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. Author manuscript; available in PMC: 2013 Feb 5.
Published in final edited form as: J Neurobiol. 2005 Aug;64(2):145–156. doi: 10.1002/neu.20134

Differential Trk Expression in Explant and Dissociated Trigeminal Ganglion Cell Cultures

Bariş Genç 1, Emel Ulupinar 1, Reha S Erzurumlu 1,*
PMCID: PMC3564663  NIHMSID: NIHMS428534  PMID: 15828064

Abstract

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.

Keywords: TrkA, TrkB, TrkC, explant cultures, dissociated cell cultures

INTRODUCTION

Trigeminal (TG) and dorsal root (DRG) ganglia are composed of a heterogeneous population of neurons expressing a variety of transmembrane receptors and molecular markers (Mu et al., 1993; Ichikawa et al., 1994; Wright and Snider, 1995). Specific classes of sensory neurons in these ganglia depend on different target-derived neurotrophins for survival (Davies, 1994; Snider, 1994; Fariñas et al., 1996; Snider and Silos-Santiago, 1996; Liebl et al., 1997; Chao, 2003). Small diameter neurons express TrkA, and depend on NGF, whereas large diameter, TrkC expressing neurons depend on NT-3 for survival (Mu et al., 1993). Survival promoting effects of neurotrophins have been most extensively studied using dissociated TG or DRG cell culture assays, and by quantitative analyses of neuronal populations in sensory ganglia of mice with targeted mutations of different neurotrophin or Trk genes (Barde et al., 1980; Davies et al., 1986; Buchman and Davies, 1993; Buj-Bello et al., 1994; Crowley et al., 1994; Ernfors et al., 1994a,b; Fariñas et al., 1994; Jones et al., 1994; Snider, 1994; Tessarollo et al., 1994; Davies et al., 1995; Piñón et al., 1996; Snider and Silos-Santiago, 1996; White et al., 1996; Liebl et al., 1997). Along with their survival promoting effects, NGF and NT-3 signaling lead to differential axonal morphogenesis of embryonic rodent TG and DRG neurons (Lentz et al., 1999; Ulupınar et al., 2000; Özdinler et al., 2004). In dissociated cell cultures, or wholemount explant cultures of TG with intact connections to the brainstem, NGF induces axonal growth in the form of elongation, while NT-3 promotes precocious arborization of trigeminal axons (Ulupınar et al., 2000). When applied locally to the developing trigeminal tract, NGF induces defasciculation and diversion of trigeminal axons from the tract, whereas NT-3 induces interstitial branching and formation of neuritic tangles around the beads (Özdinler et al., 2004). In order to better understand survival promoting and axonal morphogenetic effects of neurotrophins signaling through different Trks, it is important to chart out Trk expression in sensory neurons subjected to different culture conditions and how they compare to in vivo Trk expression at developmentally equivalent stages. In the present study, we used combinations of single and double TrkA/TrkB/TrkC immunostaining in explant and dissociated E15 TG cultures grown in serum-free medium with or without neurotrophin supplement. We also took advantage of Bax knockout (KO) mice that do not require neurotrophins for sensory neuron survival (White et al., 1998; Lentz et al., 1999), and repeated our dissociated cell cultures with Bax null TG. We obtained strikingly different results from explant versus dissociated cell cultures of the TG. In explant cultures, different classes of TG cells express specific Trk receptors similar to their in vivo counterparts. In contrast, under all conditions tested, dissociation of TG cells induced rapid co-expression of all Trk receptors. Dissociated cells switched from one neurotrophin to another displayed characteristics of neurons continuously exposed to a mixture of both neurotrophins. These observations caution about the interpretation of survival and axonal effects of NGF family of neurotrophins using dissociated cultures of primary sensory ganglia.

