Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Gastroenterology. 2019 May 7;157(2):507–521.e4. doi: 10.1053/j.gastro.2019.04.022

Effects of Serotonin and Slow-release 5-HTP on Gastrointestinal Motility in a Mouse Model of Depression

Narek Israelyan 1, Andrew Del Colle 1,2, Zhishan Li 1,3, Yeji Park 1,2, Albert Xing 1, Jacob PR Jacobsen 4, Ruth Ann Luna 5, Dane D Jensen 6, Moneek Madra 1,2, Virginia Saurman 1, Ray Rahim 1,3, Rocco Latorre 6, Kimberly Law 1, William Carson 4, Nigel W Bunnett 6, Marc G Caron 4, Kara G Margolis 1
PMCID: PMC6650329  NIHMSID: NIHMS1527806  PMID: 31071306

Abstract

Background & Aims:

Mood disorders and constipation are often comorbid yet their shared etiologies have rarely been explored. The neurotransmitter serotonin (5-HT) regulates central nervous system and enteric nervous system (ENS) development and long-term functions, including gastrointestinal (GI) motility and mood. Defects in neuron production of 5-HT might therefore result in brain and intestinal dysfunction. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis. A variant of TPH2 that encodes the R441H substitution (TPH2-R441H) was identified in individuals with severe depression. We studied mice with an analogous mutation (TPH2-R439H), which results in a 60%–80% decrease in levels of 5-HT in central nervous system and behaviors associated with depression in humans. Feeding chow that contains 5-HTP slow release (5-HTP SR) to TPH2-R439H mice restores levels of 5-HT in the central nervous system and reduces depressive-like behaviors.

Methods:

We compared the effects of feeding chow, with or without 5-HTP SR, to mice with the TPH2-R439H mutation and without this mutation (control mice). Myenteric and submucosal plexuses were isolated from all 4 groups of mice and immunocytochemistry was used to quantify total enteric neurons, serotonergic neurons, and 5-HT–dependent subsets of neurons. We performed calcium imaging experiments to evaluate responses of enteric neurons to tryptamineevoked release of endogenous 5-HT. In live mice we measured total GI transit, gastric emptying, small intestinal transit, and propulsive colorectal motility. To measure colonic migrating motor complexes (CMMCs), we isolated colons and constructed spatiotemporal maps along the proximo-distal length to quantify the frequency, velocity, and length of CMMCs. We measured villus height, crypt perimeter, and relative densities of enterochromaffin and enteroendocrine cells in small intestinal tissue.

Results:

Levels of 5-HT were significantly lower in enteric neurons from TPH2-R439H mice than from control mice. TPH2-R439H mice had abnormalities in ENS development and ENS-mediated GI functions, including reduced motility and intestinal epithelial growth. Total GI transit and propulsive colorectal motility were slower in TPH2-R439H mice than controls and CMMCs were slower and less frequent. Villus height and crypt perimeter were significantly decreased in colon tissues from TPH2-R439H mice, compared with controls. Administration of 5-HTP SR to adult TPH2-R439H mice restored 5-HT to enteric neurons and reversed these abnormalities. Adult TPH2-R439H mice given oral 5-HTP SR had normalized numbers of enteric neurons, total GI transit, and colonic motility. Intestinal tissue from these mice had normal measures of CMMCs and enteric epithelial growth

Conclusions:

In studies of TPH2-R439H mice, we found evidence for reduced release of 5-HT from enteric neurons that results in defects in ENS development and GI motility. Our findings indicate that neuron production of 5-HT links constipation with mood dysfunction. Administration of 5-HTP SR to mice restored 5-HT to the ENS and normalized GI motility and growth of the enteric epithelium. 5-HTP SR might be used to treat patients with intestinal dysfunction associated with low levels of 5-HT.

Keywords: gut–brain disease, mood disorders, GI dysfunction, CNS

Graphical Abstract

graphic file with name nihms-1527806-f0001.jpg

Introduction:

Depression and constipation are common, debilitating conditions that affect, respectively, up to 8% and 27% of the national population1, 2. Although these disorders can occur independently, preliminary studies have shown that depression may be comorbid with constipation; The prevalence of major depression in people with chronic constipation has been reported to be as high as 33% 3, 4 On a larger scale, meta-analyses have demonstrated that both anxiety and constipation are comorbid with all forms of IBS, including individuals with the constipation-predominant subtype5-7. Constipation has also been shown to be the leading comorbidity in depressed individuals3.

Diseases that affect the brain, including autism spectrum disorder, Parkinson’s, and Alzheimer’s disease, are often associated with gastrointestinal (GI) disturbances8. Shared pathophysiological mechanisms may explain the frequent co-existence of GI and central nervous system (CNS) pathologies in these conditions. Shared pathophysiological mechanisms should not be surprising given that the CNS and the enteric nervous system (ENS) share many of the same neurotransmitters and mechanisms of neurotransmission9.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter in the CNS and ENS10. Enteric 5-HT participates in paracrine, endocrine, and neurocrine signaling10. Tryptophan hydroxylase 1 and 2 (TPH1; TPH2) are the rate-limiting enzymes involved in 5-HT biosynthesis in the enteric mucosa (enterochromaffin [EC] cells) and ENS, respectively9, 11. It is TPH2-derived, neuronal 5-HT, however, that specifically stimulates ENS neurogenesis in general and especially that of late-developing neuronal subsets12, 13. Neuronal 5-HT also enhances GI motility and intestinal mucosal growth12-14. Notably, TPH2 is also required for CNS-derived 5-HT production15. Because TPH2 is essential for neuronal 5-HT biosynthesis in both the ENS and the CNS, it is likely to be involved in processes that concomitantly regulate CNS and ENS development and function. The involvement of TPH2 in the biosynthesis of 5-HT in both the ENS and CNS is compatible with the idea that a TPH2 deficiency underlies 5-HT-related comorbidities of brain and gut8.

Selective serotonin reuptake-inhibitors (SSRIs) are a first line-treatment for depression. SSRI treatment, however, induces remission in only a third of depressed patients16, 17. The side effects of select chronic SSRI therapies, furthermore, may worsen constipation18. Thus, patients with depression are often faced with limited treatment options and prominent GI dysfunction.

Multiple polymorphisms of TPH2 are found in individuals with depression19-22. One of these, a single nucleotide polymorphism (SNP) in TPH2 that replaces a highly conserved residue (Arg441) with histidine (R441H), was identified in a cohort of patients with SSRI-resistant unipolar depression22. Individuals in this cohort also anecdotally exhibited symptoms consistent with anxiety and a worsened depressive state upon treatment with an SSRI22.

