C .J. Helke, D. Verdier-Pinard Brain Research 884 2000 206 –212
207
afferent neurons of the nodose and petrosal ganglia trans- petrosal ganglia from 15 rats were pooled in 2 ml of F14
mit visceral sensory information from specialized sensory growth medium supplemented with 10 heat-inactivated
endings of the vagus nerve to the nucleus tractus solitarius horse serum F-14 complete medium, F-14CM, freed of
[1,7,31]. Differences in embryologic origin, transmitter nerve trunks and capsular connective tissue, treated twice
contents, and presence of specific neurotrophin receptors for 1.5 h at 378C with 0.25 collagenase, and treated for
[36] predict distinct differences in the neurotrophin respon- 45 min with 0.25 trypsin in F-14. Following trituration
siveness of mature vagal afferent and efferent neurons by 8–10 passages through the barrel of fire-polished
compared to somatic sensory neurons or sympathetic pasteur pipet, a single-cell suspension was obtained, and
neurons. phase-bright neurons 15–50 mm diameter were counted
Neurons of the nodose and petrosal ganglia do show with a hemocytometer. Centrifugation and resuspension of
plasticity in the phenotype of neurotransmitters and neuro- the neuronal pellet removed cellular debris.
peptides in response to injury or environmental change Neuronal viability was tested with Trypan blue exclu-
[6,8,26,38]. The ability of inhibition of the axoplasmic sion. Neuronal enrichment was achieved by a differential
transport in the vagus nerve, to alter the numbers of VIP-, adhesion procedure wherein the cell suspension was pre-
TH- and CGRP-ir and mRNA containing neurons in the plated overnight in a 35 mm polyornithine 500 mg ml-
nodose ganglion of the adult rat [37] suggests that axonal coated culture dish. After 12–14 h, the non-neuronal cells
transport of a target-derived regulatory influence, such as a were firmly attached to the culture dish, the weakly
neurotrophin, alters the expression of these neurochemi- attached neurons were gently dislodged with media. Fur-
cals. ther neuronal enrichment was achieved by centrifugation,
Specific neurotrophin receptors p75, TrkA, TrkC are the pellet of viable neurons was resuspended in 0.8–1 ml
present on mature nodose and petrosal ganglia neurons F14CM. Cell counts and viability testing with Trypan blue
[4,34,35]. Moreover, NGF, NT-3 and NT-4 but not were done. Enriched neurons from 30 pairs of ganglia 15
BDNF are transported retrogradely by mature afferent rats were seeded 1000 neurons well in 600 ml in
vagal neurons to the nodose ganglion [4]. These data and F14CM in four-well |20 wells experiment Nunc Lab-
the competition profiles for the transport of NT-3 and NGF Tek chamber slides Nalge Nunc, Rochester, NY, USA
[4] are consistent with the presence of TrkA and TrkC and double-coated with 500 mg ml polyornithine and 5 mg ml
the absence of TrkB in the nodose ganglion. However, laminin, and incubated with 800 ml of Ham’s F-14CM
little is known about neurotrophic factors and the regula- maintained at 378C in 3.5 CO –96.5 air. Media was
2
tion of expression of neurochemicals in mature placode- changed at 2–3 day intervals.
derived visceral sensory neurons of the nodose ganglion. Human recombinant NGF and NT-4 were supplied by
To determine the effects of neurotrophins on TH, VIP Regeneron Tarrytown, NY, USA. Human recombinant
and CGRP in visceral afferent neurons, human recombi- NT-3 was provided by Amgen Thousand Oaks, CA,
nant NGF, NT-3 and NT-4 were applied to dissociated, USA. Neurotrophins 10, 50 and 100 ng ml were added
enriched, cultures of mature nodose and petrosal ganglia on the day of plating and for the entire time in culture 5
neurons. The numbers of TH-, VIP- and CGRP-immuno- days. In some experiments, neutralizing antibodies to
labeled cells were assessed after 5 days in culture in the NGF monoclonal antibody to mouse-NGF, catalog No.
