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The mechanisms and possible sites of acetylcholine release during chick primary
sensory neuron differentiation
V. Corsetti1, C. Mozzetta1, S. Biagioni, G. Augusti Tocco, A.M. Tata⁎
Dept. of Biology and Biotechnologies Charles Darwin, Research Center of Neurobiology, Daniel Bovet, “Sapienza” University of Rome, Italy
a b s t r a c ta r t i c l ei n f o
Received 21 February 2012
Accepted 13 August 2012
Vesicular acetylcholine transporter
Aims: In this study, we evaluated the ability of differentiating embryonic chick DRG neurons to release and
respond to acetylcholine (ACh). In particular, we investigated the neuronal soma and neurites as sites of
ACh release, as well as the mechanism(s) underlying this release.
Main methods: ACh release from DRG explants in the Campenot chambers was measured by a chemilumines-
cent assay. Real-time PCR analysis was used to evaluate the expression of ChAT, VAChT, mediatophore and
muscarinic receptor subtypes in DRGs at different developmental stages.
Key findings: We found that ACh is released both within the central and lateral compartments of the Campenot
chambers, indicating that ACh might be released from both the neuronal soma and fibers. Moreover, we ob-
served that the expression of the ChAT and mediatophore increases during sensory neuron differentiation and
during the post-hatching period, whereas VAChT expression decreases throughout development. Lastly, the ki-
netics of the m2 and m3 transcripts appeared to change differentially compared to the m4 transcript during the
same developmental period.
Significance: The data obtained demonstrate that the DRG sensory neurons are able to release ACh and to re-
spond to ACh stimulation. ACh is released both by the soma and neurite compartments. The contribution of
the mediatophore to ACh release appears to be more significant than that of VAChT, suggesting that the
non-vesicular release of ACh might represent the preferential mechanism of ACh release in DRG neurons and
possibly in non-cholinergic systems.
© 2012 Elsevier Inc. All rights reserved.
It was proposed that, in addition to their classical role in synaptic
transmission, neurotransmitters (NTs) have been proposed to play
functional roles both during neurogenesis and during adult life
(Buznikov et al., 1996; Biagioni et al., 2002; Karczmar, 2007; Bovetti
et al., 2011; Young et al., 2011; Abreu-Villaça et al., 2011).
Cholinergic components have been identified in non-neuronal cells
as part of autocrine or paracrine signaling systems (Loreti et al., 2006;
Kawashima and Fujii, 2008). Thus, NTs appear to be “old” molecules
that are present in many organisms and that participate in signaling
mechanisms that control variouscellularfunctionssuchasproliferation
and migration. Their role in neurogenesis has been proposed on the
basis of their early appearance during development, which extends
well into the advanced stages of synaptogenesis (Bovetti et al., 2011;
Young et al., 2011).
The role of acetylcholine (ACh) as a cofactor of neuronal differen-
tiation has been investigated in mouse neuroblastoma cell lines and
dorsal root ganglia (DRGs) (Biagioni et al., 2002; Salani et al., 2009).
DRG neurons are not a single population; they display both morpho-
logical and biochemical differences in relation to their ability to re-
ceive diverse physico-chemical signals from all body structures, thus
ensuring responses to peripheral stimuli and their integration (Salt
and Hill, 1983). Despite their different properties, DRG neurons
share the unique morphology of pseudounipolar neurons, character-
ized by a single process proximal to the soma that bifurcates into a
peripheral and a central branch. The neurons (large light and small
dark) and satellite cells present in DRG, as well as their differentia-
tion, have been described in detail (Pannese, 1974; Hanani, 2005).
Cholinergic markers, such as ChAT and AChE, are expressed in DRGs
from early developmental stages (Tata et al., 1994). Acetylcholine pro-
motes the expression of specific neuronal markers (e.g., neurofilament
proteins, NF) and the outgrowth of neurites via the activation of the
ACh muscarinic receptor (Tata et al., 2003). Moreover, the DRG neurons
express ChAT, AChE, and the ACh vesicular transporter (VAChT), and
they release ACh during development and in adult (Tata et al., 2004;
ry perception. Notably, electrophysiological studies have demonstrated
Life Sciences 91 (2012) 783–788
⁎ Corresponding author at: Dept of Biology and Biotechnologies Charles Darwin,
Research Center of Neurobiology Daniel Bovet, Sapienza, University of Rome, P.le A.