The murine mutation in TPH2 that is analogous to R441H, R439H, was transgenically engineered into a murine model. CNS 5-HT levels were found to be decreased by 60-80% in TPH2-R439H mice and the animals also demonstrated behaviors associated with anxiety and depression. Immobility time in the tail suspension test was greater in TPH2-R439H mice than in WT animals and latency to feed was also decreased in TPH2-R439 mice during novelty-suppressed feeding 23-25. The phenotype of the TPH2-R439H mice thus suggests that the constitutively decreased TPH2 activity in these animals interferes with the 5-HT signaling required for normal CNS function. As in the case of the depressed patients, treatment of the TPH2-R439H mice with the SSRI, fluoxetine, also failed to relieve depression-like behaviors.

Because TPH2 is present in the intestine as well as the brain, it is possible that the TPH2-R439H polymorphism interferes with enteric 5-HT biosynthesis and causes ENS development and function to become abnormal. Rare variants in TPH2 have also been noted in individuals with depression and IBS 26-30. Simultaneous deficiencies in 5-HT in both the ENS and the CNS may thus be a link between constipation and depression.

If reduced neuronal 5-HT production results in functional deficits, a reasonable treatment might be to bypass TPH2 and utilize 5-hydroxytryptophan (5-HTP) to normalize ENS and CNS 5-HT. 5-HT cannot be employed by itself to treat these conditions because of its rapid inactivation and failure to cross the blood-brain and blood-myenteric plexus barriers31. 5-HTP is derived from the TPH2-mediated hydroxylation of tryptophan and is rapidly decarboxylated to form 5-HT. In human studies, acute adjunctive 5-HTP has been reported to enhance the effects of SSRIs on neuroendocrine biomarkers of elevated extracellular 5-HT 32; moreover, in TPH2-R439H animals, administration of 5-HTP alleviated the constitutive CNS 5-HT deficiency33. The rapid absorption and elimination of 5-HTP, however, reduces its ability to maintain the sustained levels of 5-HT necessary for effective treatment of either constipation or depression. Approaches to deliver 5-HTP as slow-release (5-HTP SR) are therefore being pursued to achieve sustained 5-HTP exposure. In contrast to native immediate release 5-HTP, 5-HTP administered in the mouse chow (SR) maintains therapeutically relevant levels of plasma 5-HTP and enhances CNS 5-HT function in the TPH2-R439H mice33. The administration of 5-HTP SR also results in a decreased latency to feed in the novelty-suppressed feeding test in TPH2-R439H mice and, when combined with an SSRI, results in a significantly decreased amount of marble burying, a behavior that is sensitive to serotonergic manipulation (Jacobsen et al, submitted).

We tested the hypotheses that: (1) The TPH2-mediated 5-HT deficit that leads to depressive-like behavior also contributes to abnormalities in: (a) enteric neuronal 5-HT production; (b) ENS development; (c) ENS-mediated GI functions (GI motility and enteric epithelial growth) and (2) that these abnormalities can be treated through mechanism-guided intervention with 5-HTP SR.

Observations demonstrate that enteric neuronal 5-HT is deficient in TPH2-R439H mice and, as a result, enteric neuronal development, GI motility, and enteric mucosal growth are all abnormal in these animals. Strikingly, each of these abnormalities were corrected in adult TPH2-R439H mice with 5-HTP SR. Our observations thus reveal that ENS development and long-term function, in addition to those of the CNS, are exquisitely sensitive to neuronal 5-HT signaling and that these abnormalities, including ENS neurogenesis, can be reversed with a slow-release delivery of 5-HTP during adulthood. 5-HTP SR may thus serve as a novel form of treatment for 5-HT-related disorders that simultaneously affect the brain and the intestine.

Methods:

Animals.

Male and female TPH2-R439H mice (c57BL6/J–129S6/SvEvTac background) were bred at Duke and shipped to CUMC or bred at CUMC34. Experiments were conducted with homozygous WT and TPH2-R439H littermates. 5-HTP IR was administered i.p. in a dose response fashion, from 1-100 mg/kg. 5-HTP SR (≅1 g/kg/day; 6.7mg 5-HTP/gram chow; created by mixing the 5-HTP in finely crushed chow with binder (cellulose) and drying as food pellets) was administered from ≅6-10 weeks of age in the same chow that control mice received without 5-HTP SR, as done previously16.

ARRIVE guidelines and approvals.

All studies were approved by the IACUC at CUMC.

Calcium imaging.

Calcium imaging was studied in myenteric neurons of ileum as described35. LMMP preparations were dissected from distal ileum and mounted in an imaging chamber. Preparations were incubated with Dispase (1 U/ml) and collagenase Type II (150 U/m) in oxygenated DMEM/F-12 media (Gibco) for 15 min, washed with Krebs solution, and loaded with Fluo-4 (4.0 μM) for 45 min. The imaging chamber was mounted on a widefield microscope (Leica) and viewed with a 20x objective (NA). Images were captured with LASX software (frame rate:1 image/5 seconds). Fluo-4 fluorescence was recorded for 30 seconds to obtain a baseline. Tryptamine hydrochloride (1 mM, Sigma-Aldrich) was then superfused into the chamber, response was recorded for 2 min followed by washout with Krebs. After a second 30 second baseline period, tryptamine (1 mM) was again superfused and response recorded for 2 min. Finally, preparations were washed with Krebs, a last 30 second baseline was obtained, and preparations were superfused with KCl (75 mM) as a positive control to ensure that the tissue was still capable of responding to a depolarizing stimulus. The response to KCl was recorded for 2 min. An additional five male WT mice were used to study the effects 5-hydroxy-L-tryptophan (5-HTP) on the recovery of 5-HT contents in the enteric neurons after tryptamine depletion. The method employed for calcium imaging is similar to that described above except that after the initial tryptamine perfusion, 5-HTP (1 μM, Sigma-Aldrich) was superfused into the imaging chamber for 15 min between the tissue responses to the first and second superfusions of tryptamine. Images were analyzed with ImageJ software. Regions of interest (ROIs) were carefully selected and the fluorescent intensities that tryptamine/KCl induced were compared to those of their resting baseline fluorescence using an established formula36. The differences between the fluorescence intensities of different groups were analyzed by unpaired t tests and 1-way ANOVA.

