depression. CONCLUSIONS: Our results show that depression reactivates inflammation inmice that have quiescent colitis and this vulnerability is reversed by a tricyclic antidepressant.These findings suggest that an episode of depression could influence the relapse IBD patientand that anti-depressants may play a role in attenuating both the depression and the severityof inflammation.

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Tryptophan Catabolism in Irritable Bowel Syndrome: Relationship toCytokines, Severity and Psychiatric Co-MorbidityPeter Fitzgerald, Gerard Clarke, Paul Scully, John F. Cryan, Fergus Shanahan, Eamonn M.Quigley, Eugene Cassidy, Timothy G. Dinan

Introduction: Irritable bowel syndrome (IBS) is a functional disorder which has been linkedwith abnormal serotonin functioning. It has a high psychiatric co-morbidity with depressiveand anxiety disorders, conditions which result from a central serotonergic deficit. Tryptophan(TRP) forms the substrate for serotonin biosynthesis, but alternatively TRP can also becatabolised to kynurenine (KYN). Tryptophan catabolism along the kynurenine pathway isregulated by the enzyme indoleamine 2,3-dioxygenase (IDO), and therefore the ratio ofkynurenine to tryptophan provides a measurement of IDO activity. The main inducer ofIDO is the pro-inflammatory cytokine, interferon-gamma (IFN-γ). Aims: The primary aimof this study was to investigate tryptophan catabolism in IBS, and to relate such catabolismto immune activation and psychiatric co-morbidity. We hypothesised that there would beincreased IFN-γ levels in IBS, and consequently, increased shunting of tryptophan alongthe kynurenine pathway at the expense of serotonin biosynthesis. Method: 42 female IBSsubjects and 22 female healthy controls had plasma kynurenine and tryptophan concentra-tions measured using an HPLC method. Interferon-γ was assayed using an electro-chemilu-minescence multiplex system imager. Co-morbid depressive and anxiety disorders wereidentified using a valid and reliable self-report tool, the Patient Health Questionnaire (PHQ),and the severity of IBS was assessed using a summary score of 4-point ordinal scales inaccordance with a previously published method. Results: Of the 42 IBS subjects, 17 hadsevere IBS, 16 reported moderate symptoms and 9 reported mild IBS symptomatology.There was a significant positive correlation between IBS severity and Kyn/Trp ratio(r = 0.47,p=0.002). Those with severe IBS symptoms demonstrated increased tryptophan catabolismalong the kynurenine pathway (i.e. increased Kyn/Trp ratio)compared to those with mild-to-moderate IBS and healthy controls(p = 0.004); and those with severe IBS were over twiceas likely to have depression or anxiety compared to those with less severe symptoms (RR =2.2;95% C.I 1.2-3.9). No difference in IFN-γ levels was observed between the IBS patientsand controls; however IFN-γ was positively correlated with Kyn:Trp ratio in the IBS group(r = 0.58, p = 0.005) and this was not seen in the controls (r = - 0.05, p = 0.8). Conclusion: Ourresults indicate an increased sensitivity to IFN-γ in the regulation of tryptophan catabolism inIBS. This may predispose to the increased rate of Kyn production observed in severe IBScases and to the high co-morbidity with depressive and anxiety disorders in this group.

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Differences in Localization of TRPV1 Channels and Contractile Effect ofCapsaicin in Mouse Isolated Lower Gastrointestinal Tract: Higher Abundanceand Sensitivity of TRPV1 Channels in Rectum and Distal Colon Than inTransverse and Proximal ColonKenjiro Matsumoto, Kimihito Tashima, Syunji Horie

