Brain-Derived Neurotrophic Factor Augments Peristalsis by Augmenting 5-HT and Calcitonin Gene-Related Peptide Release

Background & Aims: Brain-derived neurotrophic factor (BDNF) acts rapidly to modulate synaptic neurotransmission in the brain. Although present in neurons, glial cells, and mucosal cells of the colon, and in higher concentrations than in brain, the action of BDNF in gut have not been characterized. The aim of this study was to identify the role of BDNF in mediating the peristaltic reflex. Methods: BDNF and a specific antiserum were examined for their effects on the peristaltic reflex and release of the sensory mediators serotonin and calcitonin gene-related peptide in rat colon. The peristaltic reflex and release of serotonin and calcitonin gene-related peptide were also examined in genetically modified mice (BDNF+/−) with reduced levels of BDNF. Results: Endogenous brain-derived neurotrophic factor was released into the sensory compartment in a stimulus-dependent manner during the peristaltic reflex induced by mucosal stimulation but not muscle stretch. BDNF stimulated and immunoneutralization of endogenous BDNF reduced ascending contraction and descending relaxation of circular muscle and release of serotonin and calcitonin gene-related peptide during the peristaltic reflex induced by mucosal stimulation but not muscle stretch. The peristaltic reflex and release of serotonin and calcitonin gene-related peptide during the peristaltic reflex induced by mucosal stimulation but not muscle stretch were significantly reduced in BDNF+/− mice. Conclusions: Endogenous BDNF enhances the peristaltic reflex by augmenting the release of serotonin and calcitonin gene-related peptide that mediate the sensory limb of the reflex induced by mucosal stimulation. Background & Aims: Brain-derived neurotrophic factor (BDNF) acts rapidly to modulate synaptic neurotransmission in the brain. Although present in neurons, glial cells, and mucosal cells of the colon, and in higher concentrations than in brain, the action of BDNF in gut have not been characterized. The aim of this study was to identify the role of BDNF in mediating the peristaltic reflex. Methods: BDNF and a specific antiserum were examined for their effects on the peristaltic reflex and release of the sensory mediators serotonin and calcitonin gene-related peptide in rat colon. The peristaltic reflex and release of serotonin and calcitonin gene-related peptide were also examined in genetically modified mice (BDNF+/−) with reduced levels of BDNF. Results: Endogenous brain-derived neurotrophic factor was released into the sensory compartment in a stimulus-dependent manner during the peristaltic reflex induced by mucosal stimulation but not muscle stretch. BDNF stimulated and immunoneutralization of endogenous BDNF reduced ascending contraction and descending relaxation of circular muscle and release of serotonin and calcitonin gene-related peptide during the peristaltic reflex induced by mucosal stimulation but not muscle stretch. The peristaltic reflex and release of serotonin and calcitonin gene-related peptide during the peristaltic reflex induced by mucosal stimulation but not muscle stretch were significantly reduced in BDNF+/− mice. Conclusions: Endogenous BDNF enhances the peristaltic reflex by augmenting the release of serotonin and calcitonin gene-related peptide that mediate the sensory limb of the reflex induced by mucosal stimulation. Neurotrophins are target-derived growth factors that regulate the survival, phenotypic differentiation, and axonal growth of neurons in the central, peripheral, and enteric nervous system. These classical actions are prolonged or long-term effects because they occur over hours to days and are delayed in onset because they are often dependent on transcriptional or translational changes leading to protein synthesis. Recently, however, rapid or acute actions of neurotrophins that occur over milliseconds to seconds have been identified in the central nervous system (reviewed in Berninger and Poo1Berninger B. Poo M. Fast actions of neurotrophic factors.Curr Opin Neurobiol. 