presence of three doses of each neurotrophin. Neurofila- 1087 754, used at 0.5 mg ml, Boehringer Mannheim or
ment 200-ir labeling was used to label sensory neurons [3] NT-3 chicken polyclonal IgY to human recombinant NT-
and assess the survival of neurons after 5 days in culture 3, catalog No. G1651, used at 2 mg ml; Promega,
with and without exogenous neurotrophins. Madison, WI, USA were added to the wells on the day of
plating and for the entire time in culture 5 days. Cultured nodose and petrosal ganglia cells were fixed on
2. Materials and methods the Lab-Tek chamber slides with 4 paraformaldehyde
pH 7.4; 800 ml well for 20 min at room temperature, Adult male Sprague–Dawley rats weighing 225–250 g
rinsed with phosphate buffered saline pH 7.4, incubated were obtained from Taconic Farms Germantown, NY,
60 min at room temperature with blocking serum [0.3 USA, and given food and water ad libitum on a 12 h
Triton X-100 and 5 normal goat serum in phosphate- light–12 h dark cycle. Rats were anesthetized with
buffered saline PBS]. After rinsing, primary antibodies halothane 2 in oxygen and decapitated. The bilateral
diluted in 0.3 Triton X-100 in PBS were added for nodose and petrosal ganglia were rapidly and aseptically
overnight incubation at room temperature. The primary dissected and separated from the jugular ganglion. Because
antibodies used were: rabbit polyclonal anti-TH 1:1000; separate preparations of nodose ganglia and petrosal
Eugene Tech, Avendale, NJ, USA; rabbit polyclonal anti- ganglia yielded too few neurons, the nodose and petrosal
VIP serum 1:1000, Incstar, Stillwater, MN, USA; rabbit ganglia both placode-derived visceral afferent ganglia
polyclonal anti-CGRP 1:1000, Cambridge Research Bio- were pooled for these experiments. For each preparation of
chemicals, Atlantic Beach, NJ, USA, polyclonal rabbit cultured adult ganglia neurons, 30 pairs of nodose and
anti-NPY antiserum 1:1000 Peninsula Labs., Belmont,
208 C
CA, USA; rabbit anti-neurofilament 200 NF-200 IgG control using one-way analysis of variance ANOVA and
1:500, Sigma, St. Louis, MO, USA. The antisera were multiple comparison test.
previously tested and characterized for specificity [3,5,6,9]. Immunolabeling was visualized using the appropriate
secondary antibodies, avidin–biotin–peroxidase reagent 3. Results
VectaStain Elite ABC kit, and the chromagen, Vip Vector Labs.. After chambers and gaskets were removed
Preliminary studies showed that dissociated neuron-en- from the slides, the slides were rinsed, dried, and cover
riched cultures of mature rat nodose and petrosal ganglia slipped with DPX mountant Fluka, New York, NY, USA,
were viable Fig. 1. Survival of neurons was apparent for and viewed with a Leitz Diaplan microscope equipped for
at least 7 days in culture, although by 7 days in the absence photomicroscopy and digital image capture.
of anti-mitotic agents there was an increased number of The average numbers and percentages of immuno-
non-neuronal cells thus neurons were routinely harvested reactive neurons labeled for each agent were counted for
and processed for immunocytochemistry after 5 days in each sample well. The total number of immunolabeled
culture. TH-, VIP-, CGRP- and NF-200-ir were present and neurons with visible nuclei per well were counted. The
readily detectable in nodose petrosal ganglia neurons after percentage of immunolabeled neurons number of im-
5 days in culture Fig. 1. munoreactive neurons with visible nuclei divided by the
total number of neurons with visible nuclei3100 in 6–8 3.1. NGF
randomly selected fields of view within each culture well were determined and averaged to obtain a single value for
Each concentration of NGF 10, 50 and 100 ng ml that well. Data are presented as means6standard error of
applied to nodose petrosal ganglia neuron-enriched cul- the mean S.E.M.. Each value presented is averaged from
tures and present throughout the 5 days in culture, similar- a minimum of three culture chamber wells from at least
ly elevated the numbers and percentages of TH-ir neurons two distinct preparations of cultured adult ganglia neurons.
Fig. 2A. In the presence of 100 ng ml NGF, the numbers Statistical significance was set at P,0.05 compared to
219624 versus 303628 TH-ir neurons |1000 neurons
Fig. 1. Photomicrographs of immunolabeled dissociated neuron-enriched cultures of nodose petrosal ganglia after 5 days in culture. A TH-ir neurons, B VIP-ir neurons, C CGRP-ir neurons, D NF-200-ir neurons. The calibration bar represents 50 mm.
C .J. Helke, D. Verdier-Pinard Brain Research 884 2000 206 –212
209
plated, control and NGF, respectively and the percentage 19 versus 27 TH-ir neurons as of neurons in
culture well, control and NGF, respectively of cultured neurons that were immunolabeled for TH-ir increased
more than a third. The addition of an NGF neutralizing antibody 0.5 mg ml completely attenuated the effects of
100 ng ml NGF on TH-ir Fig. 2A.