METHODS

Animals

Timed pregnant Sprague-Dawley rats (Taconic Farms, NY) were anaesthetized, and embryos were removed by caesarian section at E15 (day of sperm positivity was designated as E0). Three embryos were fixed in 2% paraformaldehyde and sections through the heads were processed for immunohistochemistry to visualize Trk receptor expression patterns at E15 in rat TG in vivo. Bax KO mouse embryos were obtained from crosses of Bax KO females to Bax heterozygote males (Jackson Laboratories, Bar Harbor, ME), and embryos were removed by caesarian section at E13. TGs were used to set up dissociated cultures from individual embryos, and tail tissue was used to extract DNA for genotyping of each embryo. Genotyping was performed by PCR using the primers R661: GTT GAC CAG AGT GGC GTA GG, R662: CCG CTT CCA TTG CTC AGC GG, R663: GAG CTG ATC AGA ACC ATC ATG specific for the Bax locus. Only cultures from Bax null TGs were recorded for results. All protocols used in this study were approved by the LSUHSC Institutional Animal Care and Use Committee (IACUC) and conformed to the NIH guidelines for use of experimental animals.

Explant Cultures

Wholemount explant cultures were prepared as described previously (Ulupınar et al., 2000). Briefly, TG with its intact central connections to the brainstem were dissected out as an open-book preparation, and placed on Millicell membrane inserts (Millipore, Bedford, MA) with the ventral side down. Culture inserts were placed in six-well plates containing serum-free medium at the bottom of the wells, supplemented with or without 50 ng/ml NGF (Regeneron, Tarrytown, NY; Chemicon, Temecula, CA), NT-3 (Regeneron; Chemicon) or BDNF (Regeneron) (three samples each; cultures were repeated three times). In some cultures, a mixture of NGF and NT-3 was applied together (50 ng/ml each). Cultures were grown for 3 days, fixed in 2% paraformaldehyde, and sectioned at 10 μm in a cryostat for immunohistochemistry. In one set of experiments, the central connections to the brainstem were severed with a scalpel just prior to starting the cultures, isolating the TG from the brainstem.

Dissociated Cell Cultures

E15 rat TG were dissected out, washed in calcium magnesium free Hank’s balanced salt solution (Invitrogen, Carlsbad, CA), digested with 0.05% trypsin (Sigma, St. Louis, MO) at 37°C for 30 min, washed in SFM supplemented with 10% fetal calf serum, and rinsed in SFM several times before triturating using fire-polished glass pipettes. Dissociated cells were plated on polyornithine (Sigma)-laminin (Invitrogen) coated glass coverslips in six-well plates at a concentration of 1000 cells/well. Culture medium was supplemented with 50 ng/ml NGF, NT-3, or both. Control cultures were set without any neurotrophin supplement for incubation times shorter than 1 day. Cultures were removed from the incubator at 2 h, 6 h, 24 h, and 3 days after plating, and fixed in 2% paraformaldehyde. In another set of experiments, E13 TG from Bax KO mice were used to set up dissociated cultures grown without any neurotrophins in the media for 3 days. At the end of the culture period, cells were fixed in 2% paraformaldehyde, and processed for immunohistochemistry. For each condition, three sets of cultures were set up at different times.

Neurotrophin Switch

TG neurons from E15 embryos were dissociated and plated as described above. For the first 24 h of the culture period, cells were kept in the presence of 10 ng/ml NGF or NT-3, after which they were gently washed three times with SFM (20 min each) and switched to 50 ng/ml of the other neurotrophin for the rest of the 3-day culture period. One group of cells were initially cultured with NGF and switched to NT-3, while another group was cultured in NT-3 first, followed by NGF. For one set of experiments, cells were traced by camera lucida drawing before the switch, and the same cells were located at the end of the culture period and retraced. For another set of experiments, cells were fixed at the end of 1 day or 3 days in culture and used for quantitative analysis. Soma size, primary neurite length, and number of branches from the primary neurite were measured and statistically analyzed as described previously (Ulupınar et al., 2000).