1996; 6: 324-330Crossref PubMed Scopus (160) Google Scholar and Kovalchuk et al2Kovalchuk Y. Holthoff K. Konnerth A. Neurotrophin action on a rapid timescale.Curr Opin Neurobiol. 2004; 14: 558-563Crossref PubMed Scopus (79) Google Scholar). These typically do not require protein synthesis and are neurotransmitter- or neuromodulator-like effects that are most evident as enhanced synaptic transmission, release of neurotransmitter, or change in synaptic function (ie, synaptic plasticity). Of the several neurotrophins identified, brain-derived neurotrophic factor (BDNF) has been associated most often with the rapid type effects. BDNF is widely distributed within neurons of the central nervous system, including hippocampus, hypothalamus, septum, cerebral cortex, corpus striatum, cerebellum, amygdala, and various medulary nuclei as well as a subset of primary sensory neurons of the dorsal root ganglia. BDNF is synthesized as proBDNF, packaged in the golgi, transported in an anterograde manner to the nerve terminal, stored in presynaptic vesicles, secreted from terminals in a stimulus-dependent manner, and acts at both pre- and postsynaptic sites to produce its rapid effects.3Malcangio M. Lessmann A. A common thread for pain and memory in synapses? Brain-derived neurotrophic factor and trkB receptors.Trends Pharmacol Sci. 2004; 24: 116-121Abstract Full Text Full Text PDF Scopus (132) Google Scholar, 4Tapia-Arancibia L. Rage F. Givalois L. Arancibia S. Physiology of BDNF focus on hypothalamic function.Front Neuroendocrinol. 2004; 25: 77-107Crossref PubMed Scopus (282) Google Scholar Thus, BDNF fulfills all the criteria for a neurotransmitter. The rapid effects of BDNF in the central nervous system have been reviewed extensively2Kovalchuk Y. Holthoff K. Konnerth A. Neurotrophin action on a rapid timescale.Curr Opin Neurobiol. 2004; 14: 558-563Crossref PubMed Scopus (79) Google Scholar, 3Malcangio M. Lessmann A. A common thread for pain and memory in synapses? Brain-derived neurotrophic factor and trkB receptors.Trends Pharmacol Sci. 2004; 24: 116-121Abstract Full Text Full Text PDF Scopus (132) Google Scholar, 4Tapia-Arancibia L. Rage F. Givalois L. Arancibia S. Physiology of BDNF focus on hypothalamic function.Front Neuroendocrinol. 2004; 25: 77-107Crossref PubMed Scopus (282) Google Scholar; however, some are especially noteworthy. In the hippocampus, cortex, and cerebellum, BDNF depolarized neurons as rapidly as glutamate but at a 1000-fold lower concentration, making BDNF one of the most potent excitatory agents in the central nervous system.5Kafitz K.W. Rose C.R. Thoenen H. Konnerth A. Neurotrophin-evoked rapid excitation through TrkB receptors.Nature. 1999; 401: 918-921Crossref PubMed Scopus (471) Google Scholar In the hippocampus, one of the most well-documented rapid synaptic effects of BDNF is the induction of long-term potentiation (LTP) that is critical to learning and memory.6Xu B. Gottschalk W. Chow A. Wilson R.I. Schnell E. Zang K. Wang D. Nicoll R.A. Lu B. Reichardt L.F. The role of brain-derived neurotrophic factor receptors in the mature hippocampus modulation of long-term potentiation through a presynaptic mechanism involving TrkB.J Neurosci. 2000; 20: 6888-6897Crossref PubMed Google Scholar, 7Zakharenko S.S. Patterson S.L. Dragatsis I. Zeitlin S.O. Siegelbaum S.A. Kandel E.R. Morozov A. Presynaptic BDNF required for a presynaptic but not postsynaptic component of LTP at hippocampal CA1-CA3 synapses.Neuron. 2003; 39: 975-990Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar BDNF acts presynaptically to enhance the release of glutamate and γ-aminobutyric acid (GABA) by increasing the phosphorylation of the synaptic vesicle protein synapsin.8Jovanovic J.N. Czernik A.J. Fienberg A.A. Greengard P. Sihra T.S. Synapsins as mediators of BDNF-enhanced neurotransmitter release.Nat Neurosci. 2000; 3: 323-329Crossref PubMed Scopus (477) Google Scholar Similarly, in developing neuromuscular synapses in culture, BDNF enhances excitatory neurotransmission.9Lohof A.M. Ip N.Y. Poo M.M. Potentiation of developing neuromuscular synapses by the neurotrophin NT-3 and BDNF.Nature. 1993; 363: 350-353Crossref PubMed Scopus (680) Google Scholar, 10Boulanger L. Poo M. Gaiting of BDNF-induced synaptic potentiation by cAMP.Science. 