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

Transcription

1 GASTROENTEROLOGY 2006;130: Brain-Derived Neurotrophic Factor Augments Peristalsis by Augmenting 5-HT and Calcitonin Gene-Related Peptide Release JOHN R. GRIDER, BARBARA E. PILAND, MELISA A. GULICK, and LI YA QIAO Departments of Physiology and Internal Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 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 generelated 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 Poo 1 and Kovalchuk et al 2 ). These typically do not require protein synthesis and are neurotransmitter- or neuromodulatorlike 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. 3,4 Thus, BDNF fulfills all the criteria for a neurotransmitter. The rapid effects of BDNF in the central nervous system have been reviewed extensively 2 4 ; 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. 5 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. 6,7 BDNF acts presynaptically to enhance the release of glutamate and -aminobutyric acid (GABA) by increasing the phosphorylation of the synaptic vesicle protein synapsin. 8 Similarly, in developing neuromuscular synapses in culture, BDNF enhances excitatory neurotransmission Oneofthe more interesting effects of BDNF with regard to synaptic plasticity is the rapid conversion of sympathetic neurons Abbreviations used in this paper: 5HT, 5-hydroxytryptamine; BDNF, brain-derived neurotrophic factor; CGRP, calcitonin gene-related peptide; EC, enterchromaffin; SP, substance P by the American Gastroenterological Association Institute /06/$32.00 doi: /j.gastro

2 772 GRIDER ET AL GASTROENTEROLOGY Vol. 130, No. 3 from excitatory to inhibitory neurotransmission in sympathetic nerve-cardiomyocyte cultures. 12 These rapid effects of BDNF are mediated by its cognate tyrosine kinase receptor, Trk B, and/or the nonspecific neurotrophin receptor p75 NTR. BDNF and the Trk B and p75 NTR 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 ,20 Depending on the species, BDNF and Trk B are also present in glial cells and mucosal cells. 13,15,17,19,20 22 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. 23 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. 24 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. Materials and Methods Measurement of Peristaltic Reflex in Compartmented Flat-Sheet Segments of Rat and Mouse 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 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 KH 2 PO 4, 2.5 mmol/l CaCl 2, 1.2 mmol/l MgSO 4, 25 mmol/l NaH 2 CO 3, and 11 mmol/l glucose. In experiments in which the medium was collected for measurement of CGRP or BDNF, it contained additionally 10 mol/l amastatin, 1 mol/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 mol/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. Experimental Protocols 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.

3 March 2006 AUGMENTATION OF PERISTALTIC REFLEX BY BDNF 773 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) Figure 1. Release of BDNF during the peristaltic reflex in rat colon. Mucosal stimulation (brush strokes) but not muscle stretch (grams) caused significant increase in BDNF release into the central compartment as measured by ELISA. Values are expressed as percentage increase above a basal value of fmol 100 mg 1 min 1 and are means SEM of 4 experiments. *Denotes significance of at least P.01. Measurement of CGRP Release CGRP was measured by radioimmunoassay as described previously using antibody RIK The limit of detection of the assay was 2.6 fmol/ml, and the IC 50 was 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. Measurement of 5-HT Release 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 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. Measurement of BDNF Release 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 fmol/ml of original sample. The antibody reacts with BDNF but not CGRP, VIP, nerve growth factor, neurotrophin-3, or neurotrophin-4. Data Analysis Ascending contraction and descending relaxation were measured as grams force and expressed as grams in mice and as Materials CGRP, CGRP antiserum RIK 6009, and 125 I-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. Figure 2. Effect of BDNF on the peristaltic reflex in rat colon. Addition of 10 nmol/l BDNF to the central compartment caused a significant augmentation of the ascending contraction (left) and descending relaxation (right) component of the peristaltic reflex induced by mucosal stimulation. Responses are expressed as percentage of the maximal response obtained at 8 strokes (maximal response: g force for ascending contraction and g force for descending relaxation). Values are means SEM of 6 experiments.

