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The Journal of Neuroscience, August 1, 2001, 21(15):5620-5636

Neurotrophin-3 Is Required for the Survival-Differentiation of Subsets of Developing Enteric Neurons

Alcmène Chalazonitis1, Tuan D. Pham1, Taube P. Rothman1, Peter S. DiStefano2, Mark Bothwell3, Janet Blair-Flynn4, Lino Tessarollo4, and Michael D. Gershon1

1 Department of Anatomy and Cell Biology, Columbia University, New York, New York 10032, 2 Milennium Pharmaceuticals Inc., Cambridge, Massachusetts 02139, 3 Department of Physiology and Biophysics SJ-40, University of Washington, Seattle, Washington 98195, and 4 Neural Development Group, National Cancer Institute, Frederick, Maryland 21702


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of 125I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of 125I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta -hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Key words: neurotrophins; Trk C; neural crest; apoptosis; retrograde transport; transgenic mice; gastrointestinal tract; autonomic nervous system


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The enteric nervous system (ENS) is a neural crest derivative (Yntema and Hammond, 1954, 1955; Le Douarin and Teillet, 1973, 1974). Although this origin is shared with extraenteric ganglia (Le Douarin and Kalcheim, 1999), the ENS is structurally and functionally unique (Furness and Costa, 1987; Gershon et al., 1994; Gershon, 1998b; Furness, 2000). The mature ENS contains intrinsic primary afferent neurons (IPANs) (Kirchgessner et al., 1992, 1996; Kunze et al., 1995; Furness et al., 1998; Kunze and Furness, 1999) and microcircuits that allow it to regulate enteric motile and secretory behavior (Trendelenburg, 1917; Cooke, 1989; Cooke et al., 1997, 1999; Pan and Gershon, 2000). No other peripheral ganglia function independently of CNS control. The developmental mechanisms responsible for the unique structure and function of the ENS are not understood.

The crest-derived cell population that colonizes the bowel is multipotent (Rothman et al., 1990, 1993; Sextier-Sainte-Claire Deville et al., 1994; Lo and Anderson, 1995; Lo et al., 1997); therefore, not only cell lineages (Blaugrund et al., 1996; Gershon, 1997), but also the enteric microenvironment, play critical roles in determining the fates of crest-derived cells differentiating within the gut. Microenvironmental factors that influence the development of enteric neurons and/or glia include glial cell line-derived neurotrophic factor (GDNF) (Schuchardt et al., 1994; Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996; Cacalano et al., 1998; Chalazonitis et al., 1998b; Hearn et al., 1998; Heuckeroth et al., 1998), neurturin (Heuckeroth et al., 1999), neurotrophin-3 (NT-3) (Chalazonitis et al., 1994), a still-to-be-identified neuropoietic cytokine that binds to the alpha  component of the ciliary neurotrophic factor (CNTF) receptor (Chalazonitis et al., 1998a) [which may be the newly discovered CNTFII (Elson et al., 2000; Lesser and Lo, 2000)], endothelin-3 (Baynash et al., 1994; Hearn et al., 1998; Wu et al., 1999), serotonin (5-HT) (Fiorica-Howells et al., 2000), and laminin-1 (Chalazonitis et al., 1997). The timing, sequence, and combinations of growth-differentiation factors to which precursor cells are exposed all affect the responses of enteric neural and glial progenitors (Chalazonitis et al., 1998b; Gershon, 1998a).

The physiological significance of the promotion by NT-3 of enteric neuronal-glial development has not yet been established. An ENS is present in mice that lack NT-3 (Fariñas et al., 1994; Tessarollo et al., 1994), TrkC (Klein et al., 1994), or p75NTR (Lee et al., 1992). At least some enteric neurons can thus develop and survive independently of NT-3; however, defects in specific subsets of neurons would not be obvious in the histological appearance of the ENS. The survival of mice that lack NT-3 or TrkC, moreover, is brief; therefore, the motility and/or secretion of their gastrointestinal tracts may be abnormal. NT-3 and other neurotrophins are known to be survival factors for some but not all neurons (Chalazonitis, 1996; Lewin and Barde, 1996). The current study was thus designed to test the hypotheses that NT-3 is a survival factor for subsets of enteric neurons and that, after exposure to NT-3, these subsets become NT-3 dependent.

In vitro studies were performed with enteric neurons immunoselected from the developing rat gut. In vivo observations were made with transgenic mice that overexpress NT-3 and also with animals that lack either NT-3 or TrkC. Retrograde transport of NT-3 was studied in the adult bowel because this property correlates with target-derived neurotrophic dependency (DiStefano et al., 1992). Because the effects of CNTF and NT-3 have been found previously to be interactive (Chalazonitis et al., 1998a), the abilities of these factors to influence the effect of the other on survival were also investigated.

In vitro observations supported the idea that NT-3 is a target-derived neurotrophic factor on which enteric neurons become dependent. Studies with transgenic mice, which overexpress NT-3 in the developing ENS, confirmed that NT-3 promotes enteric neuronal development in vivo; moreover, experiments with NT-3- and TrkC-deficient mice revealed the presence and regional distribution of subsets of neurons in both enteric plexuses that are NT-3 dependent. Retrograde transport of NT-3 was demonstrated in mature enteric ganglia, suggesting that NT-3 continues to function in the adult ENS, perhaps in the maintenance of neurons that become NT-3 dependent during development. Although CNTF can replace NT-3 and support NT-3-dependent enteric neurons, NT-3 dependency is exacerbated when precursors are simultaneously exposed to CNTF and NT-3. We conclude that NT-3 plays a subtle but critical role during ENS development and adulthood.

Parts of this work have been published in abstract form (Pham et al., 1996, 1999).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Fetal rats were obtained from timed pregnant dams (Harlan Sprague Dawley, Charles River, MA). The day at which a vaginal plug was found was designated as day 0 of gestation. Maternal rats were anesthetized with CO2 and killed by a thoracotomy at day 14 of gestation. The fetal bowel was dissected aseptically from 25-30 fetuses for each experiment involving tissue culture. Adult rats of either sex, which were used for the study of the retrograde transport of NT-3, were anesthetized with Metofane and exsanguinated by decapitation. The Animal Care and Use Committee of Columbia University approved all procedures.

Mice that overexpress NT-3 in the ENS were generated in a C57BL/6 genetic background by pronuclear injection of a plasmid containing sequentially a 5.8 kb fragment of the human dopamine-beta -hydroxylase (DBH) gene promoter (Mercer et al., 1991), a 0.15 kb fragment comprising intron A of the human insulin gene, a 0.96 kb fragment containing the complete translated sequence of a human NT-3 cDNA, and a 0.27 kb fragment of a mouse protamine I cDNA 3' UTR, containing the polyadenylation sequence. The line used for these studies contained 5-10 tandemly integrated copies of the transgene. The DBH promoter has been shown to direct expression of transgenes to the neural crest-derived cells that colonize the gut (Kapur et al., 1991, 1992; Rice et al., 2000). Transgene expression begins when the crest-derived cells enter the bowel and is maintained throughout development. The ENS was analyzed in comparable regions of the proximal intestines of five transgenic mice and five wild-type littermates.