1999; 284: 1982-1984Crossref PubMed Scopus (108) Google Scholar, 11Boulanger L. Poo M. Presynaptic depolarization facilitates neurotrophin-induced synaptic potentiation.Nat Neurosci. 1999; 2: 346-351Crossref PubMed Scopus (136) Google Scholar One of the more interesting effects of BDNF with regard to synaptic plasticity is the rapid conversion of sympathetic neurons from excitatory to inhibitory neurotransmission in sympathetic nerve-cardiomyocyte cultures.12Yang B. Slonimsky J.D. Birren S.A. A rapid switch in sympathetic neurotransmitter release properties mediated by the p75 receptor.Nat Neurosci. 2002; 5: 539-545Crossref PubMed Scopus (102) Google Scholar These rapid effects of BDNF are mediated by its cognate tyrosine kinase receptor, Trk B, and/or the nonspecific neurotrophin receptor p75NTR. BDNF and the Trk B and p75NTR receptors are also present in neurons of the myenteric and submucosal plexus of the gut of a variety of species, including human, rat, and mouse.13Hoehner J.C. Wester T. Pahlman S. Olsen L. Localization of neurotrophins and their high-affinity receptors during human enteric nervous system development.Gastroenterology. 1996; 110 (756–567)Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Lucini C. Maruccio L. de Girolamo P. Vega J.A. Castaldo L. Localisation of neurotrophin-containing cells in higher vertebrate intestine.Anat Embryol. 2002; 205: 135-140Crossref PubMed Scopus (33) Google Scholar, 15Radaelli G. Domeneghini C. Arrighi S. Castaldo L. Lucini C. Mascarello F. Neurotransmitters, neuromodulators, and neurotophin receptors in the gut of pantex, a hybrid sparid fish (Pagrus major x Dentex dentex). Localization in the enteric nervous and endocrine systems.Histol Histopathol. 2001; 16: 845-853PubMed Google Scholar, 16Hoehner J.C. Wester T. Pahlman S. Olsen L. Alterations in neurotrophin and neurotrophin-recptor localization in Hirschsprung’s disease.J Pediatr Surg. 1996; 31 (1542–1529)Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 17Maruccio L. Castaldo L. de Girolamo P. Lucini C. Neurotrophin and trk receptor-like immunoreactivity in the frog gastrointestinal tract.Histol Histopathol. 2004; 19: 349-356PubMed Google Scholar, 18Guarino N. Yoneda A. Shima H. Puir P. Selective neurotrophin deficiency in infantile hypertrophic pyloric stenosis.J Pediatr Surg. 2001; 36: 1280-1284Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Estaban I. Levanti B. Garcia-Suarez O. Germana G. Ciriaco E. Naves F.J. Vega J.A. A neuronal subpopulation in the mammalian enteric nervous system expresses TrkA and TrkC neurotrophin receptor-like proteins.Anat Rec. 1998; 251: 360-370Crossref PubMed Scopus (30) Google Scholar, 20Lommatzsch M. Braun A. Mannsfeldt A. Botchkarev V.A. Botchkareva N.V. Paus R. Fischer A. Lewin G.R. Renz H. Abundant production of brain-derived neurotrophic factor by adult visceral epithelia.Am J Pathol. 1999; 155: 1183-1193Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar Depending on the species, BDNF and Trk B are also present in glial cells and mucosal cells.13Hoehner J.C. Wester T. Pahlman S. Olsen L. Localization of neurotrophins and their high-affinity receptors during human enteric nervous system development.Gastroenterology. 1996; 110 (756–567)Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 15Radaelli G. Domeneghini C. Arrighi S. Castaldo L. Lucini C. Mascarello F. Neurotransmitters, neuromodulators, and neurotophin receptors in the gut of pantex, a hybrid sparid fish (Pagrus major x Dentex dentex). Localization in the enteric nervous and endocrine systems.Histol Histopathol. 2001; 16: 845-853PubMed Google Scholar, 17Maruccio L. Castaldo L. de Girolamo P. Lucini C. Neurotrophin and trk receptor-like immunoreactivity in the frog gastrointestinal tract.Histol Histopathol. 2004; 19: 349-356PubMed Google Scholar, 19Estaban I. Levanti B. Garcia-Suarez O. Germana G. Ciriaco E. Naves F.J. Vega J.A. A neuronal subpopulation in the mammalian enteric nervous system expresses TrkA and TrkC neurotrophin receptor-like proteins.Anat Rec. 1998; 251: 360-370Crossref PubMed Scopus (30) Google Scholar, 20Lommatzsch M. Braun A. Mannsfeldt A. Botchkarev V.A. Botchkareva N.V. Paus R. Fischer A. Lewin G.R. Renz H. Abundant production of brain-derived neurotrophic factor by adult visceral epithelia.Am J Pathol. 