In the presence of 100 ng ml NGF, a reduction in the numbers 265622 versus 182617 VIP-ir neurons |1000
neurons plated, control and NGF, respectively and per- centage 24 versus 16 VIP-ir neurons in culture well,
control and NGF, respectively of VIP-ir nodose petrosal ganglia neurons was noted Fig. 2A. A smaller but
significant decline in VIP-ir neurons was noted with 10 ng ml but not 50 ng ml NGF. The addition of an NGF
neutralizing antibody 0.5 mg ml partially attenuated the effects of 100 ng ml NGF on VIP-ir neurons Fig. 2A.
The number and percentages of CGRP-ir neurons were unchanged by any concentration of NGF Fig. 2A. The
number of NF-200-ir nodose petrosal ganglia neurons did not significantly change in the presence of any dose of
NGF over 5 days in culture data for 100 ng ml shown in Fig. 4.
3.2. NT-3 Human recombinant NT-3 10, 50, 100 ng ml did not
alter the numbers or percentages of nodose petrosal gang- lion neurons immunolabeled for TH, or CGRP after 5 days
in culture Fig. 2B. Nor did the addition of an NT-3
Fig. 2. Bar graphs showing the percent of nodose petrosal ganglia
neutralizing antibody affect the numbers of TH-ir neurons
neurons immunoreactive for TH, VIP and CGRP after 5 days in cultures
Fig. 2B.
treated with A NGF at 0, 50, 100 ng ml or 100 ng ml in the presence of
Each dose of NT-3 10, 50 100 ng ml significantly
an NGF neutralizing antibody 1AB, B NT-3 at 0, 10, 50, 100 ng ml or 100 ng ml in the presence of an NT-3 neutralizing antibody 1AB.
increased the numbers and percentages of VIP-ir nodose
Neurotrophins and neutralizing antibodies were present from the time of
petrosal ganglia neurons in the cultures at 5 days. The 100
plating, throughout the 5-day culture period. a5P,0.05 compared to
ng ml concentration of NT-3, increased the numbers of
control, b5P,0.05 compared to corresponding 100 ng ml data.
VIP-ir neurons from 244618 to 347627 neurons |1000 neurons plated, control and NT-3 treated, respectively and
the percentage of VIP-ir neurons from 19 to 26 of total neurons in culture well, control and NT-3 treated,
respectively Fig. 2B. The addition of an NT-3 neutraliz- ing antibody 2 mg ml completely attenuated the effects
of 100 ng ml NT-3 on VIP-ir neurons Fig. 2B. The number and percentages of CGRP-ir neurons Fig. 2B or
NF-200-ir neurons Fig. 4 did not change in the presence of any dose of NT-3 over 5 days in culture.
3.3. NT-4 Addition of human recombinant NT-4 10, 50 100
ng ml to cultures of nodose petrosal ganglia neurons throughout the 5-day culture time, had no significant
Fig. 3. Bar graphs showing the percent of nodose petrosal ganglia
effects on the numbers or percentages of TH, VIP, CGRP
neurons immunoreactive for TH, VIP and CGRP after 5 days in cultures treated with NT-4 at 0, 10, 50, 100 ng ml.
or NF-200-ir neurons Figs. 3 and 4.
210 C
decrease in TH and an increase in VIP neurons [6,37,38], the current data are consistent with a role for endogenous
NGF in vivo to maintain normal neurotransmitter pheno- type in nodose petrosal ganglia neurons. These data,
coupled with the presence of TrkA mRNA and of the
125
retrograde transport of I-NGF by vagal afferent neurons,
indicate an important role for NGF in the functions including maintenance of normal transmitter phenotype
of these visceral afferent neurons. Moreover, these data coupled with the well established insensitivity of develop-
ing nodose ganglion neurons to the survival promoting actions of NGF at a time when the neurons do require
trophic support [14], suggest a developmental change in the responsiveness of these neurons. Our data also demon-
strate differences in the response between mature placode-
Fig. 4. Bar graphs showing the numbers of NF-200-ir neurons per 1000
derived visceral afferent and neural crest-derived DRG
cells plated in each well in dissociated, enriched 5-day cultures of
neurons. In distinction to the NGF-induced alterations in
nodose petrosal ganglia in the absence of neurotrophin control, and the
TH- and VIP-ir in nodose petrosal ganglia neurons; in
presence of 100 ng ml of NGF, NT-3 or NT-4.
adult DRG neurons in culture, NGF 50 ng ml increases SP- and CGRP-ir but does not alter VIP-ir in culture
mature DRG neurons do not have TH [17,23]. Verge et al. [33] showed however that NGF infusion reduced the
4. Discussion numbers of VIP-ir neurons in the DRG after injury. In