Immunocytochemistry.

Myenteric and submucosal plexuses were examined in whole mounts of laminar preparations of gut wall fixed with 0.1M phosphate-buffered formaldehyde (1.5 h) and washed in phosphate-buffered saline12, 13. Preparations were exposed to primary antibodies used to detect total neurons (human anti ANNA-1; gift from V. Lennon37), dopamine (sheep anti-TH, Sigma) and/or GABA (rabbit anti-GABA, Millipore). Preparations were incubated with primary antibodies for 48-72 hours, visualized with species-specific secondary antibodies (Alexa Fluor™ 350, 488, or 594; 1:200) and mounted13. A computer-controlled motorized stage was used to scan images (20× objective) covering a 10-mm2 area. Collected images were computer-processed (Volocity 6.0, Improvision/Perkin-Elmer) to quantify immunoreactive cells (cells/mm2 ganglionic area). Axonal fluorescence was measured (ImageJ 2.0) to quantify mean pixel intensity of traced neurites, with neurite background serving as a fluorescence baseline.

Total gastrointestinal transit time (TGIT).

Carmine red (300 μl; 6% in 0.5% methylcellulose ; Sigma-Aldrich) was orally gavaged. TGIT was the interval between gavage and carmine red appearance in stool13.

Colonic propulsion.

Mice were anesthetized with isoflurane (Baxter). A fire-polished glass rod placed a 3mm glass bead into the colon 2 cm from the anal verge13. Time required to expel the bead is an estimate of colorectal propulsion.

Gastric emptying and small intestine transit13.

Rhodamine B dextran (100 μl;10 mg/ml in 2% methylcellulose; Invitrogen) was orally gavaged through a 21-gauge feeding needle. Animals were euthanized 15 min after gavage; the stomach, small intestine, cecum, and colon were collected. The small intestine was divided into 10. The colon (used to obtain total recovered rhodamine) was divided in half. Each tissue was placed in 4 ml 0.9% NaCl, homogenized, and centrifuged (2000×g). Fluorescence was measured in 1 ml of supernatant (VersaFluor; Bio-Rad). Gastric emptying and small intestinal transit were calculated by established formulas13.

Colonic migrating motor complexes (CMMC) measured in vitro.

We thank Joel Bornstein for adaptation of this technique38. Each colon was mounted in an organ bath, with luminal and serosal compartments superfused with oxygenated Krebs’ solution, and intraluminal- and back-pressures maintained at +2 cm H2O. Contractile activity was imaged with a Logitech Quickcam pro-camera positioned 7-8 cm above the gut. Preparations were equilibrated and six 15 min videos were captured. Spatiotemporal maps along the proximo-distal length of colon were constructed to quantify the frequency, velocity and length of CMMCs (diameter constrictions that propagated for ≥50% length of the preparation) 13. For studies involving 5-HTP IR, a dose-response curve was conducted whereby colons were exposed to concentrations from 1-10 μM.

Quantitation of transcripts.

Real-time PCR methods to quantify transcripts encoding TPH2 and SERT (Applied Biosystems) have been described13.

Parameters of mucosal maintenance.

Intestine was fixed overnight (described above), paraffin-embedded, sectioned (10 μm), and stained with hematoxylin and eosin. Computer-assisted imaging (Volocity 6.0/ImageJ 2.0) was employed to measure villus height and crypt perimeter14.

Epithelial staining.

EC cells and EE cells were quantified after 5-HT and chromogranin A immunostaining (Immunostar and Abcam, respectively, 1:1000) as above13.

Statistical Analysis.

The student’s unpaired t test and 1-way ANOVA with Bonferroni correction were used to compare single and multiple means, respectively.

Results:

The TPH2-R439H polymorphism results in decreased levels of enteric neuronal 5-HT and impedes ENS development.

TPH2 is required for 5-HT production in enteric neurons12. Immunocytochemistry and calcium (Ca2+) channel imaging were thus used to assess the 5-HT content of the ENS and enteric neurons, respectively, in TPH2-R439H mice and WT littermates. Significantly fewer 5-HT-immunoreactive nerve cell bodies were detected in TPH2-R439H mice than in WT controls (Figure 1, Supp. Table 1). This deficit was manifest both in total numbers of serotonergic neurons and as the proportion of total enteric neurons that were serotonergic (Figures 1A,1B). Because enteric serotonergic neurons are a relatively small proportion of ENS neurons, but project extensively within the bowel, computer-assisted imaging was employed to estimate the proportion of the total area of the myenteric plexus occupied by 5-HT-immunoreactive neurites. The mean fluorescence intensity of serotonergic neurites was significantly less in TPH2-R439H mice than in WT mice (Figure 1C; Supp. Table 1). These observations suggest that the TPH2-R439H polymorphism significantly reduces 5-HT in the ENS, which causes a reduction both in the numbers of neurons and in neurites that can be detected.

Figure 1. Levels of 5-HT are diminished in the enteric nervous system of TPH2-R439H mice.

Figure 1.

Open in a new tab

(n = 3-5/group). (A) Total and serotonergic neurons as a proportion of myenteric area (ileum). (B) Percentage of serotonergic neurons as a proportion of total neurons. (C) Mean axonal fluorescence intensity of neurites (myenteric plexus). (D,G) 5-HT immunoreactivity (green). (E,H) Total neurons (blue). (F,I) Coincident immunoreactivity between 5-HT and total neurons. Student’s unpaired t test used to compare groups. Data represent the mean ± SEM. Scale bar: 100μm. (J) Graphical representation of how enteric serotonergic neurons take up tryptamine, which causes 5-HT release, thus eliciting Ca2+ transients in the ENS. (K,L,M) Ca2+ traces of neurons showing tissue responses of tryptamine 1 (T1) and tryptamine 2 (T2), compared to one another and to KCl, in WT, R439H mice and WT mice exposed to 5-HTP superfusion. Arrows indicate delivery of T1, T2, and KCl, respectively. (N,O,P) Fluorescence intensities in ΔF/F of ROIs following drug delivery, as percent of KCl (positive control). (Q,R,S;ROI) Fluorescent micrographs showing changes of Fluo-4 intensities in ROIs after drug delivery of T1 (Q2,R2,S2), T2 (Q3,R3,S3) and KCl (Q4,R4,S4). R5 represents the heat map calibration bar. Scale bar: 50 μm.