1999; 155: 1183-1193Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 21Esteban I. Hannestad J. Levanti B. Del Valle M.E. Naves F.J. Vega J.A. Neurotrophin receptor proteins immunoreactivity in human gastrointestinal endocrine cells.Br Res Bull. 1995; 38: 539-543Crossref PubMed Scopus (28) Google Scholar, 22Shibayama E. Koizumi H. Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.Am J Pathol. 1996; 148: 1807-1818PubMed Google Scholar Although the classical effects of BDNF on growth and development of enteric neurons have been examined to some degree, there have been only limited studies of the effect of BDNF on gut function. Two pharmacologic studies suggest that exogenous BDNF has an excitatory effect on the colon. In the rat, BDNF increases the frequency, amplitude, and duration of colonic spike bursts.23Chai N.L. Dong L. Li Z.F. Du K.X. Wang J.H. Yan L.K. Dong X.L. Effects of neurotrophins on gastrointestinal myoelectric activities of rats.World J Gastroenterol. 2003; 9: 1874-1877Crossref PubMed Scopus (36) Google Scholar Coulie et al showed that, in humans, recombinant human BDNF produced in Escherichia coli (r-metHuBDNF) dose-dependently accelerated colonic transit and increased stool frequency without changing stool consistency.24Coulie B. Szarka L.A. Camilleri M. Burton D.D. McKinzie S. Stambler N. Cedarbaum J.M. Recombinant human neurotrophic factors accelerate colonic transit and relieve constipation in humans.Gastroenterology. 2000; 119: 41-50Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar The authors postulated that these effects on transit were most likely mediated by rapid effects of BDNF on transmitter release rather than the classical trophic actions of BDNF. These studies, along with the presence of BDNF and its receptors in the gut, raise the possibility that endogenous BDNF may play a physiologic role in colonic peristalsis. In the present study, we have examined the effect of exogenous BDNF on the colonic peristaltic reflex and on the release of serotonin (5-hydroxytryptamine; 5-HT) and calcitonin gene-related peptide (CGRP), which are key components in the initiation of the peristaltic reflex. The role of endogenous BDNF was examined by immunoneutralization of BDNF and by the use of heterozygous knockout mice (BDNF+/−) that have reduced levels of BDNF. The results indicate that BDNF acts by augmenting the release of 5-HT from mucosal enterochromaffin (EC) cells and CGRP from enteric sensory neurons. The resulting augmentation of the sensory limb of the peristaltic reflex leads to enhancement of the peristaltic reflex that underlies propulsive peristalsis in the colon. The peristaltic reflex was measured in a 3- to 5-cm segment of middle to distal colon of rat or mouse. Segments were opened to form a flat sheet and pinned mucosal side up in a 3-compartment organ bath as described previously.25Grider J.R. Neurotransmitters mediating the intestinal peristaltic reflex in the mouse.J Pharmacol Exp Ther. 2003; 307: 460-467Crossref PubMed Scopus (138) Google Scholar, 26Grider J.R. CGRP as a transmitter in the sensory pathway mediating peristaltic reflex.Am J Physiol. 1994; 266: G1139-G1145PubMed Google Scholar, 27Grider J.R. Jin J.G. Distinct populations of sensory neurons mediate the peristaltic reflex elicited by muscle stretch and mucosal stimulation.J Neurosci. 1994; 14: 2854-2860PubMed Google Scholar The compartments were separated by vertical partitions, sealed with vacuum grease, and contained Krebs-bicarbonate medium composed of 118 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L KH2PO4, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, 25 mmol/L NaH2CO3, and 11 mmol/L glucose. In experiments in which the medium was collected for measurement of CGRP or BDNF, it contained additionally 10 μmol/L amastatin, 1 μmol/L phosphoramidon, and 0.1% bovine serum albumin. In experiments in which the medium was collected for measurement of 5-HT, it contained additionally 10 μmol/L pargyline. The