BACKGROUND & AIMS: Transient receptor potential vanilloid subtype 1 (TRPV1) is anonselective cation channels expressed in primary sensory neurons, and is activated by lowpH, heat and capsaicin, a constituent of chili peppers. In lower gastrointestinal tract, TRPV1channels are involved in visceral pain. TRPV1 channels are reported to be increased incolonic nerve fibers of patients with inflammatory bowel disease and irritable bowel syn-drome. However, the distribution and the roles of afferent neurons expressing TRPV1 inmotor function of lower gastrointestinal tract remain to be fully elucidated. In the presentstudy, we investigated the differences in immunohistochemical distribution of TRPV1 chan-nels and contractile effect of capsaicin on smooth muscle in lower gastrointestinal tract.METHODS: The rectum and distal, transverse and proximal colon were surgically isolatedfrom male, ddY mice. The longitudinal change in smooth muscle tone was isotonicallymeasured using Magnus apparatus. The expression of mRNA of TRPV1 was examined byRT-PCR. Extensive TRPV1-immunoreactivity was detected by using immunohistochemicalstaining with fluorescein-conjugated tyramide amplification. RESULTS: RT-PCR analysisrevealed that mRNA expression of TRPV1 channels in all part of the lower gastrointestinaltract. The localization of TRPV1 was studied in the lower gastrointestinal tract by an immu-nohistochemical technique. Numerous TRPV1-immunoactive nerve fibers were found insubmucosal layer, smooth muscle layer and myenteric nerve plexus. The large numbers ofTRPV1-immunoreactive axons were observed in rectum and distal colon. In contrast, theTRPV1-immunoreactive axons were sparsely distributed in transverse and proximal colon.The density of TRPV1-immunoreactive axons was much lower in transverse and proximalcolon than in rectum and distal colon. In the Manus experiment with rectum and distal colon,capsaicin induced transient contraction followed by long-lasting contraction. Meanwhile, intransverse and proximal colon, only transient contraction was observed. The reactivity ofsmooth muscle in rectum and distal colon was more sensitive than those in transverse andproximal colon. Capsaicin-induced contraction was significantly inhibited by tachykininreceptor antagonists, especially the tachykinin NK2 antagonist GR159897. CONCLUSIONS:The present results suggest that TRPV1-expressing sensory nerves facilitate lower gastrointes-tinal motility, especially through the release of neurokinin A. Differences in the distributionof TRPV1 and contractile effect of capsaicin, suggest the part-dependent role of TRPV1-expressing sensory nerves from proximal colon to rectum.

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The Effect of Acute Tryptophan Depletion (ATD) On the Activity andConnectivity of An Emotional Arousal Network During Visceral PainJennifer S. Labus, Emeran A. Mayer, Tessa Kilkens, Elisabeth A. Evers, Robert-JanBrummer, Walter H. Backes, Michiel A. van Nieuwenhoven

Background and Aims: ATD temporarily reduces serotonin (5-HT) synthesis in the brainand has been to shown to produce disinhibition of central arousal circuits, and alterationsin visceral perception and emotional memory in IBS patients (Kilkens et al. 2004). Wehypothesized that ATD-induced reduction in 5-HT synthesis would alter activity in anemotional arousal network previously shown to be involved in central pain amplification.Methods: BOLD responses of 12 healthy women were assessed using fMRI (1.5 T) during6 low and 6 high, individualized rectal balloon distensions (INF) and 12 non-INF or restperiods, following ATD by oral administration of a tryptophan-depleted drink, or placebo(PL). Multivariate spatiotemporal partial least squares (ST-PLS) was applied to test theinteraction of treatment (ATD,PL) with a distributed pattern of brain activity discriminatingthe low and high INF conditions. Structural equation modeling (SEM) tested for groupdifferences in the effective connectivity of the emotional arousal network. Results: ST-PLSrevealed a network of brain responses that differentiated low and high INF and showedstronger engagement during ATD compared to PL. The network accounted for about 73%of the variance in the data analyzed, and permutation testing revealed significance at p<.01.This network included regions comprising the emotional arousal network (medial orbitalfrontal cortex (mOFC), rostral (rACC), and subgenual anterior cingulate cortices (sACC),and amygdala) as well as thalamus, insula, ventral tegmental area and periaqueductal grey.While most regions generally demonstrated sustained activity, amygdala and thalamic activitywas only evident during the first 3 seconds (1st scan). Subjects receiving ATD showed thegreatest engagement of the emotional arousal circuitry during high INF. Specifically, networkanalyses with SEM revealed significantly greater positive coupling between iACC →sACCand sACC→ Amyg circuits (p values<.05) in ATD as compared to PL during the high INF.Conclusions: Together with our previous demonstration of ATD on visceral pain perception,these findings are consistent with the concept that acute lowering of 5-HT levels results ingreater engagement of a central arousal network which is involved in central pain amplifica-tion. Support Contributed By NIH grants: K08 DK 071626 (JSL) NR04881, P50 DK64539(EAM), R24 AT002681 (EAM), DK 64539 (EAM)

