al. 1987; Furness et al. 2000). Indeed, the submucosal ganglia in the guinea-pig are remarkably simple, containing only four types of neuron (Furness et al. 2003), these being classes of secretomotor neurons, vasomotor neurons and intrinsic primary afferent neurons, compared with 14 types in the pig, including secretomotor neurons, vasomotor neurons, intrinsic primary afferent neurons, muscle motor neurons, interneurons and intestinofugal neurons (Timmermans et al. 2001). Mouse submucosal ganglia have a simplicity of structure similar to that of the guinea-pig and may have a further simplification of organisation in that we have not been able to find evidence for the presence of type-II putative intrinsic primary afferent neurons, in these ganglia. In the myenteric plexus of the mouse (Mao et al. 2006), as in the guinea-pig (Furness et al. 2004a), the putative intrinsic primary afferent neurons have type-II morphology and AH electrophysiological characteristics. However, an electrophysiological study of mouse submucosal neurons, although in the colon, has found that none of the neurons exhibit electrophysiological features suggestive of AH neurons (Wong et al. 2008); similarly, we have not found type-II neurons or neurons with the chemical signatures (NF-M/ChAT/CGRP) that mark AH/type-II neurons in the myenteric ganglia of the mouse small intestine. In particular, no nerve cells have been found that are immunoreactive for NF-M, which is an excellent marker of the type-II neurons in the myenteric ganglia of the mouse small intestine (Qu et al. 2008).
The present study has also identified nerve fibres that exhibit TH/NPY immunoreactivity, that are of extrinsic origin and that innervate submucosal ganglia and submucosal arterioles. These are almost certainly the fibres that supply the sympathetic noradrenergic innervation of these targets. In the guinea-pig, the sympathetic vasoconstrictor neurons also contain NPY (Furness et al. 1983), as is the case in most mammals. However, in contrast to the mouse, the sympathetic neurons that innervate submucosal neurons of guinea-pigs contain somatostatin (Costa and Furness 1984). Other sources of innervation of the submucosal neurons, e.g. connections from the myenteric ganglia, need to be investigated, particularly as innervation by calretinin-immunoreactive fibres seems to be lacking, even though calretinin is present in 92% of the submucosal nerve cells. Moreover, there is no innervation by VIP fibres, although 51% of nerve cells were VIP-immunoreactive. This has some consistency with the submucosal ganglia of the guinea-pig small intestine, where the motor neurons in the ganglia (90% of the neurons) do not provide collaterals to other submucosal neurons (Furness et al. 2003).
A small proportion of neurons (6%–8%) have not been identified neurochemically. We have not used colchicine in an attempt to enhance neuronal immunoreactivity for peptides because immunoreactivity for each of the peptides that has been localised is clearly seen. In future, the range of neurochemical markers that are targeted should be expanded in order to identify this remaining group of neurons. Furthermore, the exploration of other markers might show that the types that have been identified can be sub-divided.
We thus conclude that the three neuron types that account for most neurons in the submucosal ganglia of the mouse small intestine are VIP-immunoreactive non-cholinergic secretomotor neurons (~30% of neurons), VIP-immunoreactive non-cholinergic vasomotor neurons (~20% of neurons), and cholinergic secretomotor neurons, many of which also contain CGRP and somatostatin (~45% of neurons). Based on our investigation of cell morphology and neurochemistry, no evidence for the presence of intrinsic primary afferent neurons in submucosal ganglia of the mouse has been obtained.