Mice carrying targeted deletions of genes encoding NT-3 (Tessarollo et al., 1994) or TrkC (Tessarollo et al., 1997) were derived in a mixed 129/Sv-C57BL/6 genetic background and subsequently backcrossed for 10 generations into a C57BL/6 background. The guts of wild-type, heterozygote, and mutants were removed and processed for determining the total numbers of neurons in the enteric plexuses and for the immunocytochemical identification of classes of enteric neurons (see below). Comparisons were made in corresponding regions of the bowel between wild-type, heterozygotes, and homozygotes animals of the same strains. Analyses were made on six wild-type, four TrkC +/-, four TrkC -/-, three NT-3 +/-, and two NT-3 -/- mice [ranging in age from postnatal day 0 (P0) to P22].

Immunoselection. Crest- and noncrest-derived cells were isolated from dissociated fetal bowel by a process of positive and negative immunoselection with antibodies to human natural killer cell antigen HNK-1 (Erickson et al., 1989; Pomeranz et al., 1993; Chalazonitis et al., 1994) or p75NTR (Baetge et al., 1990a; Chalazonitis et al., 1997, 1998a,b) as described previously. HNK-1 antibodies were derived from cells purchased from American Type Culture Collection (Rockville, MD). Antibodies to p75NTR (clone IgG192) were donated by Regeneron Pharmaceuticals (Tarrytown, NY). Magnetic beads coated with species-specific secondary antibodies that were used for immunoselection were obtained from Miltenyi Biotec Inc. (Mt. Auburn, CA). In some experiments, which were designed to study whether noncrest-derived cells are a source of endogenous NT-3 that affects the in vitro development of enteric neurons, mixed cultures of dissociated cells from the fetal bowel were investigated without previous separation by immunoselection.

Tissue culture. Cells were plated in tissue culture dishes that had been coated previously with rat tail collagen and mouse laminin (10 µg/ml) and cultured in a defined medium (Ziller et al., 1983) as described previously (Chalazonitis et al., 1994). The plating density was 2.75 × 105 cells/35-mm-diameter tissue culture dish (catalog #3001; Becton Dickinson, Franklin Lakes, NJ) or 1.2 × 105 cells/20 mm2 chamber slide (catalog #177429; Nunc, Naperville, IL). Cultures of each type were maintained in triplicate. NT-3 was always applied at a concentration found previously to be supramaximal (40 ng/ml) (Chalazonitis et al., 1994). CNTF was also applied at a concentration (10 ng/ml) at which it is known to be supramaximal (Chalazonitis et al., 1998a). Both NT-3 and CNTF were supplied by Regeneron. A blocking antibody to NT-3 (Gaese et al., 1994) was supplied by Dr. Ilse Bartke (Boehringer Mannheim, Pinsberg, Germany). Controls consisted of equivalent cultures exposed to the vehicle in which the growth factors were dissolved (basic Brazeau medium containing 0.5% BSA). Experiments were terminated by fixing cultures with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M PBS.

Immunocytochemistry in cultures. Fixed cultures were permeabilized by incubation with 0.1 M PBS containing 0.1% Triton X-100 (Sigma, St. Louis, MO). Primary antibodies were applied overnight at room temperature as described previously (Chalazonitis et al., 1994, 1997, 1998a). Three reagents were used to identify neurons: (1) polyclonal antibodies to the intermediate neurofilament protein peripherin (diluted at 1:750; gift of Dr. Lloyd Greene, Department of Pathology, Columbia University) (Portier et al., 1984; Aletta et al., 1988), (2) a mixture of monoclonal antibodies to the 68, 160, and 200 kDa components of the neurofilament triplet (each diluted 1:100; Sigma), and (3) polyclonal antibodies to rat neuron-specific enolase (NSE) (diluted 1:1000; Polysciences, Warrington, PA). Neurons that could be NT-3 responsive were identified with polyclonal antibodies to TrkC (diluted 1:100; gift of Dr. B. Hempstead, Cornell Medical College, New York, NY). These antibodies were raised against a peptide (amino acids 639-653) in the cytoplasmic domain of rat TrkC. The antibodies react with isoforms of TrkC that contain an active kinase but not with TrkB or TrkA (Donovan et al., 1996). Preimmune sera (diluted 1:100) were used as a control for the antibodies to TrkC. Sites in which primary antibodies were bound were localized with affinity-purified species-specific secondary antibodies, including biotinylated goat anti-rabbit or anti-mouse IgG. The secondary antibodies were visualized with avidin coupled to HRP (ABC Elite kit; Vector Laboratories, Burlingame, CA) or alkaline phosphatase (Vectastain SK#5300; Vector Laboratories). After rinsing, the cultures were exposed to reagents to visualize the sites of immunoreactivity with either alkaline phosphatase or with peroxidase diaminobenzidine (Vectastain SK#4100) according to the procedure of the manufacturer and as described previously (Chalazonitis et al., 1994, 1997, 1998a,b).

Immunocytochemistry in situ. Freshly removed bowel was opened, flattened, and pinned to a wax support. The tissue was then fixed with 4% formaldehyde (from paraformaldehyde) in saline buffered with 0.1 M sodium phosphate to pH 7.4. The gut was then mechanically dissected into layers to obtain laminar preparations of the submucosa (containing the submucosal plexus) and the muscularis externa with adherent myenteric plexus. The laminar preparations were then rinsed and stained or immunostained as whole mounts. To count the total number of neurons, preparations were stained for 1 hr at 37°C with cuprolinic blue (0.5% in 0.05 M sodium acetate buffer, pH 5.6, containing 1.0 M MgCl2). This reagent, in the presence of Mg2+ ions, selectively demonstrates single-stranded RNA and thus enables the entire set of enteric neurons, which are rich in ribosomes, to be visualized without interference from the relatively ribosome-poor surrounding cells (Heinicke et al., 1987; Holst and Powley, 1995; Karaosmanoglu et al., 1996). All preparations were treated for 10 min with H2O2 (0.3%) in PBS, washed again with PBS, and blocked for 30 min with 4% goat serum in PBS containing 0.3% Triton X-100. Primary antibodies, listed in Table 1, were then applied (diluted in blocking solution) to the sections for 72 hr at 4°C. Sites of antibody binding were detected with goat anti-rabbit antibodies coupled to horseradish peroxidase (Kirkegaard & Perry, Gaithersburg, MD). Peroxidase activity was visualized with H2O2 and 3,3'-diaminobenzidene (DAB) with or without nickel intensification.