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Spinal NMDA NR-1 Expression Following Resolution of TNBS ColitisQiQi Zhou, Donald Price, Robert Caudle, G Nicholas Verne

Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in thedevelopment of neural plasticity and hyperalgesia following inflammation or tissue injury.It is unclear, if changes in spinal-NMDA receptor expression occur following resolution ofcolitis. Objectives: To investigate the role of NMDA NR1receptors in the spinal cord (L4-S1; T10-L1) of rats following resolution of TNBS colitis. We hypothesize that there will bealtered expression of NMDA NR1 subunits in TNBS treated rats compared to controls.Methods: Male Sprague-Dawley rats (150g-250g) were treated with either intracolonic 20mg/rat trinitrobenzene sulfonic acid (TNBS, Sigma Chemical Co.) in 50% ethanol (n=29) or anequivalent volume of saline (n=8). Sixteen weeks following induction of colitis, all ratsunderwent nociceptive visceral and somatic pain testing. (Zhou et al., 2007). Animals weresacrificed and their spinal cord (L4-S1; T10-L1) was retrieved from 3 groups of rats followingnociceptive visceral and thermal pain testing. Group 1: saline control group (SC) (n=3);group 2: recovered rats (RC) from TNBS injection (n=3); group 3: hypersensitive rats (HC)from TNBS injection (n=3). Immunohistochemistry techniques were used to investigatespinal-NMDA receptor expression in the spinal cord (L4-S1; T10-L1) of all rats. Results:The NMDA NR1-N1,C1, and C2 expression were found in lamina I & II of spinal cord ofT10-L1 and L4-S1 in HS rats but not SC and RC rats. Conclusions: Selective changes inthe expression of NR1 splice variants of the NMDA receptor occur after TNBS inducedcolitis was healed in the rat. These results suggest a role of NMDA receptors NR1 splicevariance in the development of neuronal plasticity and resulting visceral hyperalgesia/painin the spinal cord.

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RET Activation Drives Neuronal Phenotype in CNS-Derived Neural Stem CellsMaria-Adelaide Micci, Kristen M. Kahrig, Pankaj J. Pasricha

Introduction Neural stem cell (NSC) transplantation is a promising technique for thetreatment of disorders of the peripheral (PNS) and central nervous system (CNS). It hasbeen shown that CNS-derived NSC can successfully engraft in the gut of a mouse modelof gastroparesis and differentiate into nNOS expressing neurons and partially rescue gastricfunction. However, much needs to be learnt about the mechanisms responsible for differenti-ation and survival of NSC in the gut and whether these processes require classical factorssuch as GDNF and its cognate receptor, RET. Methods CNS-NSC were transfected with aconstitutively active RET mutant gene (RET-PTC2) and were either (a) cultured in standarddifferentiating conditions (NB27 medium) or (b) co-cultured with a fresh preparation ofmouse longitudinal muscle-myenteric plexus (LMMP) placed inside transwells (no-contact).Subsequently, cell proliferation and phenotypic fate were assesses using a variety of tech-niques. Results Activation of the RET pathway in transfected CNS-NSC was confirmed bythe phosporylation of RET as well as of ERK and Akt. This was associated with a significantincrease in proliferation in CNS-NSC as compared to CNS-NSC transfected with the controlvector (abs @ 490 nm were 0.097+/-0.004 in RET-PTC2 transfected CNS-NSC vs 0.078+/-0.003 in ctrl cells; N=3 P<0.05). The expression of the neuronal markers βIII-tubulin andPGP9.5 was also significantly increased in CNS-NSC transfected with RET-PTC2 (1.20+/-0.05 and 1.38+/- 0.11 folds respectively by Western blotting, N=3 P<0.05) along with adecrease in the glial marker GFAP (0.67+/-0.10 folds vs ctrl by Western blotting, N=3P<0.05). Further, the expression of nNOS and peripherin were also increased (1.20 and1.30 folds respectively by qRT-PCR). When co-cultured with mouse LMMP, CNS-NSC

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