4 774 GRIDER ET AL GASTROENTEROLOGY Vol. 130, No. 3 Figure 3. Effect of BDNF antiserum on the peristaltic reflex in rat colon. Addition of BDNF antiserum (final dilution 1:100) to the central compartment caused a significant inhibition of the ascending contraction (left) and descending relaxation (right) component of the peristaltic reflex induced by mucosal stimulation. Responses are expressed as percentage of the maximal response obtained at 8 strokes (maximal response: g force for ascending contraction and g force for descending relaxation). Values are means SEM of 5 experiments. Results Release of BDNF During the Peristaltic Reflex 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 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). Effect of BDNF and BDNF Antiserum on the Peristaltic Reflex 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). Effect of BDNF and BDNF Antiserum on the Release of 5-HT and CGRP During the Peristaltic Reflex 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-HT 4 receptors on CGRP-containing primary afferent neurons. Thus, initiation of the peristaltic reflex is mediated by an increase in both 5-HT and CGRP 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 Figure 4. Effect of BDNF on the release of 5-HT 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 5-HT into the central compartment during peristalsis induced by mucosal stimulation. Responses are expressed as percentage above a basal level of pmol 100 mg 1 min 1. Values are means SEM of 5 experiments. *Denotes significant difference from control of at least P.05.

5 March 2006 AUGMENTATION OF PERISTALTIC REFLEX BY BDNF 775 Figure 5. Effect 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 fmol 100 mg 1 min 1. Values are means SEM of 5 experiments. *Denotes significant difference from control of at least P.05. Figure 6. Effect of BDNF antiserum on the release of 5-HT 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 5-HT into the central compartment during peristalsis induced by mucosal stimulation. Responses are expressed as percentage above a basal level of pmol 100 mg 1 min 1. Values are means SEM of 4 experiments. *Denotes significant difference from control of at least P.05. mucosal stimulation did not cause a statistically significant change in the basal release of 5-HT or CGRP. 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). Peristaltic Reflex in Mice With Reduced Levels of BDNF 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 fmol/g tissue wet weight in ileal segments and fmol/g tissue wet weight in colonic segments from BDNF / mice. In samples from BDNF / mice, BDNF content was fmol/g in ileum (39% decrease from wild type) and fmol/g in colon (48% decrease from wild type), consistent with the reduced levels reported from measurements in brain of BDNF / mice 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 ( g in segments from BDNF / mice vs g in segments from BDNF / mice; P.01) to 13% reduction at 8 strokes ( g in segments from BDNF / mice vs g in segments from BDNF / mice; n.s.). Similarly, the reduction in descending relaxation ranged from 65% reduction at 2 strokes ( g in segments from BDNF / mice vs g in segments from BDNF / mice; P.01) to 13% reduction at 8 strokes ( g in segments from BDNF / mice vs 0.75 Figure 7. Effect 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 fmol 100 mg 1 min 1. Values are means SEM of 4 experiments. *Denotes significant difference from control of at least P.05.

6 776 GRIDER ET AL GASTROENTEROLOGY Vol. 130, No. 3 Figure 8. Peristaltic reflex elicited by mucosal stimulation in BDNF / and BDNF / mice. The ascending contraction (left) and descending relaxation (right) component of the peristaltic reflex induced by mucosal stimulation were significantly reduced in BDNF / mice. Responses are expressed as grams force above or below resting tone. Values are means SEM of 3 experiments. *Denotes significant difference from control of at least P g in segments from BDNF / mice; n.s.). In contrast, the peristaltic reflex elicited by radial muscle stretch was similar in BDNF / and BDNF / mice (Figure 9). Release of 5-HT and CGRP During the Peristaltic Reflex in Mice With Reduced Levels of BDNF The release of 5-HT and CGRP into the central compartment during the peristaltic reflex elicited by mucosal stimulation was significantly reduced in the BDNF / mice compared with BDNF / mice (Figures 10 and 11). The basal release of 5-HT was similar in BDNF / mice (18 4 pmol 100 mg 1 min 1 ) and Figure 10. Release of 5-HT during the peristaltic reflex elicited by mucosal stimulation in BDNF / and BDNF / mice. The release of 5-HT into the central compartment during peristalsis induced by mucosal stimulation was significantly reduced in BDNF / mice. Values are expressed as percentage above a basal level of 18 4 and 21 3 pmol 100 mg 1 min 1 in BDNF / and BDNF / mice, respectively. Values are means SEM of 3 experiments. *Denotes significant difference from control of at least P.05. in BDNF / mice (21 3 pmol 100 mg 1 min 1 ). Mucosal stroking caused a stimulus-dependent increase in 5-HT release in BDNF / and BDNF / mice; however, the release of 5-HT was significantly less in the BDNF / mice compared with that in BDNF / mice (Figure 10). The reduction in 5-HT release in segments from BDNF / mice ranged from 34% 8% (P.05) at 2 strokes to 23% 6% (P.05) at 8 strokes. The basal release of CGRP was similar in BDNF / mice ( fmol 100 mg 1 min 1 ) and in BDNF / mice ( fmol 100 mg 1 min 1 ). Mucosal stroking Figure 9. Peristaltic reflex elicited by muscle stretch in BDNF / and BDNF / mice. The ascending contraction (left) and descending relaxation (right) component of the peristaltic reflex induced by muscle stretch were not different in BDNF / and BDNF / mice. Responses are expressed as grams force above or below resting tone. Values are means SEM of 3 experiments. Figure 11. Release of CGRP during the peristaltic reflex elicited by mucosal stimulation in BDNF / and BDNF / mice. The release of CGRP into the central compartment during peristalsis induced by mucosal stimulation was significantly reduced in BDNF / mice. Values are expressed as percentage above a basal level of and fmol 100 mg 1 min 1 in BDNF / and BDNF / mice, respectively. Values are means SEM of 3 experiments. *Denotes significant difference from control of at least P.05.