To evaluate the functional efficacy of releasable endogenous 5-HT, we utilized Ca2+ imaging. Tryptamine has been shown to release endogenous 5-HT from enteric serotonergic neurons 39. Enteric serotonergic neurons take up tryptamine, which displaces 5-HT in intracellular synaptic vesicles, causing 5-HT release and thus eliciting Ca2+ transients in the ENS (Figure 1J). This release enables tryptamine to mimic the effects of 5-HT transiently, but the effects of tryptamine are lost because of the depletion of endogenous stores of 5-HT. In the current experiments, Ca2+ transients in follower cells were employed to detect the effects of tryptamine-released 5-HT (Figure 1K-S). Tryptamine-induced Ca2+ transients were quantified in enteric neurons. Analysis of Ca2+ fluorescence in regions of interest (ROIs; Figure 1Q1,1R1,1S1) of image stacks in TPH2-R439H and WT mice revealed that, in all groups, an initial application of tryptamine reproducibly led to a cytoplasmic Ca2+ transient in enteric neurons (Figure 1K-P, 1Q2, 1R2, 1S2; T1). In contrast, no such calcium transient was evoked by a second application of tryptamine (Figure 1K,1L,1N,1O,1Q3,1R3; T2); nevertheless, enteric neurons were still capable of responding because cytoplasmic Ca2+ continued to be increased in response to depolarization with potassium chloride (Figure 1K-S,1Q4,1R4 1S4; KCl). To confirm that tryptamine-induced Ca2+ signaling in enteric neurons is indeed 5-HT-dependent, in a different set of experiments we applied 1 μM 5-HTP after the initial exposure to tryptamine (Fig. 1M,1P,1S). When this was done, a second application of tryptamine evoked a Ca2+ transient equivalent to the first tryptamine response (Fig. 1M (T2),1P,1S3). As previously reported39, therefore, repletion of tryptamine-depleted stores of endogenous 5-HT provides transmitter for subsequent applications of tryptamine to release. These observations verify that tryptamine-induced neuronal firing is a measure of the availability of releasable endogenous stores of 5-HT39. The average amplitudes of the Ca2+ transients evoked by an initial application of tryptamine were also compared in WT and TPH2-R439H mice. The tryptamine responses were significantly greater in WT than in TPH2-R439H animals (1K,1L,1N,1O,1Q1,1R1; Supp. Table 1), suggesting that endogenous stores of releasable 5-HT are significantly lower in enteric neurons of TPH2-R439H mice than in WT animals.

To determine whether the TPH2-R439H polymorphism leads to a decrease in overall enteric neurogenesis, we quantified the numbers of enteric neurons in gut wall whole mounts. Total neurons were significantly less abundant in male and female TPH2-R439H mice than in WT mice in both the myenteric (Figure 2A,2I,2L; Supp. Table 2A) and submucosal plexuses (Figure 2C) of the ileum and the colon (Figures 2E,2G,2O,2R; Supp. Table 2B). The development of enteric dopaminergic (tyrosine hydroxylase [TH]-immunoreactive) and GABAergic (γ-aminobutyric acid [GABA]-immunoreactive) neurons were studied as examples of late-born neurons, the development of which are known to be especially 5-HT-dependent13. GABAergic and dopaminergic neurons were quantified in the myenteric or submucosal plexus, respectively, where most are located. In TPH2-R439H mice, GABAergic neurons were deficient in absolute numbers (Figures 2A,2B,2E) and also as a proportion of total neurons (Figures 2B,2F,2J,2M). TH-expressing neurons were deficient in absolute numbers (Figures 2C,2D,2G,2H). These data suggest that the TPH2-R439H polymorphism leads to both a deficiency of releasable enteric neuronal 5-HT and a non-sex-dependent ENS hypoplasia, which is especially manifest in the late-born subsets of neurons that are dependent on 5-HT for their genesis/survival.

Figure 2. Numbers of total and late-born enteric neurons are decreased in TPH2-R439H mice.

Figure 2.

Open in a new tab

(n = 3-4/group). (A,E) Total and GABAergic neurons as a proportion of area in myenteric plexus of (A) ileum and (E) colon. (B,F) GABAergic neurons as a proportion of total neurons. (C,G) Total and dopaminergic neurons as a proportion of area in submucosal plexus of (C) ileum and (G) colon. (D,H) Dopaminergic neurons as a proportion of total neurons. Myenteric plexus from ileum of WT (I–K) and R439H (L–N) mice. (I,L) Total neurons (green). (J,M) GABAergic neurons (red). (K,N) Coincident immunoreactivity between total (green) and GABAergic (red) neurons. Submucosal plexus from colon of WT (O–Q) and R439H (R–T) mice. (O,R) Total neurons (blue). (P,S) Dopaminergic neurons (green). (Q,T) Coincident immunoreactivity between total (blue) and dopaminergic (green) neurons. Student’s unpaired t test used to compare groups. Data represent the mean ± SEM. Scale bars: 25μm

The TPH2-R439H polymorphism slows GI transit and disrupts the peristaltic reflex.

To determine whether the TPH2-R439H polymorphism affects GI motility, we measured total GI transit time (TGIT), propulsive colorectal motility, gastric emptying, and small intestinal transit in vivo. Colonic migrating motor complexes (CMMCs), which are ENS-dependent40, were investigated in vitro to determine whether the observed changes in motility in TPH2-R439H mice are due to an intrinsic defect of the ENS. In the isolated preparations of colon in which CMMCs are studied, the extrinsic innervation of the gut is severed. TGIT (Figure 3A; Supp. Table 3), colorectal motility (Figure 3B), and small intestinal transit (Figure 3D, measured as the geometric center of dye in the small intestine) were significantly slower in TPH2-R439H mice than in WT littermates. Gastric emptying (Figure 3C) was not affected by genotype.

Figure 3. In vivo and in vitro intestinal motility are slower in TPH2-R439H than WT mice.

Figure 3.

Open in a new tab

(n = 12-14/group; 1-3 trials). (A) Total GI transit. (B) Colonic motility. (C) Gastric emptying. (D) Small intestinal transit. (E–F) Spatiotemporal maps shows CMMCs (white arrows) in isolated colons of WT (E) and TPH2-R439H (F) mice (n = 8-10/group). The ordinate represents time, and the abscissa represents oral-to-anal distance. (G) CMMC frequency and (H) CMMC velocity were measured following construction of spatiotemporal maps. Student’s unpaired t test was used to compare groups. Data represent the mean ± SEM.