                              
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  		Table 1.   Antibodies used to identify enteric neurons in whole mounts

Identification of apoptotic, dying, or dead cells. The terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated UTP nick end labeling (TUNEL) method was used to identify apoptotic cells (TACS kit, used according to the directions of the manufacturer; Trevigen Inc., Gaithersburg, MD). This method uses TdT and biotinylated nucleotides to label the 3' hydroxyl groups of cleaved DNA. Briefly, the cultures were permeabilized with saponin, and endogenous peroxidase was blocked with periodic acid. The TdT-catalyzed incorporation of biotinylated nucleotides into cleaved DNA was performed in the presence of Mn2+, which is optimal for the TUNEL method in neuronal systems. Incorporated biotinylated nucleotides were visualized by using streptavidin conjugated to HRP (Vectastain #SK4100).

Dying and dead cells were identified using an ethidium homodimer (Live/Dead; Molecular Probes, Eugene, OR). This reagent binds to DNA but does not permeate the plasma membranes of living cells. Once cells begin to die, their plasma membrane becomes compromised and permeant to the ethidium homodimer, which then stains the nuclei of the dying or dead cells. Briefly, cultures were rinsed with a Ca2+-, Mg2+-free buffer and then incubated in the same buffer with the ethidium homodimer (6 µM) for 1 hr. After this incubation, cultures were washed and fixed with 4% formaldehyde (from paraformaldehyde) for the immunocytochemical identification of neurons.

Axonal transport of NT-3. NT-3 was iodinated as described previously (DiStefano et al., 1992). The specific activities of radioactive 125I-NT-3 ranged from 2458 to 3834 cpm/fmol. The bioactivity of the 125I-NT-3 was 90-99% compared with nonlabeled NT-3, as determined by neurite outgrowth in chicken dorsal root ganglion explants (DiStefano et al., 1992). Loops of adult rat small intestine were removed from the animals and immediately immersed in oxygenated Krebs' solution at room temperature. The lumen of the intestine was then thoroughly purged with Krebs' solution, and the bowel was transferred to iced Krebs' solution. Intestinal segments (50 mm in length) were opened along the mesenteric border and pinned flat as open rectangles on a substrate of silicone elastomer (Sylgard; Dow Corning, Midland, MI). When injections were to be made into the mucosa, the preparations were pinned out with the mucosal surface facing up. When injections were to be made into myenteric ganglia, the preparations were pinned out with the serosal surface facing up. The immobilized preparations were continuously superfused at 10-15 ml/min with oxygenated Krebs' solution at 37°C, except during injections. Injections were performed with beveled glass micropipettes with a tip size of 8-12 µm. Pipettes were positioned with a Narishige (Tokyo, Japan) micromanipulator under microscopic control using differential interference contrast optics to allow the boundary between the epithelium and the lamina propria to be visualized in mucosal preparations and myenteric ganglia to be visualized in the muscularis externa. 125I-NT-3 (40 ng/ml in 0.5%BSA and PBS) was injected into either the mucosa (lamina propria) or individual ganglia of the myenteric plexus (see Fig. 12A,C,D). Solutions were ejected from the micropipettes with pressure pulses (9-12 psi; pulse duration, 0.2-1 sec) delivered by a multichannel Picospritzer (General Valve, Fairfield, NJ). A retrograde fluorescent tracer, Fluoro-Gold (4%; Fluorochrome Inc. Englewood, CO), or the beta  subunit of cholera toxin (beta -CTx) conjugated to fluorescein isothiocyanate (FITC) (1%; List Biologic, Campbell, CA) was coinjected with 125I-NT-3. These tracers were used to identify the full set of neurons that extended axonal projections to the injection site. Only a subset of such neurons would be expected to take up 125I-NT-3 and display retrograde labeling of nerve cell bodies. Neurons were thus considered to be labeled by the retrograde transport of 125I-NT-3 if they were double labeled by 125I-NT-3 and Fluoro-Gold or beta -CTx. Specificity of transport was determined by coinjecting a 40- to 260-fold excess of nonlabeled NT-3, NGF, or BDNF with 125I-NT-3. Retrograde transport of 125I-NT-3 was considered to be specific if it was blocked by NT-3 but not by NGF or BDNF. After injections, the tissue was transferred to a sterile vial and incubated overnight at 37°C in 10 ml of Eagle's MEM (Life Technologies, Gaithersburg, MD), supplemented with HEPES buffer (15 mM) and penicillin-streptomycin (1%) to allow time for retrograde axonal transport.

After overnight culture, the segments of bowel were again pinned flat and fixed with freshly prepared 4% formaldehyde (from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.4, for 4 hr at room temperature or overnight at 4°C. The gut was then dissected into laminar preparations of mucosa-submucosa, and the longitudinal smooth muscle with adherent myenteric plexus and tissues were permeabilized with 1.0% Triton X-100. beta -CTx was demonstrated by immunocytochemistry [rabbit primary antibodies, diluted 1:500 (List Biologic); FITC-labeled goat anti-rabbit secondary antibodies). Fluoro-Gold was localized by virtue of its native fluorescence.

To locate 125I-NT-3 in tissues, preparations were mounted onto glass slides coated with chromium-alum gelatin (0.5 gm/l). Tissues were then dehydrated by passage through a graded series of ethanols to diethyl ether and air dried. A thin film of carbon was evaporated (with a Denton evaporator) over the slides to prevent nonspecific chemographic artifactual labeling (Gershon and Sherman, 1987). The slides were coated with nuclear track emulsion [Ilford L4 or Kodak NTB-2 (Eastman Kodak, Rochester, NY), diluted 1:1 with distilled H2O]. The emulsion-coated slides were exposed for 3-4 weeks in a light-tight box under an atmosphere of dry CO2, developed [Kodak D-19 developer (Eastman Kodak) for 5 min], and fixed. After an aqueous wash, the preparations were dehydrated with ethanol, cleared in xylene, and coverslipped in a nonfluorescent mounting medium.

Statistical analyses. Total cells and cells in cultures that were specifically identified with reagents described above were counted as described previously (Chalazonitis et al., 1994, 1997, 1998b). The numbers of cells were normalized by expression as a percentage of cells found in cultures treated with vehicle or as a percentage of cells found in cultures treated for 1 week with NT-3. When the ENS was examined in situ, the neuronal density as a function of area (in square millimeters) was quantified by taking as a measurement the mean of the pooled counts of neurons in 10 fields covering 1.254 mm2. The numbers of such measurements ranged from 4 to 32 per gut. Artifactually damaged tissue was not examined. Means were compared by ANOVA (STATVIEW 4.1 program for the Macintosh computer; SAS Institute, Cary, NC).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A subset of neurons that develop in response to NT-3 becomes NT-3 dependent