7 March 2006 AUGMENTATION OF PERISTALTIC REFLEX BY BDNF 777 caused a stimulus-dependent increase in CGRP release in BDNF / and BDNF / mice; however, the release of CGRP was significantly less in the BDNF / mice compared with that in BDNF / mice (Figure 11). The reduction in CGRP release in segments from BDNF / mice ranged from 38% 5% (P.05) at 2 strokes to 25% 7% (P.05) at 8 strokes. Discussion The present study indicates that exogenous BDNF acts to augment the peristaltic reflex by enhancing the release of 5-HT and CGRP. Furthermore, these studies indicate that endogenous BDNF plays a physiologic role in mediating the peristaltic reflex. This conclusion is based on several findings: (1) mucosal stimulation caused a stimulus-dependent release of BDNF, (2) immunoneutralization of endogenous BDNF reduced the peristaltic reflex and the concomitant release of 5-HT and CGRP, and (3) the peristaltic reflex and release of 5-HT and CGRP are reduced in BDNF / mice that are deficient in BDNF. The peristaltic reflex is mediated by an integrated circuit of intrinsic neurons in the myenteric and submucosal plexus, which includes excitatory motor neurons that mediate orad or ascending contraction and caudad or descending relaxation, ascending and descending interneurons, and sensory or afferent neurons that initiate the peristaltic reflex in response to luminal stimuli or muscle stretch. In the present study, we have used a 3-compartment preparation that allows separate measurement of orad and caudad responses and of the accompanying release of transmitter from motor and sensory compartments as described previously In this preparation, BDNF was released only into the central compartment in which the stimulus was applied, indicating that endogenous BDNF might play a role in the sensory but not motor limb of the peristaltic reflex. Previous studies have shown that the sensory limb of the peristaltic reflex differs for different stimuli. 27 Muscle stretch activates an extrinsic CGRP-containing sensory neuron with cell body in the dorsal root ganglion. 26 Chemical or mechanical stimulation of the mucosa activates a more complex intrinsic pathway initiated by the release of 5-HT from EC cells HT then diffuses to adjacent intrinsic afferent nerve terminals at which it activates 5-HT4/5-HT1p receptors and stimulates that neuron to release CGRP from terminals in the myenteric plexus. These terminals connect to ascending and descending interneurons that facilitate or inhibit activity of motor neurons. In the present study, BDNF was released in proportion to the intensity of mucosal stimulation (ie, number of strokes) but not in response to muscle stretch (Figure 1). This suggests that BDNF was not released from extrinsic sensory neurons and that endogenous BDNF plays a role exclusively in the peristaltic reflex initiated by mucosal stimulation. Consistent with this notion, application of exogenous BDNF to the mucosa enhanced the peristaltic reflex elicited by mucosal stroking (Figure 2) but not muscle stretch and caused an increase in the release of 5-HT and CGRP (Figures 4 and 5). The participation of endogenous BDNF in the initiation of the mucosal reflex is evident by the finding that immunoneutralization of BDNF inhibited the peristaltic reflex induced by mucosal stroking (Figure 3) as well as the release of 5-HT and CGRP into the central compartment (Figures 6 and 7). The physiologic role of BDNF in the peristaltic reflex was further tested in a genetically modified mouse in which the levels of endogenous BDNF were reduced. Previous studies had shown that the neural circuit mediating the peristaltic reflex in mice was identical to those in rat and human. 25 Heterozygotic (BDNF / ) knockout mice were used in the present study because homozygotic mice (BDNF / ) do not survive more than a few days postpartum. In segments of ileum and colon from BDNF / mice, BDNF levels were reduced 39% 48% compared with BDNF / mice, consistent with the reduction in BDNF reported for brain regions In BDNF / mice, the peristaltic reflex and release of 5-HT and CGRP elicited by mucosal stimulation were significantly reduced (Figures 8, 10, and 11) compared with BDNF / mice. These results strongly support the notion that endogenous BDNF acts to facilitate the sensory limb of the peristaltic reflex under normal conditions. The fact that the reflex elicited by muscle stretch was similar in BDNF / and BDNF / mice further supports the notion that the sensory pathways initiating the response to muscle stretch and to mucosal stimulation are distinct. 27 Addition of BDNF to the mucosa in the central compartment of the 3-compartment preparation at the 10 nmol/l concentration used in this study did not initiate the peristaltic reflex or stimulate the release of 5-HT or CGRP. This lack of effect of BDNF in the absence of concomitant mucosal stimulation suggests that the primary role of BDNF is to augment release of 5-HT and CGRP, thereby leading to an augmentation of ongoing peristalsis. This modulatory role of BDNF is similar to that noted in other systems. For example, in rat brain striatal slices, BDNF alone had no effect on transmitter release but augmented depolarization-induced release of