Spatiotemporal maps of CMMCs were constructed and analyzed (Figures 3E,3F). CMMC frequency and velocity (Figures 3H,3G) were each significantly lower in TPH2-R439H than in WT mice. Of note, the decrease in CMMC frequency and velocity was apparent in both male and female mice (Supp. Table 3). These observations suggest that the ENS hypoplasia that occurs in TPH2-R439H mice impairs generation and conduction of peristaltic reflexes and thus causes GI transit to slow in a non-sex specific manner.

Immediate-release 5-HTP (5-HTP IR) increases in vivo motility and enhances CMMCs.

To confirm that 5-HTP can normalize motility and CMMCs in TPH2-R439H mice, we administered 5-HTP IR to WT and TPH2-R439H mice. 5-HTP IR has previously been shown to increase intestinal motility in mice41. In WT mice, doses of 1, 3, 10, 30 and 100 mg/kg of 5-HTP IR (i.p.) significantly enhanced both TGIT and colonic motility (Figures 4A,4B; Supp. Table 4A). The effect of 5-HTP IR on CMMC generation (frequency) and propagation (velocity) was also analyzed. The concentration-effect relationship was compared for intralumenal and extralumenal applications of 5-HTP IR in WT and TPH2-R439H mice. Interestingly, exposure of the TPH2-R439H mouse colon to 1μM of extra- or intralumenal 5-HTP IR caused significant increases both in CMMC frequency and velocity without changing either parameter in WT mice (Figures 4C,4D; Supp. Table 4B,4C). These observations suggest that 5-HTP IR can normalize TGIT and colonic motility in TPH2-R439H animals by increasing both the generation and propagation of peristaltic reflexes, therefore acting through mechanisms that are ENS-mediated. At the appropriate concentration (1.0 μM), 5-HTP IR can thus restore the releasable pool of endogenous 5-HT without causing excessive motility.

Figure 4. Immediate-release 5-HTP (5-HTP IR) modulates in vivo motility and in vitro peristaltic contractions.

Figure 4.

Open in a new tab

(n = 6-10/group). (A) Total GI transit and (B) colonic motility after administration of 1, 3, 10, 30 or 100 mg/kg of intraperitoneal 5-HTP. CMMC frequency and velocity before and after receiving a single extraluminal (C,D) or intraluminal (E,F) dose of 1μM 5-HTP (n = 4-6/group). Spatiotemporal maps showing CMMCs (white arrow) in isolated colons in WT and R439H mice before (G,I) and after (H,J) administration of 1μM intraluminal 5-HTP. Student’s unpaired t test and 1-way ANOVA were used to compare groups, respectively, to compare single and multiple means. Data represent the mean ± SEM.

Administration of slow-release 5-HTP (5-HTP SR) during adulthood rescues the ENS of TPH2-R439H mice.

Although 5-HTP IR can elevate 5-HT levels, its’ rapid elimination diminishes its utility as a therapeutic agent because pharmacologically active levels are not maintained33. The incorporation of 5-HTP within the mouse chow (5-HTP SR) significantly enhances the 5-HTP concentrations in plasma and in the CNS (Jacobsen et al, submitted). We therefore determined whether oral 5-HTP SR also rectifies the abnormalities of the ENS of TPH2-R439H mice and thus also their functional deficits. Mice received ~1 g/kg/day of 5-HTP SR (Jacobsen et al, submitted), incorporated into mouse chow, from 6-7 weeks of age, for 4 weeks. Strikingly, oral 5-HTP SR administration increased the number of total, GABA- and TH-expressing neurons in the myenteric and/or submucosal plexuses of adult TPH2-R439H mice to numbers that were equivalent to those of untreated adult WT mice (Figures 5A-F; Supp. Table 5). 5-HTP SR did not affect the numbers of total neurons, nor those of any of the neuronal phenotypes studied, in WT animals (Figures 5B,5C,5E,5F). 5-HTP SR rescued in vivo TGIT (Figure 6A; Supp. Table 6) and colonic motility (Figure 6B) in TPH2-R439H mice, yet did not affect their gastric emptying or small intestinal transit (Figures 6C,6D). 5-HTP SR treatment also normalized TPH2-R439H–associated decreases in CMMC frequency and velocity (Figures 6E-J). 5-HTP SR did not significantly affect any motility parameter in WT mice (Figure 6A-F). The normalization of GI motility induced by 5-HTP SR in the isolated colon (CMMCs) suggests that the phenomenon is a result of the normalization of the intrinsic function of the ENS itself.

Figure 5. Administration of slow-release 5-HTP during adulthood rescues mice from the ENS hypoplasia associated with the TPH2-R439H mutation.

Figure 5.

Open in a new tab

(n = 3-4/group). (A) Total neurons (green) and (B) GABAergic neurons (red) in the myenteric plexus of ileum. (D) Total neurons and (E) dopaminergic neurons in the submucosal plexus of ileum. (C) GABAergic and (F) Dopaminergic neurons as a proportion of total neurons. Myenteric plexus from ileum of (G–I) WT, (J-L) WT+5-HTP SR, (M-O) TPH2-R439H, and (P–R) TPH2-R439H+5-HTP SR. (I, L, O, R) Coincident immunoreactivity between total and GABAergic neurons. 1-way ANOVA and Fisher’s LSD test were used to compare groups. Data represent the mean ± SEM. Scale bars: 25μm.

Figure 6. Administration of slow-release 5-HTP during adulthood reverses motility abnormalities associated with the TPH2-R439H mutation.

Figure 6.

Open in a new tab

(n = 10-14/group, 1-3 trials). (A) Total GI transit. (B) Colonic motility. (C) Gastric emptying. (D) Small intestinal transit. (E, F) CMMC frequency and velocity. (n = 5-9/group). (G–J) Spatiotemporal maps showing CMMCs (white arrow) in isolated preparations of colon of (G) control WT and (H) control TPH2-R439H mice and (H) WT and (J) TPH2-R439H mice receiving 5-HTP SR. The ordinate represents time. The abscissa represents oral-to-anal distance. 1-way ANOVA and Fisher’s LSD test were used to compare groups. Data represent the mean ± SEM.

Intestinal epithelial growth is decreased in TPH2-R439H mice and normalized after 5-HTP SR administration.