Moro, 5‐00185 Roma, Italy. Tel.: +39 06 49912637; fax: +39 06 49912351.
E-mail address: firstname.lastname@example.org (A.M. Tata).
1These authors equally contributed to the work presented here.
0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/lifescie
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ulating CGRP release from the DRG neurons (Bernardini et al., 2001a,
phase, characterized by processes that are related to the final stages of
and peripheral targets have been established in a previous phase, as de-
scribed by Hamburger and Levi-Montalcini (1949). Our investigation
addressed the three following important DRG neuron-related issues:
a) the site of ACh release (to address this issue, we cultured DRGs in
Campenot chambers, which allow the isolation of the soma and fibers
in separate compartments); b) the expression of mediatophore, a trans-
membrane protein capable of the ACh translocation across the plasma
membrane, independently of vesicle-based ACh release, that has been
described previously in the vertebrate neurons (Israel and Dunant,
1999) and in other cell types (Fujii et al., 2012); and c) the levels of ex-
pression of the muscarinic receptor subtypes.
Materials and methods
Chick embryos were handled in accordance with the guidelines of
the European Communities Council Directive (86/609/EEC of 24 No-
vember 1986) and the Italian National law DL/116/92. After decapita-
tion of the embryos, DRGs were dissected, collected, rapidly frozen
and stored at −80 °C for subsequent mRNA analysis. For the in vitro
studies, the DRG neurons dissected from the E12 embryos were im-
mediately placed in cultures as described below.
The developmental stages were established and defined according
to the tables detailed by Hamburger and Hamilton (1951). DRG neu-
rons collected on days E12 (stage 32) and E18 (stage 44) of develop-
ment and day 7 after hatching (P7) were used. The DRG neurons were
dissected and pooled from at least 10 to 20 embryos/young chicks
from each stage, depending on the type of analysis.
Campenot chambers were assembled in 35 mm collagen-coated
dishes after parallel grooves had been made on the collagen in order to
guide fiber growth, as described previously (Campenot, 1977) (Fig. 1).
The chambers were sealed onto the dishes with a thin layer of silicone
to maintain thepartition between the central andlateral compartments.
Modified Eagle's medium (MEM) supplemented with 5% fetal bovine
serum (FBS) and 0.6% Methocel (Sigma)was added tothe centralcham-
berandincubatedfor2–3 hat37 °C.Next,mediumwasaddedtothelat-
eral compartments and the dishes maintained at 37 °C overnight. DRGs
dissected from E12 embryos were then plated as explants into the cen-
tral compartment. The explants were maintained in culture for 7 days
to allow neurite extension into the lateral compartments, as guided by
the grooves on the collagen.
ACh release was evaluated using the choline oxidase chemilumi-
nescent procedure (Israel and Lesbats, 1981). The medium was re-
moved and each compartment was washed with a saline solution
(136 mM NaCl, 5.6 mM KCl, 1.2 mM, MgCl2, 6 mM CaCl2in 10 mM
Tris buffer, pH 8.6) in order to remove pre-existing traces of choline
in the medium.
Saline solution (250 μl) was added to each compartment and the
dishes were incubated at 37 °C for three consecutive periods (5 min
each); during the second incubation period, KCl (final concentration,
80 mM) was added to trigger ACh release. Saline solution (250 μl)
was collected before, during, and after stimulation and added to a reac-
tion mixture (500 μl) containing 10 μl luminol (1 mM stock solution),
Fig. 1. DRG explants plated in Campenot chambers: (top left) is a schematic representation of a Campenot chamber, where (A) indicates the central chamber and (B) indicates the
lateral chambers. Shown in the center is a photographic field of the E12 DRG explants plated in the central chamber and fibers present in the lateral compartments. The nuclei are
labeled with Hoechst 33258. Shown in the middle are the neurons and fibers labeled with an α-NF145 primary antibody and a FITC-conjugated secondary antibody-FITC conjugated
(25×). Shown at the bottom is a higher magnification of the lateral compartment where the fibers are more evident (160×).