Immunohistochemistry

For TrkA/TrkC double immunohistochemistry, samples were blocked in 10% normal donkey serum, and incubated overnight in a cocktail of rabbit anti-TrkA and goat anti-TrkC antibodies (gift of L. Reichardt). On the next day, samples were washed extensively, and a cocktail of fluorescently labeled secondary antibodies (CY3-conjugated donkey anti-rabbit, and FITC-labeled donkey anti-goat (both from Chemicon) was applied. Dissociated cultures were counterstained with nuclear dye DAPI (Sigma). Control experiments were performed by single labeling of antigens through omission of one or both of the primary antibodies in double labeling experiments. Same regions from samples were photographed under a Nikon epifluorescence microscope using rhodamine, FITC, and UV filters with a Cool-Snap camera. Images were adjusted for brightness and contrast, and rhodamine and FITC filter images were merged when appropriate using the Adobe Photoshop program; no other alterations were made. For TrkB immunohistochemistry, samples were blocked in 10% normal goat serum, and incubated overnight in a rabbit anti-TrkB antibody (Santa Cruz Biotech, Santa Cruz, CA) solution. On the next day, samples were treated with CY3 conjugated goat anti-rabbit antibody (Chemicon), and photographed under a Nikon epifluorescence microscope with a rhodamine filter set using the CoolSnap camera. TrkA/TrkB and TrkB/TrkC double immunohistochemistry was performed as described above, using a cocktail of rabbit anti-TrkA (gift of L. Reichardt) and goat anti-TrkB (Santa Cruz Bio-tech) for TrkA/TrkB, and a cocktail of rabbit anti-TrkB (Santa Cruz Biotech) and goat anti-TrkC (gift of L. Reichardt) for TrkB/TrkC immunohistochemistry. Both TrkB antibodies were raised using the same epitope, and label the full-length (kinase +) isoform of the TrkB protein according to the manufacturers description.

RESULTS

In the rat, TG neurons are born between E9.5–14 (Forbes and Welt, 1981; Rhoades et al., 1991). Peripheral and central processes begin invading their respective targets by E12 (Erzurumlu and Killackey, 1983; Erzurumlu and Jhaveri, 1992). By E15, the central trigeminal tract is laid down by unbranched TG axons, while in the snout peripheral axons form follicular nerves branching around individual follicles (Erzurumlu and Killackey, 1983). Peripheral and central targets of primary sensory neurons express neurotrophic factors during target innervation (Arumäe et al., 1993; Vogel, 1993; Elkabes et al., 1994; Fariñas et al., 1996). Sensory neurons in turn express p75NTR and Trk receptors and compete for the limited supply of available neurotrophins for survival (Vogel, 1993; Davies, 1997; Miller and Kaplan, 2001). Various in vitro studies suggest that following a brief period of neurotrophin independence after birth, TG neurons become dependent on BDNF for survival and switch to NGF dependence in later stages (Buchman and Davies, 1993; Davies, 1994; Paul and Davies, 1995; Piñón et al., 1996; White et al., 1996; Davies, 1997; Enokido et al., 1999). Trk receptor expression and co-expression has been detected in early stages of mouse TG development, but by E13.5 (equivalent to developmental stage E15 in the rat), Trk receptor expression becomes restricted so that each neuron expresses only one Trk receptor (Fariñas et al., 1998; Huang et al., 1999).

Trk immunostaining in E15 rat embryos clearly delineates the TG and its central and peripheral projections [Fig. 1(A)]. High power images of the TG reveals that TrkA expression is restricted to small diameter cells, while TrkC is expressed by large diameter cells without any overlap [Fig. 1(B)], in agreement with a previous report for the mouse (Huang et al., 1999). Similarly, no overlap is detected in TrkA and TrkB expression. However, almost all TrkB immunopositive cells were also immunopositive for TrkC, but not all TrkC immunopositive cells were TrkB immunopositive [Fig. 1(B)].

Figure 1.

Figure 1

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Trk receptors are expressed by distinct subclasses of sensory neurons in E15 rat embryo. Immunolabeling for Trk receptors clearly delineates TG and its central and peripheral projections in coronal (A) sections of E15 rat embryo. High power images of TG (B) reveal that TrkA and TrkC, as well as TrkA and TrkB, are expressed by different subclasses of sensory neurons. In contrast, there is considerable overlap between TrkB and TrkC expression, such that almost all TrkB expressing neurons also express TrkC. Merged images show cells in the same field photographed with rhodamine and FITC filters consecutively, and yellow cells represent co-expression. Arrow points to a TrkC-immunopositive neuron that does not express TrkB. Scale bar = 1 mm (A), 30 μm (B).

