Expression and developmental regulation of oxytocin (OT) and oxytocin receptors (OTR) in the enteric nervous system (ENS) and intestinal epithelium

Abstract

Although oxytocin (OT) and oxytocin receptor (OTR) are known for roles in parturition and milk let-down, they are not hypothalamus-restricted. OT is important in nurturing and opposition to stress. Transcripts encoding OT and OTR have been reported in adult human gut, and OT affects intestinal motility. We tested the hypotheses that OT is endogenous to the enteric nervous system (ENS) and that OTR signaling may participate in enteric neurophysiology. Reverse transcriptase polymerase chain reaction confirmed OT and OTR transcripts in adult mouse and rat gut and in precursors of enteric neurons immunoselected from fetal rats. Enteric OT and OTR expression continued through adulthood but was developmentally regulated, peaking at postnatal day 7. Coincidence of the immunoreactivities of OTR and the neural marker Hu was 100% in the P3 and 71% in the adult myenteric plexus, when submucosal neurons were also OTR-immunoreactive. Co-localization with NeuN established that intrinsic primary afferent neurons are OTR-expressing. Because OTR transcripts and protein were detected in the nodose ganglia, OT signaling might also affect extrinsic primary afferent neurons. Although OT immunoreactivity was found only in approximately 1% of myenteric neurons, extensive OT-immunoreactive varicosities surrounded many others. Villus enterocytes were OTR-immunoreactive through postnatal day 17; however, by postnatal day 19, immunoreactivity waned to become restricted to crypts and concentrated at crypt-villus junctions. Immunoelectron microscopy revealed plasmalemmal OTR at enterocyte adherens junctions. We suggest that OT and OTR signaling might be important in ENS development and function and might play roles in visceral sensory perception and neural modulation of epithelial biology.

Figure 1

Transcripts encoding OTR are expressed in the rodent gut and nodose ganglion. A: Reverse transcriptase polymerase chain reaction (RT-PCR) showing OTR transcripts in rat brain and in isolated samples of rat intestinal mucosa. Br, brain; Mu, mucosa. B: OTR transcripts were detectable in whole mouse gut and in isolated neural crest-derived cells immunoselected from the E16 mouse gut with antibodies to the p⁷⁵ neurotrophin receptor (p⁷⁵). G, gut. C: Messenger RNA (mRNA) encoding OTR found by RT-PCR in the nodose ganglion. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal RT-PCR control. N, nodose. D–F: OTR immunoreactivity and retrograde labeling 1 week after intra-abdominal administration of Fluoro-Gold (FG) were simultaneously demonstrated in rat nodose ganglia. D: OTR immunoreactivity. E: FG immunolabeling. F: Merged image. The arrow indicates an example of a neuron doubly labeled with antibodies to OTR and FG. G: Transcripts encoding OT are developmentally regulated in the rat large intestine. Real-time PCR was employed to quantify transcripts encoding OT as a function of age from E19 to P56. Expression peaks at P7. H: Transcripts encoding OTR are developmentally regulated in the rat large intestine. Real-time PCR was employed to quantify transcripts encoding OTR as a function of age from E19 to P56. Expression peaks at P7 where it is coincident with that of OT. A magenta-green copy of this image is available as Supplementary Figure 2. Scale bar = 50 µm in F (applies to D–F).

Figure 2

Neurons are OTR-immunoreactive in the developing (P3) myenteric plexus. Whole mount preparations of longitudinal muscle with adherent myenteric plexus. A–C: Duodenum. D–F: Colon. A: Duodenum; OTR immunoreactivity. B: Duodenum; Hu immunoreactivity identifying neurons. C: Duodenum; merged image. Note that OTR and Hu immunoreactivities are coincident. D: Colon; OTR immunoreactivity. E: Colon; Hu immunoreactivity identifying neurons. F: Colon; merged image. Note that OTR and Hu immunoreactivities are coincident. A magenta-green copy of this image is available as Supplementary Figure 3. Scale bar = 20 µm in C (applies to A–C) and F (applies to D–F).

Figure 3

OTR immunoreactivity is developmentally regulated and in adult myenteric plexus is expressed in IPANs. Whole mount preparations of longitudinal muscle with adherent myenteric plexus. A–C: P3 rat colon. D–F: Adult rat colon. A: OTR immunoreactivity. B: NeuN immunoreactivity; uniform immunostaining of developing neurons precludes using NeuN to identify IPANs in the developing gut. C: Merged image. D: OTR immunoreactivity. E: NeuN immunoreactivity is present in both the cytoplasm and nucleus of IPANs. F: Merged image. IPANs are included in the OTR-immunoreactive subset of myenteric neurons. A magenta-green copy of this image is available as Supplementary Figure 4. Scale bar = 20 µm in C (applies to A–F).