8 778 GRIDER ET AL GASTROENTEROLOGY Vol. 130, No. 3 GABA, dopamine, and 5-HT. 34 With regard to BDNF in the gut, our working hypothesis is that BDNF does not initiate the peristaltic reflex, but, rather, it is released secondarily and acts as a positive feedback agent to enhance 5-HT and CGRP release. Consistent with this hypothesis, preliminary studies show that the release of BDNF in response to mucosal stimulation is blocked by a 5-HT 4 receptor antagonist. 35 The source of BDNF was not determined in the present study; however, several sources of BDNF exist in the gut. It is noteworthy that BDNF is present in the brain in higher concentrations than any other neurotrophin, and, yet, the colon contains higher concentrations than the brain. 20 Immunohistochemical studies have consistently identified BDNF in neurons of the myenteric and submucosal plexus. 13,14,16,18,20 Studies of cultured myenteric ganglia of guinea pig indicate that BDNF is costored in neurons that express CGRP and VR-1 receptors and are therefore likely to be enteric afferent neurons. 36 This is consistent with the colocalization of BDNF and CGRP in sensory neurons of the dorsal root ganglion. 37 The localization of BDNF in enteric neurons suggests that BDNF acts as a neurotransmitter or neuromodulator in the gut just as it has been postulated to do in the central nervous system. BDNF has also been identified in glial cells and in endothelial cells, although these locations are more variable. 14,16,17,20 Thus, BDNF could be released from enteric neurons and act to stimulate release of CGRP in an autocrine manner or could be released from mucosal cells to act in a paracrine manner or both. Regardless of the source of BDNF, the release of CGRP and 5-HT is augmented by BDNF as a result of interaction with Trk B and/or p75 NTR receptors. The augmentation of CGRP release could be the result of a direct effect of BDNF on CGRP-containing enteric neurons, or it could be indirect as a result of BDNF-induced release of 5-HT, which acts, in turn, to release CGRP. A direct mechanism is a distinct possibility because Trk B and p75 NTR receptors are present on enteric neurons, although the phenotype of these neurons has not been identified. 13,16 20 A direct effect is also supported by preliminary studies showing that exogenous BDNF is capable of eliciting release of CGRP from pure cultures of enteric neurons and from colonic muscle strips devoid of 5-HT containing EC cells. 35 Strong evidence also supports an indirect mechanism for BDNF-induced CGRP release. Trk B receptors are present on colonic mucosal cells from human, rat, and mouse among other species. Of particular note, the Trk B-immunostained mucosal cells are likely to be neuroendocrine cells, based on their location in the crypts along the basement membrane and costaining with chromogranin A. 15,17,19,21,22 Although the specific neuroendocrine cell type expressing Trk B has not been identified, it is likely that the 5-HT-containing EC cells of the colonic mucosa that initiate the peristaltic reflex induced by mucosal stimulation express Trk B because Esteban et al have estimated that more than 60% of all endocrine cells in human large intestinal mucosa express Trk B. 19 The presence of receptors for BDNF on neuroendocrine cells supports the notion that BDNF released from either enteric neurons innervating the mucosa or from adjacent BDNF-containing mucosal cells can regulate the release of bioactive agents, including 5-HT, from mucosal neuroendocrine cells. The interplay of BDNF and 5-HT in the gut is interesting in light of the interactions between these endogenous agents in the central nervous system. Thus, in the neocortex, 5-HT causes an increase in levels of BDNF messenger RNA, whereas it has the opposite effect in the hippocampus. 38 Conversely, BDNF enhances 5-HT release from corpus striatum. 34 BDNF strongly stimulates the sprouting and regrowth of serotonergic neurons in brain. 39,40 Antidepressant drugs and inhibitors of the serotonin reuptake transporter (SERT), such as fluoxetine, increase BDNF levels and Trk B receptor activity in various brain regions. 41,42 Strong evidence suggests that BDNF and its interrelationship with 5-HT in regions of the brain may play a critical role in the development and treatment of depression and other pathologies of the central nervous system (reviewed in Xu et al 6 and Altar 40 ). The results of the present study demonstrating a similar interaction between 5-HT and BDNF in the gut, suggest that the relationship between these agents may be more widespread and might partially explain the ability of fluoxetine to enhance colonic motility. 43 The role of BDNF in enhancing the peristaltic reflex is consistent with previous studies of the effects of r-hubdnf and a BDNF analog, r-met-hubdnf, in human subjects, in which they have been evaluated for potential therapeutic use in the treatment of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. 44,45 These studies consistently note that the main adverse effects of BDNF in humans are increased gut motility, diarrhea, and increased bowel urgency. In the only trial specifically designed to examine the effect of BDNF on gut motility, Coulie et al 24 showed that r-met-hubdnf dose dependently accelerated overall colonic transit in normal patients and in patients with constipation. Normal subjects also demonstrated increased proximal colonic emptying and increased gastric and intestinal transit. This increased transit was also accompanied by