Crest-derived cells were immunoselected with antibodies to p75NTR and cultured for 7 d. We have shown previously that, under these conditions, NT-3 promotes the development of enteric neurons (Chalazonitis et al., 1994). To determine whether the neurons that are induced to develop in the presence of NT-3 become dependent on it for survival, we exposed crest-derived cells to NT-3 and investigated the effects of NT-3 withdrawal. In one set of these cultures of isolated crest-derived cells, NT-3 was present for the full 7 d. In a second set, NT-3 was withdrawn for the final 2 d of the 7 d culture period. The ethidium homodimer was added to the medium of both sets of cultures for 1 hr to label cells in each that were dead or dying. Both sets of cultures were then fixed and immunostained with antibodies to NSE to identify postmitotic neurons (Schmechel et al., 1980; Maxwell et al., 1982; Baetge et al., 1990b). For each culture, the total number of cells, the number of neurons, and the number of dying or dead cells were determined (Fig. 1A). Substantially fewer neurons were present in the cultures from which NT-3 was withdrawn; however, the withdrawal of NT-3 was not accompanied by a significant change in either the total number of cells or the number of cells that were dying or dead. A larger number of neurons might have been present in the cultures when NT-3 was present for 7 rather than 5 d of culture because the continued presence of NT-3 promoted the development of neurons during the final 2 d of the culture period. Alternatively, cells that develop in response to NT-3 might have become NT-3 dependent and died when NT-3 was withdrawn. Because the numbers of total cells and those that were dying or dead did not change significantly after NT-3 withdrawal, cell death, if it occurs as a result of NT-3 deprivation, would have to take place in only a limited subset of the crest-derived cells in the cultures.



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  		Figure 1.   Withdrawal of NT-3 causes neurons to be lost from cultures of enteric crest-derived cells. A, The total number of cells, neurons, and dying or dead cells were determined in cultures grown for 7 d, in either the continuous presence of NT-3 or for 5 d with NT-3 followed by a 2 d period during which NT-3 was withdrawn. Neurons were identified by using NSE immunoreactivity as a marker. Dying or dead cells were identified by using uptake of an ethidium homodimer as a marker. The number of neurons present in the cultures from which NT-3 was withdrawn for 48 hr was significantly less than that in the cultures that were continuously exposed to NT-3 for 7 d. In contrast, the withdrawal of NT-3 did not lead to a significant reduction in the total number of cells present after 7 d in vitro, nor did it lead to a significant increase in the number of dying or dead cells. B, The numbers of cells committed to a neuronal lineage were counted in cultures exposed continuously to NT-3 for 8 d, for 5 d, or for 5 d followed by a 3 d period during which NT-3 was withdrawn. Peripherin immunoreactivity was used to identify cells committed to a neuronal lineage. The number of neural cells present in the cultures after 5 d of exposure to NT-3 did not differ significantly from that present at 8 d; however, significantly fewer neural cells remained in the cultures at 8 d when NT-3 was withdrawn for the final 72 hr in vitro. n.s., Not significant.

To determine whether the loss of a subset of cells accounts for the decrement in neuron numbers that followed the withdrawal of NT-3, crest-derived cells were again immunoselected with antibodies to p75NTR and plated in three sets of cultures. One set of cultures was maintained in the presence of NT-3 for 5 d. A second set was maintained, also in the presence of NT-3, but for 8 d. The third set of cultures was maintained for 8 d, but NT-3 was present only for the first 5 d in vitro and was withdrawn for the final 3 d of the culture period. Peripherin immunoreactivity was used as a neuronal marker to include precursors committed to the neuronal lineage, as well as terminally differentiated neurons (the proportion of cells that are peripherin-immunoreactive is approximately two times that of the NSE-immunoreactive cell population). The number of neurons developing in each of the three sets of cultures was determined; data were normalized to the number of neurons developing in the cultures exposed to NT-3 for 8 d (9.1 ± 1.4 × 104 neurons per dish) (Fig. 1B). The number of neurons present in the cultures exposed to NT-3 for 5 d was virtually identical to that exposed to NT-3 for 8 d (Fig. 1B). In contrast, when NT-3 was withdrawn from the cultures after 5 d of exposure, the number of neurons present at 8 d was now significantly less than that found in the cultures that were continuously exposed to NT-3 for 8 d (Fig. 1). These observations suggest that the withdrawal of NT-3 for the final 3 d in vitro caused some of the neurons that had developed after 5 d in the presence of NT-3 to be lost.

NT-3 withdrawal leads to apoptosis of a subset of cells that express TrkC

Experiments were performed to determine whether the loss of neurons associated with NT-3 withdrawal was attributable to apoptosis of a subset of neurons that develop in response to NT-3. TrkC immunoreactivity (Donovan et al., 1996) was used as a marker for NT-3-responsive cells, and the TUNEL method was used to identify cells committed to apoptosis. Crest-derived cells, immunoselected with antibodies to p75NTR, were cultured in the absence or presence of NT-3. The cultures that were not exposed to NT-3 were maintained for 5.75 d. All of the cultures that were exposed to NT-3 were grown in its presence for a minimum of 5 d; the NT-3-exposed cultures were then maintained for an additional 18 or 48 hr in the absence or presence of NT-3. Eighteen hours is the earliest time that apoptosis can be detected after withdrawal of a growth factor from sympathetic neurons (Edwards and Tolkovsky, 1994). The design of the experiments, therefore, permitted the effects of NT-3 on the number of TrkC-immunoreactive cells developing in vitro, as well as the effects of NT-3 withdrawal on this cell population, to be ascertained.

After 7 d of growth in the presence of NT-3, the TrkC-immunoreactive cells were all found to be neurons (Fig. 2). Both nerve cell bodies and thin neurites displayed TrkC immunoreactivity. The TrkC-immunoreactive cells represented 5.2 ± 0.7% of the total cell population. This proportion was ~90% of the total number of cells demonstrated with NSE immunoreactivity (a marker for mature neurons) and 45% of the larger population of neuronally committed cells that is demonstrated with peripherin immunoreactivity. The number of TrkC-immunoreactive neurons increased significantly (p < 0.0001) between days 5 and 7 of culture in the continued presence of NT-3 (Fig. 3A). The development of neurons that expressed TrkC was found to depend on NT-3; very few TrkC-immunoreactive cells were present in cultures grown in its absence (Fig. 3A). The number of cells that expressed TrkC was very sensitive to the presence of NT-3 and decreased significantly (p < 0.0001) when NT-3 was withdrawn for 48 hr (Fig. 3A). The withdrawal of NT-3, moreover, dramatically increased apoptosis in the subset of cells that expressed TrkC (Figs. 3B, 4). This increase was quantified by counting the cells that were double labeled with antibodies to TrkC and by the TUNEL method. The brown nuclear reaction product indicative of apoptosis was readily distinguished from the blue alkaline phosphatase reaction product used to visualize TrkC immunoreactivity, which was cytoplasmic (Fig. 4). The increase in apoptosis of TrkC-expressing cells was evident as soon as 18 hr of withdrawal (p < 0.006) and persisted through 48 hr (p < 0.002). The withdrawal of NT-3, however, did not significantly alter the total number of cells in the cultures (Fig. 3C), nor did it increase the proportion of the total cell population in which apoptosis was detected (Fig. 3D). These data suggest that the population of neurons that undergoes apoptosis when NT-3 is withdrawn is a subset of the TrkC-immunoreactive cell population and is small relative to the total number of cells in the cultures and the background occurrence of apoptosis.