Immunostaining in wholemount explant cultures revealed a similar pattern, with essentially no cells co-expressing TrkA and TrkC, irrespective of the neurotrophin treatment of the cultures (Fig. 2). TrkA immunopositive and TrkC immunopositive neurons were separate cell groups in cultures grown in the presence of NGF, NT-3, or BDNF. To rule out the possibility of selecting for specific cell subpopulations, we set up control cultures with no neurotrophins in the culture medium (Fig. 2), as well as cultures with a mixture of NGF and NT-3 [Fig. 3(A)]. Neither multiple neurotrophin treatments, nor lack of exogenous neurotrophins in the medium altered Trk expression patterns. Analysis of Trk receptor co-expression by double immunolabeling revealed a similar pattern to that seen in embryo sections, with considerable TrkB and TrkC co-localization, but no overlap with TrkA expression [Fig. 3(A)]. In whole-mount explant cultures, TG neurons still had their central connections to brainstem intact. To check for the effect axotomy might have on Trk receptor expression in sensory neurons, we have repeated explant cultures after severing the central axons of the TG just before they entered the brainstem. Axotomy did not change the TrkA/TrkC expression patterns, and even the residual pyknotic/apoptotic cell remnants detected by our immunolabeling protocol were labeled with a single Trk receptor antibody [Fig. 3(B)].

Figure 2.

Figure 2

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Trk expression patterns are not affected by various neurotrophin treatments in explant wholemount cultures of TG and brainstem. Explants were isolated at E15, and cultured for 3 days in SFM supplemented with 50 ng/ml neurotrophin where indicated. Each panel shows high power images of sections through TG immunolabeled with antibodies against Trk receptors. Merged panel shows TrkA and TrkC staining simultaneously. There is limited co-localization of TrkA and TrkC, as evidenced by lack of yellow cells in merged images. Presence of unlabeled cells suggests that TrkB expression in the TG is not global, but limited to a subpopulation. Various neurotrophin treatments or complete absence of neurotrophins in the culture media does not change the expression pattern. Scale bar = 30 μm.

Figure 3.

Figure 3

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Trk receptor expression and co-expression pattern does not change in explant cultures (A). In explant cultures of E15 rat TG kept in the presence of a mixture of NGF and NT-3, there is no overlap in TrkA/TrkB or TrkA/TrkC expression similar to in vivo conditions. TrkB/TrkC co-expression, also reflects the in vivo conditions, such that most TrkB cells are also immunopositive for TrkC as well (yellow cells in merged images), however, not all TrkC expressing cells co-express TrkB (arrow). Axotomy does not change the Trk expression pattern in explant cultures (B). The nerve between TG and brainstem was severed with a scalpel prior to starting the cultures. TrkA and TrkC expressing TG neurons mostly remained distinct at the end of the 3-day culture period similar to in vivo conditions or explant cultures with the intact nerve. Scale bars = 30 μm.

Results from the dissociated cultures were completely different from that observed in the explant cultures or in vivo conditions. As soon as 2 h after plating, all of the cells in our dissociated cultures were immunopositive for all three Trk receptors [Fig. 4(A)]. TrkA and TrkC receptor co-expression was detected simultaneously in the same cells. There were no cells labeled with only one Trk receptor but not the other. We have not detected any background labeling or cross-reactivity in controls where one or both of the primary antibodies were omitted (data not shown). TrkB labeling was also detected in all the cells in culture as evidenced by lack of DAPI-labeled nuclei without TrkB immunolabeling. Since all cultured cells were labeled by all three antibodies in separate experiments, all three Trk receptors must be expressed by all neurons in dissociated cultures. Several previous studies used dissociated TG cells grown in medium supplemented with a specific neurotrophin in order to select only those cells that required that particular neurotrophin for survival (Barde et al., 1980; Davies et al., 1986; Buchman and Davies, 1993; Buj-Bello et al., 1994). However, specific Trk expression patterns in these cultures were never assayed. We found that in dissociated TG cultures, multiple Trk expression did not change after extended periods in culture even with various neurotrophin supplies in the medium [Fig. 4(B–E)]. Differential size distribution of sensory neurons expressing specific Trk receptors was lost upon dissociation, such that the three Trk receptors were detected in all cells spanning various soma sizes from small to large diameters [Fig. 4(C–E)]. TG neurons die in dissociated cultures within 24 h unless supplemented with neurotrophins. Therefore, we have used control samples grown in SFM alone and fixed at 2–6 h post-plating, but not for 1 day or longer periods. Multiple Trk expression was still detected in complete absence of any neurotrophins up to 6 h in culture [Fig. 4(A, B)].