9 March 2006 AUGMENTATION OF PERISTALTIC REFLEX BY BDNF 779 increased stool frequency and ease of stool passage without changing stool consistency in constipated patients. Although these studies did not examine mechanism of action, it is likely that the effects of exogenous BDNF in these human studies were the result of enhancement of the underlying peristaltic reflex, as identified in the present study. Additionally, these studies taken in combination with the present study suggest that BDNF analogs might be worth reevaluating for development of therapeutic use as promotility agents. References 1. Berninger B, Poo M. Fast actions of neurotrophic factors. Curr Opin Neurobiol 1996;6: Kovalchuk Y, Holthoff K, Konnerth A. Neurotrophin action on a rapid timescale. Curr Opin Neurobiol 2004;14: Malcangio M, Lessmann A. A common thread for pain and memory in synapses? Brain-derived neurotrophic factor and trkb receptors. 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10 780 GRIDER ET AL GASTROENTEROLOGY Vol. 130, No Luo X-G, Rush RA, Zhou X-F. Ultrastructural localization of brainderived neurotrophic factor in rat primary sensory neurons. Neurosci Res 2001;39: Vaidya VA, Marek GJ, Aghajanian GK, Duman RS. 5-HT2A receptor-mediated regulation of brain-derived neurotrophic factor mrna in the hippocampus and the neocortex. J Neurosci 1997; 17: Mamounas LA, Altar CA, Blue ME, Kaplan DR, Tessarollo L, Lyons WE. BDNF promotes the regenerative sprouting but not survival of injured serotonergic axons in the adult rat brain. J Neurosci 2000;20: Altar CA. Neurotrophins and depression. Trends Pharmacol Sci 1999;20: Molteni R, Calabrese F, Bedogni F, Tongiorgi E, Fumagalli F, Racagni G, Andrea Riva M. Chronic treatment with fluoxetine up-regulates cellular BDNF mrna expression in rat dopaminergic regions. Int J Neuropsychopharmacol EPUB July 22, 2005: Saarelainen T, Hendolin P, Lucas G, Koponen E, Sairanen M, MacDonald E, Agerman K, Haapasalo A, Nawa H, Aloyz R, Ernfors P, Castren E. Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is required for antidepressant-induced behavioral effects. J Neurosci 2003;23: Wade PR, Chen J, Jaffe B, Kassem IS, Blakely RD, Gershon MD. Localization and function of a 5-HT transporter in crypt epithelium of the gastrointestinal tract. J Neurosci 1996;16: Wellmer A, Misra VP, Sharief MK, Kopelman PG, Amand P. A double-blind placebo-controlled clinical trial of recombinant human brain-derived neurotrophic factor (rhbdnf) in diabetic polyneuropathy. J Peripher Nerv Syst 2001;6: Bradley WG and the BDNF Study Group (Phase III). A controlled trial of recombinant methionyl human BDNF in ALS. Neurology 1999;52:1427. Received September 16, Accepted December 7, Address requests for reprints to: John R. Grider, PhD, Department of Physiology, Box , Virginia Commonwealth University, Richmond, Virginia jgrider@hsc.vcu.edu; fax: (804) Supported by grant DK34153 from the National Institute of Diabetes and Digestive and Kidney Diseases.