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  		Figure 2.   TrkC immunoreactivity in cultures of crest-derived cells immunoselected from the E14 fetal rat gut with antibodies to p75NTR. The cultures were grown for 7 d in the continuous presence of NT-3. The TrkC-immunoreactive cells were all process bearing with the morphology of neurons. Both perikarya (arrows) and neurites (arrowheads) were immunostained by the antibodies to TrkC. A, Individual TrkC-immunoreactive cells can be seen, apparently randomly scattered in the culture. The cells are interconnected by TrkC-immunoreactive processes. B, C, TrkC-immunoreactive cells also form small aggregates, approximating the appearance of mini-ganglia. D, No immunostaining is seen in an analogous culture that was treated with preimmune sera instead of the antibodies to TrkC; nevertheless, non-immunoreactive clusters of neurons (arrow) and processes (arrowhead) can be discerned in the field of view. Scale bars, 50 µm.



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  		Figure 3.   The in vitro development of TrkC-immunoreactive cells is NT-3 dependent, and TrkC-immunoreactive cells selectively undergo apoptosis when NT-3 is withdrawn. Cultures were grown in the absence or presence of NT-3 for 5-7 d as indicated in the graphs. The effects of NT-3 withdrawal for 18 or 48 hr were also ascertained. For each panel, the number of days in culture, the duration of continuous exposure to NT-3, and the times of NT-3 withdrawal are indicated below the corresponding columns. A, The proportion of TrkC-immunoreactive cells (as a percentage of the total number of cells per culture) in cultures exposed to NT-3 was significantly greater than that found in cultures grown in the absence of NT-3 (p < 0.02, vehicle vs NT-3 for 5.75 d; p < 0.0001, vehicle vs NT-3 for 7 d). The proportion of TrkC-immunoreactive cells also increased significantly between 5.75 and 7 d of culture in the presence of NT-3 (p < 0.0001). The number of TrkC-immunoreactive cells present after 5.75 d in the presence of NT-3 did not change significantly when NT-3 was withdrawn for 18 hr; however, the number of TrkC-immunoreactive cells in cultures from which NT-3 was withdrawn for the final 48 hr of a 7 d culture period was significantly less than that found in corresponding cultures exposed continuously to NT-3 for 7 d (p < 0.0001). B, The proportion of TrkC-immunoreactive cells undergoing apoptosis, evaluated by the TUNEL method, was not significantly different in cultures that were not exposed to NT-3 or that were exposed to NT-3 for 5.75 or 7 d. In contrast, the withdrawal of NT-3 for 18 hr (p < 0.006, vs NT-3 for 5.75 d) or 48 hr (p < 0.002, vs NT-3 for 7 d) significantly increased the proportion of TrkC-immunoreactive cells undergoing apoptosis. C, The total number of cells in cultures was not significantly affected by the presence of NT-3 or by its withdrawal. D, The proportion of the total cell population undergoing apoptosis was not significantly affected by the presence or withdrawal of NT-3.



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  		Figure 4.   Apoptosis in TrkC-immunoreactive cells was identified by visualizing cells double labeled with antibodies to TrkC and the TUNEL method. Crest-derived cells were immunoselected from the E14 fetal rat gut with antibodies to p75NTR. A, Cells were cultured in the presence of NT-3 for 5.75 d. The blue alkaline phosphatase reaction product indicates the presence of TrkC immunoreactivity (red arrows). B, C, Cells were cultured in the presence of NT-3 for 5 d, but NT-3 was withdrawn for the final 18 hr of the 5.75 d culture period. The brown DAB reaction product in the nuclei (violet arrows) identify cells demonstrated by the TUNEL method to contain fragmented DNA and thus to be undergoing apoptosis. The TrkC-immunoreactive cell undergoing apoptosis, which is illustrated in C, is magnified in the inset to help enable the blue and brown reaction products to be distinguished. Note the neuritic processes extended by TrkC-immunoreactive cells. Scale bars, 25 µm.

Antibodies to NT-3 antagonize the in vitro promotion of enteric neuronal development by exogenous NT-3 (determination of the neutralizing concentration)

The ability of anti-NT-3 to block the NT-3-induced increment in the in vitro development of enteric neurons was studied to determine whether anti-NT-3 could be used to investigate the physiological role(s) played by endogenous NT-3. Crest-derived cells were immunoselected from the fetal rat gut with antibodies to p75NTR and cultured for 4 d in defined medium with NT-3 (10 ng/ml) in the absence or presence of anti-NT-3 (0.4-100 µg/ml). The concentration of anti-NT-3 able to completely neutralize the action of exogenous NT-3 was thus determined. Control cultures were maintained for 4 d in the presence of only vehicle or anti-NT-3 (55-100 µg/ml). The latter conditions were used to determine whether anti-NT-3 itself affects the in vitro development of enteric neurons. Peripherin immunoreactivity was used to identify cells committed to a neuronal lineage. The addition of as little as 2 µg/ml of anti-NT-3 to cultures grown in the presence of NT-3 significantly decreased the proportion of neural cells in the cultures (Fig. 5A). The action of NT-3 was maximally inhibited by ~40 µg/ml of anti-NT-3; at this concentration, the development of neurons was reduced to that seen in cultures maintained in the absence of NT-3. Addition of supramaximal concentrations (55 or 100 µg/ml) of anti-NT-3 by itself to cultures did not reduce the number of peripherin-immunoreactive cells below that found in cultures exposed only to vehicle (Fig. 5B). Thus, anti-NT-3 does not itself appear to be neurotoxic or to prevent neuronal development. Its ability to antagonize the promotion of enteric neuronal development by NT-3, therefore, is probably attributable to the neutralization of NT-3.



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  		Figure 5.   Antagonism by anti-NT-3 of the ability of exogenous NT-3 to promote the in vitro development of enteric neurons from isolated precursors. Crest-derived cells were immunoselected from the E14 fetal rat gut with antibodies to p75NTR. A, The immunoselected cells were cultured for 4 d in the presence of NT-3 (10 ng/ml) and increasing concentrations of anti-NT-3. The number of cells demonstrated by peripherin immunoreactivity to be in a neuronal lineage was counted and normalized to that developing in the presence of NT-3 alone. The NT-3-promoted development of neural cells is inhibited in a concentration-dependent manner by anti-NT-3. At concentrations of anti-NT-3 of 40 µg/ml, the number of peripherin-immunoreactive cells is reduced to the level that develops in the presence only of vehicle (compare with B). B, In the absence of anti-NT-3, more peripherin-immunoreactive cells develop in the presence of NT-3 (10 ng/ml) than in the presence of vehicle. The addition of anti-NT-3 (55 and 100 µg/ml; pooled data) did not reduce the number of peripherin-immunoreactive cells below the number seen in cultures exposed only to vehicle.