Figure 4.

Figure 4

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Dissociation leads to widespread expression of multiple Trk receptors by sensory neurons. All TG neurons begin to express all three Trk receptors as soon as 2 h after plating in dissociated cultures of E15 rat TG (A). TrkA and TrkC co-localization is detected in the same cells by simultaneous immunolabeling for both proteins. Immunostained cells were photographed under rhodamine and FITC filters, and DNA binding DAPI stain was observed under UV filter to visualize the nuclei of TG neurons. All cells were immunopositive for TrkA and TrkC, as all DAPI-labeled cells were immunolabeled. We have not detected any cells labeled with TrkA but not TrkC or vice versa. All cells in dissociated cultures were also immunopositive for TrkB. Multiple Trk expression remained unaffected by various neurotrophin treatments (50 ng/ml each) or complete absence of neurotrophins in the media for up to 6 h. Longer incubation periods did not change the multiple expression pattern (B–E). Multiple Trk expression in dissociated cultures of TG neurons is independent of soma size distribution. TrkA and TrkC proteins were co-localized in both small (C) and large diameter (D) TG neurons grown in dissociated cultures up to 3 days in the presence of various neurotrophins. TrkB expression was similarly detected in all cells spanning various soma sizes (E). Scale bars = 20 μm.

To circumvent the potential confounding variable that in our cultures addition of specific neurotrophins might lead to selective survival of subpopulations of TG neurons, we repeated our cultures with Bax null E13 mouse TG in which apoptosis due to neurotrophin withdrawal is eliminated (White et al., 1998, Lentz et al., 1999). In E13 Bax KO TG, TrkA and TrkC expression patterns were similar to WT E13 mice or E15 rat embryos, with no co-expression [Fig. 5(A)]. However, all dissociated Bax null neurons grown for 3 days without neurotrophins still expressed all the Trk receptors [Fig. 5(B,C)], further demonstrating that observed co-expression is not due to a selective survival of a subpopulation of sensory neurons with various neurotrophin treatments.

Figure 5.

Figure 5

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Multiple Trk expression in dissociated cultures is independent of survival effects of neurotrophins. TrkA and TrkC expression in Bax null E13 TG is nonoverlapping similar to WT mice (A) or E15 rat [see Fig. 1(B)]. However, upon dissociation they are co-localized in all TG neurons grown for 3 days in the absence of neurotrophins (B). TrkA and TrkC immunolabeling shows the same cell photographed under different filters following a double immunolabeling protocol. TrkB, similar to TrkA and TrkC is expressed by all Bax null TG neurons dissociated at E13 (C). Scale bars = 20 μm.

To further investigate the possibility of selective survival in dissociated cultures, we performed neurotrophin switch experiments where dissociated E15 rat TG neurons were first cultured in low concentrations of NGF or NT-3 for 24 h, after which they were switched to a higher concentration of the other neurotrophin. We have studied individual cells by tracing them before the neurotrophin switch, and identifying and tracing them again at the end of the 3-day culture period after the neurotrophin switch. Gross morphology of the cells resembled results of earlier findings from our lab (Ulupınar et al., 2000), where dissociated cells were cultured in a mixture of NGF and NT-3 for 3 days (Fig. 6). Detailed analysis of soma size, neurite length, and branch number show that cells switched from one neurotrophin to another resembled cells grown in a mixture of both neurotrophins for 3 days, and not those grown in either neurotrophin alone [Fig. 7(A–D)].

Figure 6.

Figure 6

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Camera lucida drawings of cells switched from one neurotrophin to another. Cells grown in 10 ng/ml of NGF (A) or NT-3 (B) for 24 h were traced before they were switched to 50 ng/ml concentration of the other neurotrophin. After switching from NGF to NT-3 (C), or NT-3 to NGF (D), same cells (marked by different numbers) were identified (denoted by asterisk), and retraced at the end of 3-day culture period. Note the differential axonal responses of the same cells when exposed to different neurotrophins and switched from one neurotrophin to the other. Scale bar = 100 μm.

Figure 7.