Enteric neuronal 5-HT is necessary for normal epithelial growth; myenteric serotonergic neurons innervate submucosal cholinergic neurons that stimulate proliferation of transit-amplifying cells14. Villus height and crypt depth are thus significantly decreased in TPH2KO mice14. We thus compared villus height and crypt depth (measured as crypt perimeter to enhance accuracy) in TPH2-R439H mice ± 5-HTP SR administration (Figure 7; Supp. Table 7). Both parameters were significantly lower in TPH2-R439H mice than in WT mice and increased in TPH2-R439H mice after treatment with 5-HTP SR (Figure 7A-F). The effects of TPH2-R439H extended to cell lineage. The relative densities of enterochromaffin (EC; Figures 7I-L) and enteroendocrine (EE) cells were both significantly lower in TPH2-R439H than in WT mice (Figures 7G,7H). After 5-HTP SR administration, the relative density of EC cells, but not EE cells, significantly increased in TPH2-R439H mice yet was unaltered in WT mice (Figures 7G,7H). Because no change in either EC and EE cell number was induced in WT mice after 5-HTP SR administration, these findings are consistent with the idea that there may be a maximal level above which the development of enteroendocrine lineages cease responding to increases in the neuronal drive.

Figure 7. The TPH2-R439H mutation leads to abnormalities in intestinal epithelial homeostasis that are ameliorated by 5-HTP SR administration.

Figure 7.

Open in a new tab

(n = 4-6/group; 30 sections/mouse) (A) Villus height. (B) Crypt perimeter. (C–F) hematoxylin and eosin-stained ileal sections showing an individual villus and neighboring crypts in (C) WT, (D) WT+5-HTP, (E) TPH2-R439H and (F) TPH2-R439H+5-HTP. (G,H) Numbers of EC (G) and EE (H) cells/villus area. (I–L) Ileal sections stained with bisbenzimide (DNA; blue) and 5-HT (EC cell; red) in (I) WT, (J) WT+5-HTP, (K) TPH2-R439H and (L) TPH2-R439H+5-HTP. (M,N) Transcripts of ileal TPH2 (M) and SERT (N) (n = 14-21/group). 1-way ANOVA and Fisher’s LSD test were used to compare groups. Data represents the mean ± SEM. Scale bars: 25μm.

Modulators of 5-HT homeostasis are altered in TPH2-R439H mice ± 5-HTP SR administration.

Experiments were carried out to determine whether the TPH2-R439H polymorphism altered transcription of enteric TPH2 or SERT (Slc6a4). Transcripts encoding TPH2 were not constitutively different in WT and TPH2-R439H mice. After 5-HTP SR treatment, however, transcription of TPH2 was significantly reduced in WT mice (Figure 7M; Supp. Table 7); consequently, the abundance of TPH2 transcripts was higher in TPH2-R439H mice treated with 5-HTP SR than in either WT animals or WT mice given 5-HTP SR (Figure 7M). Enteric SERT transcripts were constitutively significantly less abundant in TPH2-R439H than in WT mice (Figure 7N). Although the abundance of SERT transcripts increased significantly in WT animals after treatment with 5-HTP SR, this treatment did not alter SERT transcription in TPH2-R439H mice (Figure 7N). These observations suggest that SERT transcription is regulated in response to the availability of 5-HT.

Discussion:

GI problems and mood disorders are often comorbid. Few underlying links, however, are known. Mice with a polymorphism that alters neuronal 5-HT production, TPH2-R439H, were previously found to manifest depression- and anxiety-like behaviors similar to individuals with an analogous human SNP, TPH2-R441H22. CNS 5-HT production is known to be deficient in TPH2-R439H mice and TPH2 is responsible for 5-HT biosynthesis in both the ENS and the CNS. We therefore tested the hypothesis that TPH2 hypofunction in TPH2-R439H mice would cause 5-HT-dependent abnormalities of ENS structure and function.

As in the CNS, neuronal 5-HT was deficient in the ENS of TPH2-R439H animals. The proportion of 5-HT- immunoreactive neurons and myenteric area occupied by serotonergic neurites were both significantly less in TPH2-R439H than in WT littermates. The amplitude of Ca2+ transients evoked in response to tryptamine-released endogenous 5-HT was also significantly smaller in TPH2-R439H than in WT mice.

The TPH2-R439H ENS was hypoplastic; numbers of neurons were lower than WT in both plexuses of the small and large intestines. Because endogenous neuronal 5-HT is needed to drive enteric neurogenesis, these observations suggest that defective 5-HT signaling due to the decreased production of neuronal 5-HT in TPH2-R439H mice interferes with enteric neurogenesis12, 13. The sensitivity of late-born neurons, particularly GABA, to TPH2 activity is consistent with the idea that serotonergic neurons, which are early-born, regulate enteric neurogenesis and thus help sculpt the ENS. These observations confirm that 5-HT is an ENS growth factor and that serotonergic signaling is essential for normal neurogenesis12, 13. The data are also consistent with the hypothesis that a defect common to the ENS and CNS could be responsible for comorbid GI disturbances in depression.

GI motility in the TPH2-R439H mice was impaired both in vivo and in vitro (in isolated preparations of colon). Because CMMCs, which are ENS-dependent40, were defective in TPH2-R439H bowel, the motor abnormality is thus an intrinsic property of the ENS. This finding is important because this defect in TPH2 affects the CNS as well as the ENS. The ENS hypoplasia of TPH2-R439H mice thus has direct, functional consequences and could thus be a target for therapeutic intervention.

TPH2-derived 5-HT has previously been shown to play a critical role in regulating the proliferation of crypt epithelial cells and enteric mucosal maintenance14. Myenteric serotonergic neurons innervate submucosal cholinergic neurons that provide a muscarinic input to the mucosa, which in turn stimulates epithelial proliferation and the growth of villi and crypts14. It is likely that TPH2 hypofunction in TPH2-R439H mice decreases serotonergic transmission at submucosal synapses, which indirectly decreases the size of villi and crypts. These data suggest that ENS hypoplasia due to decreased TPH2 activity during development impairs mucosal maintenance throughout life.