The enteric mesenchyme promotes the development of enteric neurons via NT-3

Anti-NT-3 was used to test the hypothesis that NT-3 secreted by non-neuronal cells of the enteric mesenchyme promotes the development of neurons from enteric crest-derived cells. Previous experiments have demonstrated that exogenous NT-3 is able to promote enteric neuronal development in vitro (Chalazonitis et al., 1994) (also described above). In those studies, NT-3 was added to crest-derived cells that were isolated by immunoselection from their non-neuronal neighbors in the enteric mesenchyme. If crest-derived cells are not isolated from the non-neuronal cells of the enteric mesenchyme but are cultured together with them, then the crest-derived cells may be subjected to growth factors that the non-neuronal cells secrete. If the non-neuronal cells were to promote the development of enteric neurons by secreting NT-3, these effects would be expected to be antagonized by anti-NT-3. The fetal rat gut was thus dissociated at embryonic day 14 (E14), and mixed cultures of mesenchymal cells were established without immunoselecting the crest-derived subset of the population. These mixed cultures were maintained for 4 d without exogenous NT-3 but in the absence (Fig. 6A) or presence of anti-NT-3 (Fig. 6B). Peripherin immunoreactivity again served as the marker for cells committed to a neuronal lineage. Anti-NT-3 significantly decreased the development of neural cells in the mixed cultures (p < 0.05) (Fig. 6C). The presence of anti-NT-3 also reduced the complexity and size of the arborization of neurites (Fig. 6, compare A, B).



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  		Figure 6.   Anti-NT-3 inhibits the development of neurons and the arborization of neurites in cultures of mixed cells from the fetal enteric mesenchyme. The fetal bowel was dissociated, and the resulting mesenchymal cell suspension was plated without isolating the cells of neural crest origin. Cultures were grown for 4 d and fixed for the immunocytochemical demonstration of peripherin immunoreactivity. A, Control; vehicle only. Peripherin-immunoreactive cells are abundant and give rise to a profuse and complex arbor of neurites. B, Anti-NT-3 (40 µg/ml). Fewer peripherin-immunoreactive cells are present, and they give rise to relatively few neurites with a paucity of branches. C, The numbers of peripherin immunoreactive cells in each culture were quantified. Anti-NT-3 significantly inhibits the development of peripherin-immunoreactive cells in the mixed cultures.

Retrograde transport of NT-3 occurs in subsets of neurons in both plexuses of the mature rat gut

Specific uptake and retrograde transport from axon terminals to cell bodies is a characteristic feature of neurotrophins that has been reported to identify neurons that develop in response to a given neurotrophin and acquire a dependence on it that persists into adult life (DiStefano et al., 1992). The observations that NT-3 promotes the development of enteric neurons in vitro, that NT-3 dependence occurs, and that anti-NT-3 inhibits the ability of noncrest-derived cells to enhance the development of enteric neurons are all consistent with the idea that NT-3 plays a physiologically important role in the development of at least some enteric neurons. If this hypothesis is correct and dependence persists, then the axon terminals of subsets of neurons in the mature gut would be expected to take up NT-3 and transport it in the retrograde direction to cell bodies. 125I-NT-3 was thus microinjected into the target areas of a variety of enteric neurons (Gershon et al., 1994; Kunze and Furness, 1999) to determine whether specific uptake and retrograde transport of NT-3 occurs in the ENS. These targets included the mucosa, smooth muscle, myenteric ganglia, and tertiary plexus (see summary diagram in Fig. 12). The mucosa contains the terminals of secretomotor and IPANs of submucosal ganglia and also the terminals of myenteric IPANs (see diagram in Fig. 12D, inset). Myenteric ganglia contain the terminals of ascending and descending interneurons projecting from other myenteric ganglia, as well as the distal processes of submucosal IPANs. The tertiary plexus and smooth muscle both contain the terminals of myenteric motor neurons. To test the specificity of uptake and retrograde transport, 125I-NT-3 was microinjected together with an excess of nonradioactive NT-3, NGF, or BDNF. In several studies, 125I-NT-3 was microinjected into the bowel wall together with the retrograde transport markers beta -CTx or Fluoro-Gold to verify that retrograde transport was responsible for the labeling of neurons by 125I-NT-3 and to ascertain the relative proportion of axons projecting to the injection sites that took up 125I-NT-3.

When 125I-NT-3 was injected into the wall of the gut, neurons were found to have become radioautographically labeled in enteric ganglia. The locations of the labeled neurons depended on where the 125I-NT-3 was injected. When 125I-NT-3 was microinjected into the mucosa, the neurons that became radioautographically labeled were exclusively found in submucosal ganglia (Fig. 7A,B,D-I; see also the summary diagram in Fig. 12C). Mucosal injections of 125I-NT-3 did not label neurons in myenteric ganglia. When 125I-NT-3 was injected into the layer of the myenteric plexus (encompassing also the smooth muscle and tertiary plexus), labeling was found in both underlying submucosal ganglia (Fig. 7C) and myenteric ganglia distant from the injection sites (Fig. 7M-O; see also the summary diagram in Fig. 12D). At the injection sites themselves, varicose fibers labeled by 125I-NT-3 were found extending away from the masses of injected radioactive material (Fig. 7L). 125I-NT-3 labeled no varicose axons or other neurites in the distant ganglia that contained labeled neuronal perikarya. Coinjection of an excess of nonradioactive NT-3 prevented the labeling of cells by 125I-NT-3 (Fig. 7J,K). NT-3 was effective in blocking the labeling of neurons by 125I-NT-3, regardless of whether injections were placed in the mucosal or myenteric ganglia. In contrast to excess NT-3, an excess of NGF (Fig. 7D-F) or BDNF (data not shown) did not inhibit the labeling of neurons by 125I-NT-3. When Fluoro-Gold (Fig. 7D-F) or beta -CTx (Fig. 7G-I,M,N) was injected with 125I-NT-3, all of the cells that were radioautographically labeled by 125I-NT-3 contained the coinjected retrograde tracer. In each case, however, more neurons were labeled by beta -CTx or Fluoro-Gold (36 neurons) than by 125I-NT-3 (17 neurons), suggesting that only a subset of enteric neurons is capable of uptake and retrograde transport of NT-3.