V. Corsetti et al. / Life Sciences 91 (2012) 783–788
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5 μl horseradish peroxidase (2 mg/ml stock solution) and 5 μl AChE
(1000 U/ml stock solution, purified using a Sephadex G-50 column)
(see Bernardini et al., 2004). Chemiluminescence was measured using
a Lumat LB9507 (EG & G Berthold, Bad Wilbad, Germany). For each
sample, after the light emission reached a stable baseline, 50 μl choline
oxidase(50 U/mLstocksolution)was added. Attheendofeach record-
ing, an internal ACh standard was added and the light emission was
RNA extraction and real time PCR analysis
Total RNA was extracted from chick DRG neurons at different de-
velopmental stages using Trizol reagent (Invitrogen) according to
the manufacturer's protocols. The RNA concentration and integrity
were assessed by spectrophotometric analysis and agarose gel elec-
Total RNA was treated with the DNA-free kit (Ambion Inc., Milan,
Italy) to eliminate traces of DNA contamination. Two micrograms of
RNA wasreverse-transcribedusingrandomhexanucleotides asprimers
(Promega) in a 25 μl reaction volume containing 200 U of Moloney-
murine leukemia virus reverse transcriptase (M-MLV, Promega).
The expression of cholinergic markers such as ChAT, VAChT,
mediatophore, and muscarinic receptor subtypes was evaluated by
real time PCR analysis, using selective primers as indicated in Table 1.
Ten nanograms of each cDNA was used as the template for each real
time PCR reaction. SyBRGreen Jump Start Taq Ready Mix (Sigma, MA)
and the selective primers (final concentration 300 nM) were also
added to the reaction tubes and analyzed in I Cycler IQTMMulticolor
Real Time Detection System (Bio-Rad). All of the samples were run in
triplicate. The real time PCR conditions included a denaturing step at
95 °C for 3 min followed by 40 cycles at 95 °C for 30 s, 58 °C for 30 s
and 75 °C for 45 s. Two cycles (1 min/each) were included as final
steps at 72 °C (final extension). Quantification was performed using
the comparative CTmethod (CT=threshold cycle value).
value of the housekeeping gene (GAPDH) were calculated according to
the following formula: ΔCTsample=CTsample−CTGAPDH. The final results
were expressed as 2−ΔΔCT, where ΔΔCT=ΔCTsample−ΔCTcalibrator.
DRG explants were fixed for 20 min in 4% paraformaldehyde in PBS
(pH 7.4) at room temperature and incubated for 1 h in PBS containing
10% normal goat serum (NGS), 1% Bovine Serum Albumin (BSA) and
0.1% Triton X-100. Explants were incubated overnight at 4 °C in the
presence of anti-NF145 primary antibody (purified monoclonal mouse
IgG, at 1:100 dilution), followed by two washings (10 min each) in
PBS containing 1% BSA and then incubated for 90 min at room temper-
ature with the secondary antibody (FITC-conjugated Goat-anti Mouse
IgG). After washing in PBS, the nuclei were stained with 1 μg/ml
Hoechst 33258. Negative controls were generated by omitting the pri-
One-way ANOVA and Bonferroni's multiple comparison post tests
were used to evaluate the statistical significance within different sam-
ples. The results were considered statistically significant at pb0.05 (*),
pb0.01 (**) and pb0.001 (***).
Our investigation focused on specific aspects of ACh function in
DRG neurons, including the cellular site of release, the release mech-
anism and the expression levels of the muscarinic receptor subtype
during DRG sensory neuron differentiation.
The use of Campenot chambers allowed for the evaluation of the
ACh release either in the central compartment, where the explants
were plated, or in the lateral compartments, where the elongation of
the fibers occurred. Hoechst staining was largely confined within the
central compartment (Fig. 1), indicating that the neurons remained in
the explants, whereas the fibers emanating from the explants that im-
munostained for 145 kDa NF, extended into the lateral compartments.
E12 chick DRG neurons were maintained in the chambers for 7 days
to allow extensive fiber growth. Before and after KCl stimulation, saline
was collected either from the central or the two lateral compartments
and ACh released was measured as described in the Materials and
methods section. As shown in Fig. 2A, ACh release was stimulated by
lease was also observed. The release was observed in both the central
The table shows the primer sequences used in real-time PCR.