Figure 7

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Quantification of the results from neurotrophin switch experiments. Bar graphs show the soma size (A), primary neurite length (B), and the number of branches from the primary neurites (C) of cells grown in one neurotrophin for the first day, and switched to the other for the remainder of the 3-day culture period. At the end of 3 days, soma size (D), primary neurite length (E), and number of branches (F) from neurotrophin switch experiments is comparable to those of the cells grown in a mixture of both neurotrophins for 3 days, and differ from those of cells grown in either neurotrophin alone. Significance level is shown where statistics were applied: *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant. The values represent means ± S.E.M. of 200 neurons analyzed for each condition.

DISCUSSION

Co-expression of multiple Trk receptors in dissociated cultures has previously been suggested (Moshnyakov et al., 1996; Friedel et al., 1997; Cao and Shoichet, 2003). Moshnyakov et al. (1996) performed RT-PCR from single dissociated rat TG cells at E12 and E16, and studied differential gene expression. They detected single cells expressing one, two or three Trks with p75NTR, with the co-expression levels decreasing with age. However, they did not detect multiple Trk expression in all cells as in our experiments, which might have been missed due to limited sensitivity of their technique. Friedel et al. (1997) isolated RNA from explant and dissociated chick DRGs and investigated Trk expression by RT-PCR. In explant cultures, NGF treatment mainly selected for TrkA expressing cells, and NT-3 treatment selected for TrkC expressing cells. In dissociated cultures, they observed distinct survival rates and axonal morphology between NGF and NT-3 treatments, but they detected equal levels of TrkA and TrkC transcripts. While their data is in agreement with our results, their technique does not allow for detection of multiple transcripts in the same cells. Ulupınar et al. (2000) reported that dissociated small and large diameter cells all express parvalbumin, a calcium binding protein which is a marker for large diameter proprioceptive cells, suggesting dissociation changes not only Trk expression patterns, but also downstream markers as well. A detailed analysis of soma size distribution of Trk expressing TG neurons in explant cultures has been published (Ulupınar et al., 2004). Recently it has been suggested that expressing TrkC from TrkA locus is sufficient to shift sensory neurons from a nociceptive fate to a proprioceptive one (Moqrich et al., 2004). Earlier studies from our lab using dissociated and explant E15 rat TG neurons revealed an instructive role for neurotrophins in regulating axonal growth in addition to their permissive roles as survival factors (Ulupınar et al., 2000). While dissociated cells cultured in NGF had significantly longer neurites with fewer branches, cells cultured in NT-3 had shorter neurites and higher number of branches. Results of another study using Bax deficient DRGs (which do not require neurotrophins for survival) also confirm differential effects of NGF and NT-3 on axonal differentiation (Lentz et al., 1999). However, differences in survival rate and soma size distribution in the Ulupınar et al. (2000) study leads to the possibility that different neurotrophin treatments may select for cells with different intrinsic axonal growth parameters. The problem of selective survival was addressed by applying a mixture of both neurotrophins simultaneously, resulting in morphologies reflecting effects of both neurotrophins (Ulupınar et al., 2000). New data presented here on the morphometric analysis of dissociated E15 rat TG neurons switched from one neurotrophin to the other adds further support to the instructive roles of neurotrophins. Neurons were first cultured in low concentrations of one neurotrophin to intentionally “select for” cells depending on that neurotrophin, and they were switched to higher concentrations of the other neurotrophin for the rest of the culture period. If cells were selected for survival, no neurons would survive the neurotrophin switch, or if they had intrinsic programs for axonal growth, their morphology would reflect the first neurotrophin to which they were exposed. Dissociated cells show axonal characteristics of the first neurotrophin they were exposed to before the switch, however at the end of 3 days, not only they do survive, but also they respond to neurotrophin switch as if they were exposed to both neurotrophins simultaneously. These results provide a strong argument for presence of active Trk receptors for both neurotrophins on these cells, and also an active, instructive role for neurotrophins on axonal differentiation rather than just a passive, permissive role as survival factors.