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  		Figure 7.   Axons of mature enteric neurons specifically take up 125I-NT-3 and transport it in the retrograde direction to their cell bodies in enteric ganglia. 125I-NT-3 was microinjected into the mucosa or the muscularis externa-myenteric plexus and located by radioautography 4 weeks later. A, B, Submucosal ganglia after a mucosal injection of 125I-NT-3. Multiple neurons are labeled in some ganglia (A), whereas only one or two labeled neurons can be found in others (B). The stippled line in B outlines the borders of the illustrated submucosal ganglion. Arrow indicates a 125I-NT-3-labeled neuron. C, A submucosal ganglion after an injection of 125I-NT-3 into the myenteric plexus. Several neurons (arrow) are labeled. D-F, Submucosal ganglion after a mucosal coinjection of 125I-NT-3, Fluoro-Gold, and an excess of NGF. A neuron (arrow) is colabeled by 125I-NT-3 and Fluoro-Gold. NGF does not prevent the labeling of neurons by 125I-NT-3. D, Fluorescence view showing the location of Fluoro-Gold. E, Interference contrast illumination showing the location of 125I-NT-3. F, Superimposition of fluorescence and interference contrast images. G-I, Submucosal ganglion after a mucosal coinjection of 125I-NT-3 and beta -CTx. Two neurons (arrows) are colabeled by 125I-NT-3 and beta -CTx. G, Fluorescence view showing the location of beta -CTx (visualized by immunofluorescence with FITC). H, Incident dark-field illumination showing the location of 125I-NT-3. I, Superimposition of fluorescence and dark-field images. J, K, Submucosal ganglion after a myenteric coinjection of 125I-NT-3, Fluoro-Gold, and an excess of nonradioactive NT-3. The excess of nonradioactive NT-3 does not interfere with the labeling of neurons by Fluoro-Gold (J; arrow) but prevents the radioautographic labeling of neurons by 125I-NT-3 (K). J, Fluorescence view showing the location of Fluoro-Gold. K, Incident dark-field illumination. The arrow shows the location of a neuron labeled by Fluoro-Gold (visible in J but not in K). L, Site of injection of 125I-NT-3 into a myenteric ganglion. The ganglion contains a great deal of nonspecific radioautographic labeling, but 125I-NT-3-labeled varicose axons (arrows) can also be seen running away from the injection site. M-O, A myenteric ganglion is illustrated after 125I-NT-3 and beta -CTx were coinjected into a distant myenteric ganglion. A neuron (arrow) is colabeled by 125I-NT-3 and beta -CTx. M, Fluorescence view showing the location of beta -CTx (visualized by immunofluorescence with FITC). N, Incident dark-field illumination showing the location of 125I-NT-3. O, Superimposition of fluorescence and dark-field images. Scale bars, 25 µm.

Myenteric neurons are increased in number and size in transgenic mice that overexpress NT-3 directed to the ENS by the DBH promoter

Transgenic mice that overexpress NT-3 in the ENS were investigated to determine whether NT-3 affects ENS development in situ. The expression of NT-3 in these mice was under the control of the DBH promoter (DBH-NT-3 mice), which has been demonstrated to target the overexpression of a large number of molecules to the ENS, as well as to sympathetic neurons (Kapur et al., 1992; Rice et al., 2000). The DBH-NT-3 mice survive and gain weight normally. Enteric neurons were demonstrated with cuprolinic blue in dissected laminar preparations of the gut wall from wild-type and DBH-NT-3 mice (Fig. 8A,B). The neurons in the ganglia of the myenteric plexus of DBH-NT-3 mice were, on average, larger than those of wild-type animals (Table 2; Fig. 8, compare A, B). The means of the maximum chords, diameters (measured through the nucleus), perimeters, and areas were all significantly increased in the neurons of the DBH-NT-3 animals (Table 2). The number of myenteric neurons per ganglion was also substantially greater in DBH-NT-3 mice (Fig. 8C), as was the packing density of neurons within the ganglia. The packing density in DBH-NT-3 mice was 3.1 × 10-3 ± 4.2 × 10-5 neurons/µm2, whereas in wild-type animals, it was 2.9 ± 10-3 ± 4.1 × 10-5 neurons/µm2 (p < 0.03). In contrast to the myenteric plexus, the number of neurons in the submucosal plexus was not significantly different in DBH-NT-3 and wild-type mice (Fig. 8C).



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  		Figure 8.   Overexpression of NT-3 targeted to the ENS of transgenic mice by the DBH promoter is associated with increases in the size and numbers of myenteric neurons. A, B, Laminar preparations of the longitudinal muscle with adherent myenteric plexus stained with cuprolinic blue to reveal neurons. A, Wild-type mouse. B, DBH-NT-3 mouse. Scale bars, 25 µm. C, Neurons were counted in cuprolinic blue-stained preparations of the myenteric and submucosal plexuses. There are significantly more myenteric neurons per ganglion in the DBH-NT-3 mice (p < 0.001); however, the numbers of submucosal neurons ganglion are not significantly different in the two types of animal.


                              
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  		Table 2.   Sizes of neurons in the myenteric plexus of wild-type and DBH-NT-3 mice

The ENS was also examined immunocytochemically in adult transgenic mice in which the DBH promoter drives expression of bacterial beta -galactosidase (LacZ) (Kapur et al., 1992; Rice et al., 2000). The transgene was found to be expressed in neurons of both plexuses (myenteric > submucosal); however, the late-appearing calcitonin gene-related peptide (CGRP)-immunoreactive neurons, which were predominantly submucosal, were never observed to express LacZ.

The knock-out of TrkC or NT-3 causes enteric neurons to be lost from both plexuses

Subsets of neurons in the P9 postnatal gut were found to display TrkC immunoreactivity. The numbers of these cells were counted in laminar preparations of bowel wall containing the submucosal or myenteric plexuses. In the TrkC +/+ small intestine (proximal region), there were 150 ± 12 myenteric and 297 ± 28 submucosal neurons/mm2. The corresponding numbers in TrkC +/- mice were 168 ± 19 myenteric and 206 ± 21 TrkC-immunoreactive submucosal neurons/mm2. Essentially no TrkC-immunoreactive neurons were observed in either plexus in TrkC -/- mice. These observations are consistent with the ideas that mature enteric neurons express TrkC in both plexuses and confirm that this expression is lacking in TrkC -/- animals (Klein et al., 1994; Tessarollo et al., 1997). These data are also consistent with the observations (described above) that subsets of neurons in both plexuses are capable of the retrograde transport of NT-3.

Enteric neurons were counted in cuprolinic blue-stained laminar preparations of the small intestines of TrkC (-/-, +/-, and +/+) and NT-3 (-/-, +/-, and +/+) mice (Fig. 9). The numbers of neurons were significantly decreased in both the myenteric and submucosal plexuses in both the TrkC -/- and the NT-3 -/- animals. In contrast, the numbers of neurons were not significantly decreased in either the myenteric or submucosal plexuses of the heterozygous TrkC +/- or NT-3 +/- mice. In neither the TrkC -/- nor the NT-3 -/- animals was there a total loss of neurons in any region of the small intestine or a disruption of the normal pattern of organization of the plexuses. The most severe decrease in neuron numbers was in the submucosal plexus of the NT-3 -/- mice in which the numbers were approximately one-third of normal. When the myenteric plexus of the small intestine was examined regionally (Table 3), the deficits in neuronal numbers were found to be more severe proximally than distally. In fact, in the myenteric plexus of the proximal small intestine, the number of neurons was significantly lower than wild type, even in TrkC +/- mice. Surprisingly, in neither the TrkC -/- nor the NT-3 -/- mice was there a significant difference from wild-type in the number of neurons in the myenteric plexus of the most distal portion of the ileum. In the submucosal plexus of wild-type mice, the number of neurons in the middle of the small intestine was larger than in the more proximal bowel of the same animals (Table 4). In this region of the bowel, the number of submucosal neurons in the TrkC +/- mice was found to be intermediate between that of TrkC +/+ and TrkC -/- animals, but both here and in the proximal small intestine the number of neurons in the submucosal plexus was significantly lower than that found in the corresponding region of the small intestine of TrkC +/+ mice.