Figure 4

Submucosal neurons are OTR-immunoreactive. Cryostat sections; immunoreactivity visualized with HRP. A: Submucosal ganglia. B: Control (primary antibodies omitted). The arrows point to submucosal ganglia. Immunostaining obscures cell boundaries. CM, circular muscle. Scale bar = 30 µm B (applies to A,B).

Figure 5

Coincident immunoreactivities of Hu and OTR confirm that OTR-immunoreactive cells in submucosal ganglia are neurons. A: Duodenum; OTR immunoreactivity. B: Duodenum; Hu immunoreactivity identifying neurons. C: Duodenum; merged image: note that OTR and Hu immunoreactivities are coincident. A magenta-green copy of this image is available as Supplementary Figure 5. Scale bar = 20 µm in C (applies to A–C).

Figure 6

OTR immunoreactivity is present in villus enterocytes in early postnatal bowel. Sections exposed to primary antibodies at each age (A,C,E,G) are displayed in the left column. The corresponding control sections, which were not exposed to primary antibodies (B,D,F) or were exposed to antibodies that were preadsorbed with the immunizing peptide (H), are displayed in the right column. Pup ages: A,B (P0); C,D (P3); E,F (P7); G,H (P15). Scale bar = 100 µm in H (applies to A–H).

Figure 7

OTR immunoreactivity in the mucosal epithelium is developmentally regulated in the rat small intestine. A: At P0 enterocytes on the walls of villi are OTR-immunoreactive. B: If antibodies to OTR are omitted (control), the villus epithelium is not stained. C: Because of the OTR immunoreactivity of enterocytes, villi display OTR immunofluorescence. D–G: At P15 OTR immunoreactivity is modest in crypts and concentrated in enterocytes at crypt-villus junctions. Little or no OTR immunoreactivity remains in enterocytes of the walls of villi. (Compare D with A). E: Higher magnification of a portion of the field shown in D. F, F inset: The cells at the crypt-villus interface display intense OTR immunoreactivity, which is prominent in a band in the apical cytoplasm, just below the microvillus border. G: A high concentration of OTR-immunofluorescent cells is located at the crypt-villus junction (arrows). Crypt epithelial cells also display apical OTR immunoreactivity (arrowhead), although it is much less intense than that of cells at crypt-villus junctions. G inset: The apical OTR-immunoreactive band is well seen in a fluorescent image. Scale bar = 100 µm in A,B,E,G; 400 µm in C,D; 10 µm in F; 10 µm in insets to F,G.

Figure 8

OTR immunoreactivity is concentrated at apical adherens junctions in enterocytes at the crypt-villus junction in rat duodenum. A: Electron micrograph (EM) of a thin section cut tangentially through the apical OTR-immunoreactive band of cells at the crypt-villus junction. OTR immunoreactivity, demonstrated by pre-embedding staining, is visualized by the osmiophilic DAB reaction product. Although OTR immunoreactivity is present in most of the plasma membrane ringing each enterocyte (arrows), it is especially prominent in accumulation in the cytosolic dense material associated with junctions identified by the insertions of thin filaments as adherens junctions (arrowheads). Little or no immunoreactivity is seen in the plasma membrane of microvilli. B: Light micrograph of a semithin section cut tangentially through the apical cytoplasm of OTR-immunoreactive cells at a crypt-villus junction. Punctate OTR immunoreactivity fills the intercellular space, corresponding to the adherens junctions seen in the matching EM section. Scale bar = 500 nm in A; 10 µm in B.

Figure 9

OT-immunoreactive cells are rare but OT-immunoreactive varicosities are widespread throughout the myenteric plexus. Colchicine treated to maximize peptide accumulation in neuronal perikarya. A: OT immunoreactivity is punctate and limited to ganglia. B: Neurons are identified by immunostaining with antibodies to Hu. C: Merged image. Hu-immunoreactive nerve cells are outlined by OT-immunoreactive varicosities in the ganglionic neuropil. D: OT immunoreactivity in a myenteric neuron. E: Deconvoluted projection of a Z stack of images taken at 0.5-µm intervals through a myenteric ganglion in which Hu immunofluorescence is green and OT immunofluorescence is red. OT-immunoreactive varicosities are abundant in the ganglionic neuropil and some varicose neurites can be tracked (arrows). A magenta-green copy of this image is available as Supplementary Figure 6. Scale bar = 25 µm in C (applies to A–C); 30 µm in D,E.