Trk receptor expression is continuously and dynamically regulated in sensory ganglia during development (Phillips and Armanini, 1996; Fariñas, 1998; Huang et al., 1999; Rifkin et al., 2000). Although Trk co-expression is detected prior to target innervation, mouse TG neurons become restricted to only one Trk receptor after E13.5 (Huang et al., 1999). There are numerous reports of neurotrophin receptor co-expression in postnatal and adult rodents based on in situ hybridization experiments (McMahon et al., 1994; Kashiba et al., 1995; Wright and Snider, 1995; Phillips and Armanini, 1996; Jacobs and Miller, 1999; Karchewski et al., 1999). Although there are discrepancies in the exact number of cells expressing and co-expressing various Trk receptors, they all agree on the existence of a considerable amount of co-expression, as well as presence of cells that do not express any Trk receptors. Karchewski et al. (1999) points to the importance of multiple neurotrophin responsiveness, suggesting that one neurotrophin may modulate the response of a neuron to another neurotrophin, or the two may act to activate different signal transduction pathways not activated by either neurotrophin alone.

Although TrkA and TrkC antibodies used in our study were raised against the extracellular domains of the Trk receptors, TrkB antibodies used are reactive to the full-length kinase isoform of the TrkB. Considering there are no reports of presence of a truncated TrkA isoform, it is reasonable to assume that at least TrkA and TrkB immunohistochemistry represents functional full-length Trk receptors. Furthermore, our results from neurotrophin switch experiments following individual cells before and after switching from NGF to NT-3 or vice versa show that E15 TG cells survive in the presence of either neurotrophin, and respond to neurotrophin switch as if they were exposed to both neurotrophins at the same time. Presence of a uniform population of cells expressing all three Trk receptors and responding differentially to various neurotrophin treatments in dissociated cultures raises the possibility of activation of alternate signaling pathways in the same cell upon exposure to different neurotrophins. Numerous affector molecules binding Trk and p75NTR receptors and activating various cellular pathways in sensory neurons have been identified (Miller and Kaplan, 2001; Markus et al., 2002; Chao, 2003). It has recently been shown that axonal responses to neurotrophins are mediated via the Rac/Rho family of GTPases (Özdinler and Erzurumlu, 2001). Axons responding to local applications of neurotrophins using sepharose beads in explant cultures were immunopositive for Trk receptors (Özdinler et al., 2004), and TrkA and TrkB proteins have been co-localized in growth cones and distal-most portions of axons in dissociated chick DRG cultures (Tuttle and O’Leary, 1998), suggesting a possibility of Trk signaling regulating axon guidance through modification of Rac/Rho GTPases. Modulation of Rho activity by neurotrophins through the p75NTR receptor has been demonstrated in chick ciliary ganglion cultures (Yamashita et al., 1999). Growth cone turning in dissociated chick DRG axons was shown to be dependent on TrkA and p75NTR, as it was prevented by anti-TrkA antibodies and K552a, and reduced by anti- p75NTR antibody application (Gallo et al., 1997). The identity of “max factor” attracting sensory axons to the developing whisker pad has been revealed as a combination of BDNF and NT-3 (O’Connor and Tessier-Lavigne, 1999). Possibility of neurotrophins guiding neurite outgrowth by differential regulation of downstream signaling molecules has been suggested by others as well (Cao and Shoichet, 2003; Karchewski et al., 1999; Markus et al., 2002). Paves and Saarma (1997) used localized neurotrophin sources in dissociated cultures, demonstrating growth cone turning towards NGF and NT-3 sources, while BDNF treatment caused growth cone collapse. They raise the possibility of neurotrophins forming a meshwork of attractive/inhibitory cues in the periphery guiding sensory axons to their targets.

Our data clearly demonstrates that dissociation alters the Trk expression patterns in sensory ganglia such that they do not represent in vivo conditions anymore. Fariñas et al. (1998) previously raised the same point that loss of cell–cell interactions or another aspect of dissociated cultures leads to TrkC expression and NT-3 responsiveness such that in vitro results are not directly comparable to in vivo conditions. Previous literature investigating roles of neurotrophic factors in various aspects of sensory system development making use of the dissociated culture paradigm will need to be reconsidered in light of our recent findings presented here.

Acknowledgments

Contract grant sponsor: NIH/NIDCR; contract grant number: DE07734.

We thank Dr. L. Reichardt for the generous supply of TrkA and TrkC antibodies.

References

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