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  		Figure 9.   The number of neurons in both the myenteric and submucosal plexuses of the small intestine is lower in TrkC -/- and NT-3 -/- mice than in matched +/+ or +/- animals. Neurons were counted in laminar preparations stained with cuprolinic blue. In wild-type mice, the mean neuronal density was 1083 ± 60 neurons/mm2 (n = 26) in the myenteric and 733 ± 57 neurons/mm2 in the submucosal plexus (n = 13). A, Myenteric plexus of TrkC +/+ (n = 11), +/- (n = 12), and -/- (n = 10) mice. The number of neurons in -/- animals is significantly less than that of either +/+ (p < 0.01) or +/- mice (p < 0.05). B, Submucosal plexus of TrkC +/+ (n = 8), +/- (n = 8), and -/- (n = 8) mice. The number of neurons in -/- animals is significantly less than that of either +/+ (p < 0.0005) or +/- mice (p < 0.05). (The data shown in A and B were obtained from mice examined at age P9.) C, Myenteric plexus of NT-3 +/+ (n = 15), +/- (n = 15), and -/- (n = 15) mice. The number of neurons in -/- animals is significantly less than that of either +/+ (p < 0.002) or +/- mice (p < 0.001). D, Submucosal plexus of NT-3 +/+, +/-, and -/- mice. The number of neurons in -/- animals is significantly less than that of either +/+ (p < 0.0001) or +/- mice (p < 0.0001). (The data shown in C and D were obtained from mice examined at age P11.)


                              
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  		Table 3.   Regional distribution of myenteric neurons (per mm2) in the small intestines of wild-type mice and animals with targeted deletions of genes encoding TrkC or NT-3


                              
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  		Table 4.   Regional distribution of submucosal neurons (per mm2) in the small intestines of wild-type mice and animals with targeted deletions of genes encoding TrkC

Because the total number of neurons appears to be decreased in the guts of both TrkC -/- and NT-3 -/- mice, we investigated neurotransmitter-defined phenotypes of enteric neurons to determine whether these deficits could be accounted for by the absence of a particular subset of enteric neuron. Previous studies have established that multiple lineages of crest-derived precursor contribute to the formation of the ENS (Pham et al., 1991; Blaugrund et al., 1996). One of these depends on expression of the mash-1 gene, is transiently catecholaminergic (TC), and is born first during development. The other, which neither expresses nor depends on mash-1, is not TC and is born late in ontogeny (beginning after the mash-1-dependent cells are postmitotic). Studies were performed with 5-HT and CGRP, which are markers, respectively, for early-born mash-1-dependent and late-born mash-1-independent lineages of neurons. Neurons continue to be born in the mouse gut for the first 3 weeks of postnatal life (Pham et al., 1991). 5-HT and CGRP immunoreactivities were demonstrated in the bowel of control and mice lacking TrkC at P11 and P22. No significant differences were found in the density of 5-HT-immunoreactive neurons (Fig. 10A), which are exclusively myenteric (Sang and Young, 1996); however, the density of CGRP-immunoreactive cells, which are predominantly submucosal (Bornstein and Furness, 1988; Pataky et al., 1990), was significantly reduced in the TrkC -/- animals (Fig. 10B). Interestingly, this difference in the density of the late-arising CGRP-immunoreactive neurons was not apparent until P22, which follows a period of substantial addition of these cells to the ENS. The body weights of the TrkC -/- mice themselves (Fig. 10C) and the serosal area of their intestines (Fig. 10D) were each very much less than those of either TrkC +/+ or TrkC +/- animals. As a result, the absolute number of a given type of neuron in TrkC -/- mice is less than that found in control animals, even when the packing density of that type of neuron is the same in both animals. For example, the absolute number of 5-HT-immunoreactive neurons in the small intestine of P11 TrkC -/- mice (794 ± 34) is actually significantly lower that that in control mice (1826 ± 77), although the packing density is the same in both types of animal. The same is true of CGRP-immunoreactive neurons at P11. Although the packing density of these cells did not appear to be reduced compared with control, the absolute number of CGRP-immunoreactive neurons was lower in the smaller TrkC -/- bowel (7543 ± 442) than in controls (12,613 ± 438).



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  		Figure 10.   The packing density of 5-HT-immunoreactive neurons is similar in TrkC -/- and control mice, but that of CGRP-immunoreactive neurons is decreased in the TrkC -/- animals. A, Packing density of 5-HT-immunoractive neurons in the proximal small intestine at P11 and P22. The packing density does not increase significantly between P11 and P22 in the control bowel, and no significant differences are seen between TrkC -/- and control mice at any age. (Control mice include the pooled values of TrkC +/+ and +/- animals, which did not differ from one another). Values were determined by counting neurons in 80-320 fields of measurement (1 field is equivalent to 1.254 mm2). Note that all 5-HT-immunoreactive neurons are myenteric. B, Packing density of CGRP-immunoreactive neurons in the proximal small intestine at P11 and P22. The packing density increases substantially in the submucosal plexus between P11 and P22 in both the control and TrkC -/- bowel. The density of CGRP-immunoreactive cells in the submucosal plexus of TrkC -/- is significantly less than that of control mice at P22. Values were determined by counting neurons in 40-180 fields of measurement (1 field is equivalent to 1.254 mm2). Note that CGRP-immunoreactive neurons are predominantly submucosal.

A number of additional neurotransmitter-modulator-related markers were investigated in NT-3 -/- and TrkC -/- mice. Markers were demonstrated immunocytochemically as described above, and neurons were counted in submucosal and myenteric plexuses and compared with similar counts of analogous regions of the bowel of wild-type mice. These markers included tyrosine hydroxylase (TH), dopamine transporter (DAT), nitric oxide synthase (NOS), GABA, choline acetyltransferase (ChAT), and substance P. The numbers of neurons per square millimeter of each of these phenotypes was found to be variable between animals, and none differed significantly from control (data not illustrated). Unfortunately, these markers are costored in a variety of combinations in different neurons, so that, in contrast to 5-HT, none mark a single neuronal phenotype (Sang and Young, 1996). Again, however, the absolute number of all of the neurons in the gut displaying each of these markers was probably reduced in the NT-3- and TrkC-deficient mice because the bowel of the knock-out animals was severely diminished in size.

Responses to NT-3 and CNTF are interactive

The data (described above) demonstrate that subsets of enteric neurons that develop in response to NT-3 become NT-3 dependent; nevertheless, the defects found in the ENS of mice lacking NT-3 or TrkC are partial and regional. These observations suggest that other factors might be able to rescue cells that might otherwise fail to develop or die if they were deprived of NT-3. A factor that activates the tripartite neuropoietic cytokine receptor might