Brain Research 884 2000 206–212 www.elsevier.com locate bres
Interactive report
Neurotrophins alter the numbers of neurotransmitter-ir mature
1
vagal glossopharyngeal visceral afferent neurons in vitro
Cinda J. Helke , Dominik Verdier-Pinard
Department of Pharmacology and Neuroscience Program , Uniformed Services University of the Health Science, 4301 Jones Bridge Road, Bethesda,
MD 20814-4799, USA
Accepted 27 September 2000
Abstract
Mature nodose and petrosal ganglia neurons placodally derived afferent neurons of the vagal and glossopharyngeal nerves contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor NGF, neurotrophin-3 NT-3, neurotrophin-4 NT-4]. This
study evaluated neurotrophin influences on the presence of neuropeptides and or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 10–100 ng ml were applied 5 days to dissociated, enriched, cultures of mature nodose petrosal
ganglia neurons, and the neurons processed for tyrosine hydroxylase TH, vasoactive intestinal peptide VIP, calcitonin gene-related peptide CGRP and neurofilament NF-200 immunocytochemistry. Addition of NGF to nodose petrosal ganglia neuron-enriched
cultures significantly increased the number of TH-immunoreactive ir neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH
and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no
significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data
demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF
and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.
2000 Elsevier Science B.V. All rights reserved.
Theme : Sensory systems
Topic : Somatic and visceral afferents
Keywords : Nodose ganglion; Petrosal ganglion; Nerve growth factor; Neurotrophin-3; Neurotrophin-4; Vasoactive intestinal peptide; Tyrosine hydroxylase
1. Introduction rons, NGF regulates the expression of substance P and
calcitonin gene-related peptide CGRP immunoreactive An established action of neurotrophins [nerve growth
ir neurons in vitro and in vivo [2,16]. The presence of a factor NGF, brain-derived neurotrophic factor BDNF,
target-derived factor other than NGF has been proposed to neurotrophin-3 NT-3 and neurotrophin-4 NT-4] is the
induce the gene expression of VIP and galanin in DRG maintenance of normal neurotransmitter and neuropeptide
[10,24]. In sympathetic neurons, NGF effects the expres- phenotype expression in mature neurons [12,13,18,30]. In
sion of TH [19,28]. mature somatic dorsal root ganglion DRG sensory neu-
In contrast, little is known about the responsiveness to and functions of neurotrophins in other types of adult
sensory neurons such as the visceral afferent neurons of
1
Published on the World Wide Web on 23 October 2000.
the nodose and petrosal ganglia. These neurons are critical
Corresponding author. Tel.: 11-301-2953-238; fax: 11-301-2953-
to the maintenance of cardiovascular, respiratory and
220. E-mail address
: chelkeusuhs.mil C.J. Helke.
gastrointestinal functions. The placode-derived visceral
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 8 8 - 7
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