peristaltic reflex was initiated by stroking the mucosa in the central compartment with a fine brush (2 to 8 strokes at a rate of 1 stroke/s) and by radial muscle stretch applied via a hook and pulley assembly (4 and 10 g for 10 seconds each). Ascending contraction and descending relaxation were measured in the orad and caudad peripheral compartments, respectively, using force-displacement transducers attached to the circular muscle layer. In the rat colon, the effect of exogenous BDNF and BDNF antiserum were determined in separate preparations. Following a 30-minute equilibration period, the control response to mucosal stimulation or muscle stretch was determined as described above, the preparation was washed 3 times over 30 minutes, and either BDNF (10 nmol/L for 10 minutes) or BDNF antiserum (AB1513P; 1:100 dilution for 1 hour) was added to the central compartment and the mucosal or muscle stimulation repeated. In separate experiments to measure release of BDNF, 5-HT, and CGRP, the medium from each compartment was collected at the end of a 15-minute stimulus-free period (basal release), and, following a 30-minute recovery period, a single stimulus (eg, 4 mucosal strokes) was applied at 3-minute intervals over a 15-minute period for a total of 5 stimulus applications. The medium was then collected for measurement of BDNF, 5-HT, or CGRP. In some experiments, the release of 5-HT and CGRP was measured in the presence of BDNF or BDNF antiserum. Separate segments were used for collection of samples at each stimulus level, although samples for control and 1 test agent at 1 stimulus level were collected from the same segment. In the mouse colon preparations, the peristaltic reflex and release of 5-HT and CGRP were measured in heterozygous mice (BDNF+/−) in which BDNF levels are reduced and in matched wild-type litter mates (BDNF+/+). Full-thickness sections of colon and intestine from wild-type and BDNF+/− mice were also taken for measurement of BDNF following homogenization in Tissue Protein Extraction Reagent (T-PER; Pierce, Rockford, IL) containing Phosphatase Inhibitor Cocktail 1 (Sigma Chemical Co., St. Louis, MO) and Protease Inhibitor Cocktail (Sigma), and centrifugation. CGRP was measured by radioimmunoassay as described previously using antibody RIK 6006.26Grider J.R. CGRP as a transmitter in the sensory pathway mediating peristaltic reflex.Am J Physiol. 1994; 266: G1139-G1145PubMed Google Scholar The limit of detection of the assay was 2.6 fmol/mL, and the IC50 was 37.6 ± 2.1 fmol/mL of original sample. The antibody reacts with CGRP but not calcitonin, amylin, substance P (SP), neurokinin A, neurokinin B, somatostatin, vasoactive intestinal peptide (VIP), [Met]-enkephalin, or BDNF. 5-HT was chemically derivatized to N-acetyl-5-HT and measured by ELISA kit according to directions (ICN, Cosa Mesa, CA). The limit of detection of the assay was 0.3 nmol/mL, and the range of the assay was 0.3–63 pmol/mL. The N-acetyl-5-HT antibody reacts fully with N-acetyl-5-HT but not with 5-hydroxytryptophan, 5-hydroxy-3-indole acetic acid, 5-hydroxytryptophol, melatonin, SP, VIP, CGRP, or BDNF. BDNF was measured by ELISA kit according to directions (Promega, Madison, WI). All samples were first acidified to pH < 3.0 with 1 N HCl for 15 minutes and then readjusted to neutral pH before assay. The limit of detection of the assay was 0.2 fmol/mL, and the range of the assay was 0.2–557.0 fmol/mL of original sample. The antibody reacts with BDNF but not CGRP, VIP, nerve growth factor, neurotrophin-3, or neurotrophin-4. Ascending contraction and descending relaxation were measured as grams force and expressed as grams in mice and as the percentage of control response obtained with a maximal stimulus (8 strokes or 10 grams stretch) in rats. The release of 5-HT, CGRP, and BDNF into the central compartment during peristalsis was measured as pmol (5-HT) or fmol (BDNF or CGRP) per 100-mg wet tissue weight per minute (pmol or fmol · 100 mg−1 · min−1) and expressed as the percentage of basal level. The content of BDNF in muscle strips of BDNF+/+ and BDNF+/− mice was expressed as fmol/g tissue wet weight. Values were calculated as means ± SEM of