Gene Upper 5′–3′
Fig. 2. Measurement of ACh levels in E12 chick DRG explants: A) ACh levels were measured in three different compartments of the Campenot chamber. The measurements were
performed at basal conditions (prior to stimulation), in the presence of KCl stimulation, and 5 min after stimulation; B) ACh levels were also measured in E12 DRG explants that
were maintained in the absence or presence of the muscarinic antagonist atropine (10−6M). The values reported are the mean values±SEM obtained from three independent
V. Corsetti et al. / Life Sciences 91 (2012) 783–788
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and lateral compartments, indicating that the release was not confined
to the fiber terminals, but most likely occurred along the fibers.
No ACh release was observed in either compartment when sym-
pathetic ganglia explants were cultured (data not shown).
As previously reported (Tata et al., 2000), the muscarinic receptors
are expressed in the DRG neurons. To evaluate their ability to modu-
late ACh release, the experiments were performed in the presence of
the antagonist atropine (10-6M). As shown in Fig. 2B, atropine signif-
icantly reduced ACh release from the E12 DRG explants, both during
basal and KCl-stimulated conditions.
ChAT, VAChT and mediatophore expression
In addition to the classical ACh release mechanism, which is char-
acterized by ACh transport and accumulation in vesicles that subse-
quently fuse with the plasma membrane (Whittaker, 1988, 2010), a
second release mechanism that is mediated by mediatophore, a trans-
membrane protein responsible for the direct translocation of ACh
across the plasma membrane, was described by Israel and Dunant
(1999). Although DRG neurons express the ACh vesicular transporter
(VAChT) (Tata et al., 2004), our results demonstrate that ACh release
is not confined to nerve endings (see above) where vesicle accumula-
tion is expected, suggesting that mediatophore might play a role in
ACh release along the growing fibers of DRG neurons. The expression
levels of ChAT, VAChT and mediatophore in the E12, E18 and P7
DRGs were evaluated by real time PCR analysis. Fig. 3A shows that
mediatophore is coexpressed with VAChT during all developmental
stages examined, indicating a coexistence of two release mecha-
nisms. Whereas ChAT and mediatophore expression appears to be
elevated in E18 and postnatal DRG neurons compared to E12 DRG
neurons, the opposite is observed for VAChT expression. However,
the relative mRNA levels of mediatophore were consistently and sig-
nificantly higher than that of VAChT, starting from day E12 (Fig. 3B).
Muscarinic receptor expression
Previous studies have reported the presence of muscarinic recep-
tors in rat and chick DRG neurons, as well as their ability to synthesize
Fig. 3. A) Analysis of the mediatophore, VAChT and ChAT mRNA transcript expression in chick DRG at different development stages by real time PCR. The levels of the transcripts
were normalized to those of the housekeeping gene GAPDH and compared with expression levels at day E12, which served as reference samples; B) The relative expression levels of
VAChT and mediatophore transcripts at different developmental stages. The levels of the transcripts were normalized with the housekeeping gene GAPDH, and the mediatophore
mRNA transcript levels were compared to VAChT levels.
V. Corsetti et al. / Life Sciences 91 (2012) 783–788
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ACh (Bernardini et al., 1998; Tata et al., 2000; Bernardini et al., 2004),
suggesting an active autocrine regulatory system in DRG neurons. Al-
though DRG neurons are known to express several muscarinic recep-
tor subtypes, there has been no quantitative evaluation of muscarinic
receptor subtype expression during sensory neuron development.
We used real time PCR analysis to evaluate the expression of m2,
m3 and m4 muscarinic receptor transcripts. As shown in Fig. 4, all
of the muscarinic receptor subtypes examined are expressed in E12
DRG neurons. mRNA transcripts for m2 and m3 were expressed at
higher levels on day E12 than at later developmental stages and in
post-hatching periods. Conversely, the expression of m4 transcripts
increased on day E18 and then decreased on post-hatching day P7, al-
though day P7 levels remained significantly higher than those at day
Our investigation focused on the DRG (Biagioni et al., 1999), a
component of the peripheral nervous system, characterized by the
presence of primary sensory neuron subpopulations involved in the
perception of different stimuli (Ohara et al., 2009; Hanani, 2005;
Schaeffer et al., 2010). Although they predominantly use neuropep-
tides for neurotransmission, DRG neurons express all of the choliner-
gic markers from early developmental stages and are cholinoceptive,
as they express the acetylcholine receptors. The activation of musca-
rinic receptors stimulates the expression of NF proteins and neurite
outgrowth (Tata et al., 2003), suggesting a role for ACh as a modulator
of sensory neuron differentiation.
