Strain-specific genetics, anatomy and function of enteric neural serotonergic pathways in inbred mice

Abstract

Serotonin (5-HT) powerfully affects small intestinal motility and 5-HT-immunoreactive (IR) neurones are highly conserved between species. 5-HT synthesis in central neurones and gastrointestinal mucosa depends on tissue-specific isoforms of the enzyme tryptophan hydroxylase (TPH). RT-PCR identified strain-specific expression of a polymorphism (1473C/G) of the tph2 gene in longitudinal muscle-myenteric plexus preparations of C57Bl/6 and Balb/c mice. The former expressed the high-activity C allele, the latter the low-activity G allele. Confocal microscopy was used to examine close contacts between 5-HT-IR varicosities and myenteric neurones immunoreactive for neuronal nitric oxide synthase (NOS) or calretinin in these two strains. Significantly more close contacts were identified to NOS- (P < 0.05) and calretinin-IR (P < 0.01) neurones in C57Bl/6 jejunum (NOS 1.6 +/- 0.3, n = 52; calretinin 5.2 +/- 0.4, n = 54), than Balb/c jejunum (NOS 0.9 +/- 0.2, n = 78; calretinin 3.5 +/- 0.3, n = 98). Propagating contractile complexes (PCCs) were identified in the isolated jejunum by constructing spatiotemporal maps from video recordings of cannulated segments in vitro. These clusters of contractions usually arose towards the anal end and propagated orally. Regular PCCs were initiated at intraluminal pressures of 6 cmH(2)O, and abolished by tetrodotoxin (1 microm). Jejunal PCCs from C57Bl/6 mice were suppressed by a combination of granisetron (1 microm, 5-HT(3) antagonist) and SB207266 (10 nm, 5-HT(4) antagonist), but PCCs from Balb/c mice were unaffected. There were, however, no strain-specific differences in sensitivity of longitudinal muscle contractions to exogenous 5-HT or blockade of 5-HT(3) and 5-HT(4) receptors. These data associate a genetic difference with significant structural and functional consequences for enteric neural serotonergic pathways in the jejunum.

Figure 1. Endogenous 5-HT labelling is specific

Corresponding images of 5-HT- and TH-IR from the same field of view show no overlap between labels. Open arrows indicate example 5-HT-IR varicosities which are not visible in the corresponding TH image. These images were constructed by merging 2 consecutive layers from a z-series. Scale bars, 20 μm.

Figure 2. Frequency of PCCs during time controls

Balb/c (○, n= 8) and C57Bl/6 (▪, n= 6) in jejunum. Frequency of PCCs tended to be more stable in C57Bl/6 mice over time than in Balb/c mice. Frequency in jejunum from Balb/c mice tended to decrease over the period of recording, but this did not reach statistical significance (P > 0.05, ANOVA, Tukey's post hoc test).

Figure 3. Two types of contraction are revealed by spatio-temporal maps of adult mouse intestine in vitro

Aa–Da, spatio-temporal maps showing contractile activity of murine jejunum in control (A–B) and TTX (C–D). In control maps, low frequency propagating contractions (PCCs) can clearly be seen (Aa, arrowheads), but fast Fourier transform also reveals a high frequency peak at 0.70–0.75 Hz (Ab, dotted area). Low-pass filtering removes this high frequency element (Bb, dotted area), without affecting the appearance of PCCs (Ba, arrowheads). Low, but not high frequency activity is abolished by TTX (Ca and b). Low-pass filtering removes the high frequency activity from TTX maps (Da and b).

Figure 4. Measurement of PCC parameters

A, frequency (min⁻¹) and velocity (gradient, mm s⁻¹) of PCCs (arrowheads) were measured from spatio-temporal maps directly. Speed was the absolute value of the velocity. Other parameters were calculated from 2-dimensional plots of time and gut width extracted from spatio-temporal maps at a consistent point along the x-axis. The plot in B derives from the dashed line in A; the alphabetically labelled points were used to calculate additional parameters as follows: Amplitude = 100((w – x)/w); Duration = y – w; Period = z – w; Quiescence = z – y; Inhibition ratio = (z – y)/(z – w). Contractions/PCC was the number of peaks between points w and y. These points were defined as follows: x is the point of maximum contraction in a complex; w and z are the points of inflection from baseline on rising phase of consecutive contractions; y is the point of inflection to baseline on the falling phase of a contraction. Parameters were calculated from all PCCs in each spatio-temporal map, and averaged between animals.

Figure 5. Expression of tph2 C1473G polymorphism is strain specific

Standard PCR revealed allele-specific PCR products of approximately 300 bp for the C allele only in C57Bl/6 mice (A), while products for the G allele were only observed in Balb/c mice (C). The faint 523 bp control product can be seen faintly in each lane of A and C, indicating that the reactions were successful. mRNA encoding the high-activity enzyme TPH2 (C/C) was detected as a 180 bp RT-PCR product only in tissues from C57Bl/6 mice (B). mRNA encoding the low-activity enzyme TPH2 (G/G) was detected by RT-PCR in similar samples only in Balb/c mice (D). RNA degradation resulted in a lack of TPH2 mRNA in most duodenum samples. However, these samples expressed allele-specific 300 bp PCR products in the genomic DNA samples (arrowheads). Sequence analysis on PCR products from C57Bl/6 mice confirmed that the 180 bp PCR product consists of 101 nucleotides in exon X and 73 in XI of the TPH2 gene, whereas the larger 300 bp PCR product also includes 130 nucleotide sequence of intron X–XI (E). Sequencing could not confirm the allele at nucleotide position 1473 (asterisk and box) because of the primer binding at this location. Key: c, lanes showing PCR products from the c-allele-specific reactions; g, lanes showing PCR products from the g-allele-specific reactions; M, reference DNA ladder.