measurements obtained in n experiments in which separate animals were used for each experiment. Thus, n represents the number of experiments and animals for each curve. Statistical significance was evaluated using ANOVA and Student t tests (GraftPad Software, San Diego, CA) CGRP, CGRP antiserum RIK 6009, and 125I-CGRP were purchased from Bachem-Peninsula (Torrance, CA). The 5-HT ELISA kit was purchased from ICN. BDNF and the BDNF ELISA kit were purchased from Promega. The BNDF antibody used for immunoneutralization (AB1513P) was purchased from Chemicon (Temecula, CA). Phosphatase Inhibitor Cocktail 1, Protease Inhibitor Cocktail, amastatin, phosphoramidon, pargyline, and all other chemicals and reagents were purchased from Sigma Chemical Co. The heterozygous BDNF knockout mice (BDNF+/−) and wild-type littermates (BDNF+/+) were purchased from Jackson Laboratories (Bar Harbor, ME). These heterozygous mice are generated from the C57BL/6 genetic background strain and are reported by Jackson Laboratories to contain approximately half normal BDNF levels. Mucosal stimulation caused a significant increase in the release of BDNF into the central compartment at each stimulus level (Figure 1). The increase above a basal level of 1.01 ± 0.09 fmol · 100 mg−1 · min−1 ranged from 12% ± 3% increase above basal at 2 strokes (P < .01) to 82% ± 9% increase above basal at 8 strokes (P < .001). In contrast, muscle stretch did not cause BDNF release (Figure 1). There was no increase in the release of BDNF into the orad or caudad peripheral compartments during the peristaltic reflex elicited by either stimulus (data not shown). Addition of 10 nmol/L BDNF to the central compartment did not elicit a peristaltic reflex; however, addition of 10 nmol/L BDNF to the central compartment caused a significant augmentation in the peristaltic reflex elicited by mucosal stroking. The augmentation of ascending contraction ranged from 53% ± 11% at 2 strokes (P < .01) to 27% ± 6% at 8 strokes (P < .05), and the augmentation of descending relaxation ranged from 80% ± 9% at 2 strokes (P < .01) to 23% ± 4% at 8 strokes (P < .05) (Figure 2). In contrast, addition of 10 nmol/L BDNF to the central compartment had no effect on the peristaltic reflex elicited by muscle stretch (data not shown). The physiologic role of BDNF in the peristaltic reflex was examined by immunoneutralization of endogenous BDNF with a specific antiserum. Addition of BDNF antiserum AB1513P (Chemicon) to the central compartment at a final concentration of 1:100 caused a significant inhibition of the peristaltic reflex elicited by mucosal stroking at all levels of stimulation. Ascending contraction was inhibited by 51% ± 8% (P < .01) at 2 strokes to 18% ± 6% (P < .05) at 8 strokes, and descending relaxation was inhibited from 59% ± 10% (P < .05) at 2 strokes to 12% ± 6% (n.s.) at 8 strokes (Figure 3). Previous studies have shown that the peristaltic reflex induced by mucosal stimulation is initiated by release of 5-HT from mucosal EC cells which, in turn, activates 5-HT4 receptors on CGRP-containing primary afferent neurons. Thus, initiation of the peristaltic reflex is mediated by an increase in both 5-HT and CGRP.25Grider J.R. Neurotransmitters mediating the intestinal peristaltic reflex in the mouse.J Pharmacol Exp Ther. 2003; 307: 460-467Crossref PubMed Scopus (138) Google Scholar, 26Grider J.R. CGRP as a transmitter in the sensory pathway mediating peristaltic reflex.Am J Physiol. 1994; 266: G1139-G1145PubMed Google Scholar, 27Grider J.R. Jin J.G. Distinct populations of sensory neurons mediate the peristaltic reflex elicited by muscle stretch and mucosal stimulation.J Neurosci. 1994; 14: 2854-2860PubMed Google Scholar, 28Grider J.R. Kuemmerle J.F. Jin J.G. 5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP neurons.Am J Physiol. 1996; 270: G778-G782PubMed Google Scholar, 29Grider J. Foxx-Orenstein A.E. Jin J.G. 5-Hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.Gastroenterology. 1998; 115: 370-380Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 30Pan H. Gershon M.D. Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine.J Neurosci. 