Since the TPH2-R439H polymorphism diminishes 5-HT production, it follows that circumvention of the enzymatic deficiency in these animals would normalize the availability of 5-HT for neurotransmission and ameliorate their defects in ENS development and function. 5-HTP SR, unlike its immediate-release predecessor, has been found to maintain therapeutically relevant levels of CNS 5-HT in the TPH2-R439H mice16, 33. We thus tested the hypothesis that 5-HTP SR normalizes the ENS abnormalities of TPH2-R439H animals . We administered a previously validated 5-HTP SR formulation in mouse chow (Jacobsen et al, submitted). Oral 5-HTP SR was found to overcome all of the abnormalities associated with the TPH2-R439H polymorphism, including reversal of the ENS hypoplasia, deficiencies of late-developing neurons, slowing of in vivo GI transit, generation and propagation of CMMCs, as well as abnormal enteric epithelial growth in adult mice. Although 5-HT-containing axons innervate nestin-expressing cells and nestin-expressing cells give rise to adult myenteric neurons in healthy gut, 5-HTP SR did not alter enteric neuron numbers in WT mice42. This finding is not unexpected. Although 5-HTP SR administration resulted in increased neuronal 5-HT levels, the excess 5-HT is likely taken up in vesicles and/or broken down by monoamine oxidase in order to maintain homeostasis.

Depression and constipation (e.g., functional constipation and IBS-C) are prevalent, high-cost, high morbidity-causing medical conditions that co-occur1, 2,3,4 Pharmacological treatment of depression can also cause worsening of GI dysfunction18. Despite these issues, relatively little is understood about the factors linking the two conditions or how to treat them simultaneously and effectively. The current model is unique in its ability to mimic simultaneously some of the behavioral and GI phenotypes present in humans with constipation and anxiety/depression. This model therefore allowed us to evaluate the contributions of neuronal 5-HT signaling to each of these anomalies and, further, to test a novel formulation of 5-HTP (SR) for the treatment of constipation in a model previously shown to express anxiety and depressive phenotypes 16, 25.

These data add important insight to a body of literature that supports the idea that a defect in 5-HT–sensitive neurogenic pathways underlies behavioral and enteric abnormalities in disorders that affect both the brain and the intestine13. Although rare, genetic TPH2 polymorphisms have been noted in humans with depression and IBS, making the possibility of a direct association more likely26-30. Whether the effects of depression directly exert influence on ENS plasticity and/or function remain to be determined.

The striking ability of 5-HTP SR to correct TPH2-R439H-associated ENS neuronal hypoplasia and its brain and behavioral consequences in adult mice have potentially profound clinical implications for medical conditions that simultaneously affect the brain and the intestine. The data show for the first time that neurogenesis in adult intestine can be stimulated to correct abnormalities, such as those of TPH2-R439H mice, sufficiently to rescue deficiencies of enteric neuronal numbers and function that have been present since birth. The changes in neuron numbers that were noted with 5-HTP SR thus indicate that considerable plasticity is available in the ENS, which is encouraging for attempts to modulate it for therapeutic benefit. Whether 5-HTP SR might also be a helpful therapeutic strategy for the concomitant treatment of depression and constipation as well as other medical conditions associated with low neuronal 5-HT in humans requires further study.

Supplementary Material

1

Acknowledgments

Grant support: This work was supported by NIH grants DK093786 (KGM), NS15547 (KGM), MH79201 (MGC), T35AG044303 (NI), NS102722 (NWB), DE026806 (NWB), DK118971 (NWB), DoD grants PR160365 (KGM), PR170507 (NWB), AGA Student Research Fellowship Award (NI), Lundbeck Foundation (JJ) and gifts from the Phyllis and Ivan Seidenberg Family Fund for Children’s Digestive Health (KGM) and The Lennon Family (MGC).

Abbreviations:

ENS

enteric nervous system

CNS

central nervous system

5-HT

serotonin

TPH1

tryptophan hydroxylase 1

TPH2

tryptophan hydroxylase 2

5-HTP

5-hydroxytryptophan

SR

slow-release

IR

immediate-release

EC

enterochromaffin

EE

enteroendocrine

SERT

serotonin reuptake transporter

SSRI

selective serotonin reuptake inhibitor

ROI

region of interest

TGIT

total GI transit time

CMMC

colonic migrating motor complex

GABA

gamma-aminobutyric acid

TH

tyrosine hydroxylase

IBS-C

constipation-predominant irritable bowel syndrome

SNP

single nucleotide polymorphism

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of Interest Statement: JPRJ and MGC are inventors on US patents pertaining to the adjunct 5-HTP SR method-of-treatment, and hold equity in Evecxia Inc., a company founded to develop a 5-HTP SR drug for the management of serotonin related disorders. The remaining authors do not have any conflicts of interest (financial, professional or personal).

References:

  • 1.Brody DJ, Pratt LA, Hughes JP. Prevalence of Depression Among Adults Aged 20 and Over: United States, 2013-2016. NCHS Data Brief 2018:1–8. [PubMed] [Google Scholar]
  • 2.Sanchez MI, Bercik P. Epidemiology and burden of chronic constipation. Can J Gastroenterol 2011;25 Suppl B:11B–15B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hosseinzadeh ST, Poorsaadati S, Radkani B, et al. Psychological disorders in patients with chronic constipation. Gastroenterol Hepatol Bed Bench 2011;4:159–63. [PMC free article] [PubMed] [Google Scholar]
  • 4.Dipnall JF, Pasco JA, Berk M, et al. Into the Bowels of Depression: Unravelling Medical Symptoms Associated with Depression by Applying Machine-Learning Techniques to a Community Based Population Sample. PLoS One 2016;11:e0167055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Fond G, Loundou A, Hamdani N, et al. Anxiety and depression comorbidities in irritable bowel syndrome (IBS): a systematic review and meta-analysis. Eur Arch Psychiatry Clin Neurosci 2014;264:651–60. [DOI] [PubMed] [Google Scholar]
  • 6.Geng Q, Zhang QE, Wang F, et al. Comparison of comorbid depression between irritable bowel syndrome and inflammatory bowel disease: A meta-analysis of comparative studies. J Affect Disord 2018;237:37–46. [DOI] [PubMed] [Google Scholar]
  • 7.Lee C, Doo E, Choi JM, et al. The Increased Level of Depression and Anxiety in Irritable Bowel Syndrome Patients Compared with Healthy Controls: Systematic Review and Meta-analysis. J Neurogastroenterol Motil 2017;23:349–362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Rao M, Gershon MD. The bowel and beyond: the enteric nervous system in neurological disorders. Nat Rev Gastroenterol Hepatol 2016;13:517–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Gershon MD. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes 2013;20:14–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gershon MD. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Current opinion in endocrinology, diabetes, and obesity 2013;20:14–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007;132:397–414. [DOI] [PubMed] [Google Scholar]
  • 12.Li Z, Chalazonitis A, Huang YY, et al. Essential roles of enteric neuronal serotonin in gastrointestinal motility and the development/survival of enteric dopaminergic neurons. J Neurosci 2011;31:8998–9009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Margolis KG, Li Z, Stevanovic K, et al. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function. J Clin Invest 2016;126:2221–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gross ER, Gershon MD, Margolis KG, et al. Neuronal serotonin regulates growth of the intestinal mucosa in mice. Gastroenterology 2012;143:408–17 e2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Walther DJ, Peter JU, Bashammakh S, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 2003;299:76. [DOI] [PubMed] [Google Scholar]
  • 16.Jacobsen JPR, Krystal AD, Krishnan KRR, et al. Adjunctive 5-Hydroxytryptophan Slow- Release for Treatment-Resistant Depression: Clinical and Preclinical Rationale. Trends Pharmacol Sci 2016;37:933–944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Trivedi MH, Rush AJ, Wisniewski SR, et al. Evaluation of outcomes with citalopram for depression using measurement-based care in STAR*D: implications for clinical practice. Am J Psychiatry 2006;163:28–40. [DOI] [PubMed] [Google Scholar]
  • 18.Marken PA, Munro JS. Selecting a Selective Serotonin Reuptake Inhibitor: Clinically Important Distinguishing Features. Prim Care Companion J Clin Psychiatry 2000;2:205–210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wigner P, Czarny P, Synowiec E, et al. Association between single nucleotide polymorphisms of TPH1 and TPH2 genes, and depressive disorders. J Cell Mol Med 2018;22:1778–1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Tsai SJ, Hong CJ, Liou YJ, et al. Tryptophan hydroxylase 2 gene is associated with major depression and antidepressant treatment response. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:637–41. [DOI] [PubMed] [Google Scholar]
  • 21.Zill P, Baghai TC, Zwanzger P, et al. SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression. Mol Psychiatry 2004;9:1030–6. [DOI] [PubMed] [Google Scholar]
  • 22.Zhang X, Gainetdinov RR, Beaulieu JM, et al. Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 2005;45:11–6. [DOI] [PubMed] [Google Scholar]
  • 23.Sachs BD, Jacobsen JP, Thomas TL, et al. The effects of congenital brain serotonin deficiency on responses to chronic fluoxetine. Transl Psychiatry 2013;3:e291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Jacobsen JP, Medvedev IO, Caron MG. The 5-HT deficiency theory of depression: perspectives from a naturalistic 5-HT deficiency model, the tryptophan hydroxylase 2Arg439His knockin mouse. Philos Trans R Soc Lond B Biol Sci 2012;367:2444–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jacobsen JP, Siesser WB, Sachs BD, et al. Deficient serotonin neurotransmission and depression-like serotonin biomarker alterations in tryptophan hydroxylase 2 (Tph2) loss-of-function mice. Mol Psychiatry 2012;17:694–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Jun S, Kohen R, Cain KC, et al. Associations of tryptophan hydroxylase gene polymorphisms with irritable bowel syndrome. Neurogastroenterol Motil 2011;23:233–9, e116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yarandi SS, Peterson DA, Treisman GJ, et al. Modulatory Effects of Gut Microbiota on the Central Nervous System: How Gut Could Play a Role in Neuropsychiatric Health and Diseases. J Neurogastroenterol Motil 2016;22:201–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Jiang H, Ling Z, Zhang Y, et al. Altered fecal microbiota composition in patients with major depressive disorder. Brain Behav Immun 2015;48:186–94. [DOI] [PubMed] [Google Scholar]
  • 29.Lin P, Ding B, Feng C, et al. Prevotella and Klebsiella proportions in fecal microbial communities are potential characteristic parameters for patients with major depressive disorder. J Affect Disord 2017;207:300–304. [DOI] [PubMed] [Google Scholar]
  • 30.Sampson TR, Mazmanian SK. Control of brain development, function, and behavior by the microbiome. Cell Host Microbe 2015;17:565–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Margolis KG, Stevanovic K, Li Z, et al. Pharmacological reduction of mucosal but not neuronal serotonin opposes inflammation in mouse intestine. Gut 2014;63:928–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lowe SL, Yeo KP, Teng L, et al. L-5-Hydroxytryptophan augments the neuroendocrine response to a SSRI. Psychoneuroendocrinology 2006;31:473–84. [DOI] [PubMed] [Google Scholar]
  • 33.Jacobsen JP, Rudder ML, Roberts W, et al. SSRI Augmentation by 5-Hydroxytryptophan Slow Release: Mouse Pharmacodynamic Proof of Concept. Neuropsychopharmacology 2016;41:2324–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Beaulieu JM, Zhang X, Rodriguiz RM, et al. Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. Proc Natl Acad Sci U S A 2008;105:1333–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Fried DE, Gulbransen BD. In situ Ca2+ imaging of the enteric nervous system. J Vis Exp 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Takahashi A, Camacho P, Lechleiter JD, et al. Measurement of intracellular calcium. Physiol Rev 1999;79:1089–125. [DOI] [PubMed] [Google Scholar]
  • 37.King PH, Redden D, Palmgren JS, et al. Hu antigen specificities of ANNA-I autoantibodies in paraneoplastic neurological disease. J Autoimmun 1999;13:435–43. [DOI] [PubMed] [Google Scholar]
  • 38.Swaminathan M, Hill-Yardin E, Ellis M, et al. Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice. J Vis Exp 2016:53828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Takaki M, Mawe GM, Barasch JM, et al. Physiological responses of guinea-pig myenteric neurons secondary to the release of endogenous serotonin by tryptamine. Neuroscience 1985;16:223–40. [DOI] [PubMed] [Google Scholar]
  • 40.Spencer NJ, Bywater RA, Taylor GS. Evidence that myoelectric complexes in the isolated mouse colon may not be of myogenic origin. Neurosci Lett 1998;250:153–6. [DOI] [PubMed] [Google Scholar]
  • 41.Wang L, Martinez V, Kimura H, et al. 5-Hydroxytryptophan activates colonic myenteric neurons and propulsive motor function through 5-HT4 receptors in conscious mice. Am J Physiol Gastrointest Liver Physiol 2007;292:G419–28. [DOI] [PubMed] [Google Scholar]
  • 42.Kulkarni S, Micci MA, Leser J, et al. Adult enteric nervous system in health is maintained by a dynamic balance between neuronal apoptosis and neurogenesis. Proc Natl Acad Sci U S A 2017;114:E3709–E3718. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1527806-supplement-1.pdf (1MB, pdf)

RESOURCES