Figure 6. 5-HT and TH connections to jejunal neurones

A–D. 5-HT-IR (green) varicosities apposed to NOS-IR (red) cell bodies (open arrowheads) were extremely rare in both Balb/c (A and B) and C57Bl/6 (C and D) mice. Occasionally rings of 5-HT-IR varicosities were observed surrounding NOS^−ve cell bodies (A, †), but never surrounding NOS-IR cell bodies. Many NOS-IR neurones were not apposed by any 5-HT-IR varicosities (B–D). E and F, 5-HT-IR varicosites in apposition to calretinin-IR (red) neurones were relatively abundant in jejunum samples from both strains. Calices of varicosities arising from passing 5-HT-IR axons (E) were often seen in apposition to calretinin-IR cell bodies. Rings (F) of 5-HT-IR varicosities were also frequently observed surrounding calretinin-IR cell bodies. G and H, 5-HT-IR cell bodies were apposed by numerous 5-HT-IR and TH-IR (red, filled arrowheads) varicosities in Balb/c (G) and C57Bl/6 (H) jejunum. I and J, TH-IR varicosities (green) were observed in apposition to both calretinin-IR (red, I) and NOS-IR (red, J) cell bodies (filled arrowheads) in jejunum. These images were constructed by merging 1–3 planes from z-series. Scale bars, 20 μm.

Figure 7. Quantified 5-HT and TH connections to jejunal neurones

A, mean numbers of 5-HT-IR varicosities apposed to NOS-IR cell bodies were significantly larger in C57Bl/6 mice. B, this difference is reflected in the frequency distribution of appositions per NOS-IR neurone. C, calretinin-IR cell bodies in C57Bl/6 mice were apposed by significantly greater numbers of 5-HT-IR varicosities than Balb/c. D, frequency distribution of appositions per neurone revealed a trend for more calretinin-IR cell bodies in Balb/c than C57Bl/6 mice not to be apposed by 5-HT-IR varicosities, while larger numbers of varicosities tended to be observed apposing calretinin-IR neurones from C57Bl/6 mice. E, Dogiel type II (DII) and non-Dogiel type II (other) calretinin-IR neurones were apposed by 5-HT-IR varicosities in both strains. Calretinin-IR neurones of both morphologies in C57Bl/6 mice tended to be apposed by larger numbers of 5-HT varicosities than neurones in Balb/c mice, but this only reached significance in non-Dogiel type II neurones. F, the proportion of Dogiel type II neurones that were apposed by no 5-HT varicosities was higher in Balb/c than C57Bl/6 mice, and the maximum number of varicosities apposed to a Dogiel type II neurone from Balb/c mice (8) was lower than the maximum in C57Bl/6 mice (18). G, the proportion of non-Dogiel type II neurones that were apposed by no. 5-HT varicosities was also larger in Balb/c than C57Bl/6 mice. H, mean numbers of 5-HT- and TH-IR varicosities apposed to each cell body were similar between and within strains. *P < 0.05; †P < 0.01 between strains.

Figure 8. Effects of granisetron

Control PCCs (arrowheads) were observed in both C57Bl/6 (Aa) and Balb/c (Ba) mice. In granisetron, PCCs were significantly reduced in C57Bl/6 mice (Ab) but not in Balb/c mice (Bb). Granisetron-evoked reductions in frequency (C) and amplitude (D) tended to be observed in both strains when data were normalized to control. However, these reductions were only significant in C57Bl/6 mice (*P < 0.05).

Figure 9. Effects of combined 5-HT antagonists

PCCs (arrowheads) were clearly observed in control conditions in C57Bl/6 (Aa) and Balb/c (Ba) mice. Application of combined antagonists frequently converted PCCs to irregular contractile activity (Ab) or abolished contractions completely in C57Bl/6 mice. In the majority of preparations in Balb/c mice, PCCs were not significantly affected by combined antagonists (Bb). Mean frequency (C; †P < 0.01) and amplitude (D; *P < 0.05) decreased significantly in C57Bl/6 mice only.

Figure 10. Intrinsic sensitivity of murine jejunum to 5-HT

Contractions were measured in longitundinally arranged segments of murine jejunum. A, example traces from a single segment in response to carbachol (maximal contraction), and to 5-HT (100 μm) alone and following incubation with TTX. All data were normalized to carbachol controls. B, 10 and 100 μm 5-HT evoked equally strong responses in both Balb/c and C57Bl/6 mice. TTX significantly reduced the 5-HT-evoked responses in both strains, but no strain differences in the residual responses were detected. In the presence of methiothepin (100 nm) and ketanserin (30 nm), granisetron alone (1 μm; C), and in combination with SB207266 (10 nm; D) significantly reduced responses to 10 μm 5-HT in both strains. No strain differences were detected in these responses. (*P < 0.05; †P < 0.01; * and † indicate comparisons within strains).