2000; 20: 3295-3309PubMed Google Scholar Addition of 10 nmol/L BDNF to the central compartment caused a significant augmentation in the release of 5-HT and CGRP during the peristaltic reflex elicited by mucosal stroking. The augmentation of 5-HT release into the central compartment ranged from 158% ± 45% (P < .05) at 2 strokes to 46% ± 10% (P < .05) at 8 strokes (Figure 4), and the augmentation of CGRP release ranged from 86% ± 22% (P < .05) at 2 strokes to 57% ± 12% (P < .05) at 8 strokes (Figure 5). Addition of BDNF to the central compartment in the absence of mucosal stimulation did not cause a statistically significant change in the basal release of 5-HT or CGRP.Figure 5Effect of BDNF on the release of CGRP during the peristaltic reflex in rat colon. Addition of 10 nmol/L BDNF to the central compartment caused a significant augmentation of the release of CGRP into the central compartment during peristalsis induced by mucosal stimulation. Responses are expressed as percentage above a basal level of 3.9 ± 0.04 fmol · 100 mg−1 · min−1. Values are means ± SEM of 5 experiments. *Denotes significant difference from control of at least P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The physiologic role of BDNF in mediating the release of 5-HT and CGRP accompanying the peristaltic reflex was examined by immunoneutralization of endogenous BDNF. Addition of BDNF antiserum AB1513P to the central compartment at a final concentration of 1:100 significantly inhibited release of 5-HT and CGRP elicited by mucosal stroking. The inhibition of 5-HT release into the central compartment ranged from 47% ± 12% (P < .05) at 2 strokes to 29% ± 9% (P < .05) at 8 strokes (Figure 6), and the inhibition of CGRP release ranged from 48% ± 8% (P < .01) at 2 strokes to 28% ± 7% (P < .05) at 8 strokes (Figure 7).Figure 7Effect of BDNF antiserum on the release of CGRP during the peristaltic reflex in rat colon. Addition of BDNF antiserum (final dilution 1:100) to the central compartment caused a significant inhibition of the release of CGRP into the central compartment during peristalsis induced by mucosal stimulation. Responses are expressed as percentage above a basal level of 4.1 ± 0.06 fmol · 100 mg−1 · min−1. Values are means ± SEM of 4 experiments. *Denotes significant difference from control of at least P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Small full-thickness segments of ileum and colon from BDNF+/− and BDNF+/+ mice were homogenized, protein was extracted, and BDNF was measured by specific ELISA. The BDNF content was 7.9 ± 1.2 fmol/g tissue wet weight in ileal segments and 6.1 ± 0.4 fmol/g tissue wet weight in colonic segments from BDNF+/+ mice. In samples from BDNF+/−mice, BDNF content was 4.8 ± 0.2 fmol/g in ileum (39% decrease from wild type) and 3.1 ± 0.4 fmol/g in colon (48% decrease from wild type), consistent with the reduced levels reported from measurements in brain of BDNF+/− mice.31Bartoletti A. Cancedda L. Reid S.W. Tessarollo L. Poriatti V. Pizzorusso T. Maffei L. Heterozygous knock-out mice for BDNF show a pathway-specific impairment of long term potentiation but normal critical period for molecular deprivation.J Neurosci. 2002; 22: 10072-10077PubMed Google Scholar, 32Korte M. Carroll P. Wolf E. Brem G. Thoenen H. Bonhoeffer T. Hippocampal long-tem potentiation is impaired in mice lacking brain-derived neurotrophic factor.Proc Natl Acad Sci U S A. 1995; 92: 8856-8860Crossref PubMed Scopus (1196) Google Scholar, 33Lyons W.E. Mamounas L.A. Ricaurte G.A. Coppola V. Reid S.W. Bora S.H. Wihler C. Koliatsos V.E. Tessarollo L. Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities.Proc Natl Acad Sci U S A. 1999; 96: 15239-15244Crossref PubMed Scopus (716) Google Scholar The peristaltic reflex elicited by mucosal stimulation was significantly reduced in the BDNF+/− mice compared with BDNF+/+ mice (Figure 8). The reduction in ascending contraction ranged from 70% reduction at 2 strokes (0.03 ± 0.03 g in segments from BDNF+/− mice vs 0.10 ± 0.03 g in segments from BDNF+/+ mice; P < .01) to 13% reduction at 8 strokes (0.65 ± 0.04 g in segments from BDNF+/− mice vs 0.75 ± 0.05 g in segments from BDNF+/+ mice; n.s.). Similarly, the reduction in desce