on the fibers of DRG neurons (Bernardini et al., 1998, 1999), indicating
ble of releasing ACh (Bernardini et al., 2004), the site of its release was
unknown. Fig. 2 shows that ACh is released into the central and lateral
curs along the fibers. ACh released into the central compartment might
the lateral components. However, it is also possible that the release oc-
curs from the soma, as previously reported for Drosophila (Yao et al.,
2000) and Xenopus neurons (Chow and Poo, 1985). The contribution of
from the growth cone, as the release increases proportionately with fiber
length over 4 days in culture (Bernardini et al., 2004). This is the time in-
terval during which fiber elongation exceeds the number of fibers and
growth cones extending from the explants. This notion is consistent
with the finding that ACh is released along the growing fibers of the spi-
uncertain; previous reports have indicated that ACh is released by the
Schwann cells from squid giant axons (Evans et al., 1999) and from
neuron–muscle junctions in frogs (Reiser and Miledi, 1988). Thus, the
contribution of ACh release by Schwann cells, as well as by the satellite
cells surrounding neuronal soma, cannot be excluded. However because
the satellite cells of the DRG and the sympathetic ganglia share many
common properties (Hanani, 2010; Pannese, 1994), the absence of ACh
release in sympathetic ganglia cultures suggests that their contribution
to ACh release in this context might not be relevant.
Although some controversy still remains regarding the involvement
of mediatophore in the non-quantal release of ACh, a relationship be-
tween the expression levels of mediatophore and the release of ACh in
various cell types has been reported (Malo and Israel, 2003; Fujii et al.,
2012). These observations prompted us to evaluate the presence of
mediatophore in DRG neurons, although the presence of VAChT, the
protein responsible for acetylcholine transport into vesicles, has already
been reported in the DRG (Tata et al., 2004). As shown in Fig. 3,
mediatophore is present, concomitantly with VAChT, in the DRG; this
finding suggests that these two release mechanisms coexist in the DRG
neurons. However, the level of mediatophore mRNA is considerably
higher than that of VAChT suggesting that the mediatophore release
mechanism might be prevalent in the DRG neurons. VAChT activity ap-
rat DRG (Tata et al., 2004). However, as the antibody for chick DRG
mediatophore is not available, it was not possible to establish whether
the presence ofmediatophore in the DRG neurons is preferentiallyasso-
ciated with a specific neuronal subpopulation, as appears to be the case
for VAChT, and with different neuronal compartments (soma, fibers or
The levels of the two ACh release systems fluctuate differentially
during development. For instance, whereas mediatophore mRNA
levels tend to increase in DRG neurons from E12 to E18, the opposite
is true for VAChT levels. Although the ChAT and VAChT genes coexist
within the same “cholinergic locus”, they can undergo independent
transcriptional regulation due to the presence of multiple promoters
(Cervini et al., 1995), which might lead to the uncoordinated expres-
sion of the two genes, as appears to be the case in DRG.
The muscarinic receptors are present both in rat and in chick DRG
neurons (Tata et al., 2000), and analysis by real time PCR allowed for
the evaluation of changes in muscarinic receptor subtypes during
DRG neuron differentiation. Our data indicate that m2 and m3 tran-
scripts are present at early developmental stages and decrease at
later stages, including the post-hatching period. Conversely, the m4
transcripts appear to be more abundantly expressed during the
post-hatching period. Rat satellite and Schwann cells express musca-
rinic receptors (Tata et al., 1999; Loreti et al., 2006), and their activa-
tion modulates glial cell proliferation (Loreti et al., 2007). Because the
total RNA used for our experiments included RNA that was derived
from neurons, satellite and Schwann cells, it is not possible to estab-
lish whether the variation in muscarinic receptor subtype expression
that we observed during DRG neuronal development, was influenced
by variable expression in the glial cells (see Bernardini et al., 1998,
1999; Loreti et al., 2006). Thus, the muscarinic receptors present in
the DRG neurons appear to contribute to ACh release. Notably, the
use of the muscarinic antagonist atropine counteracted the release
of ACh in E12 DRG explants, suggesting that the release of ACh in
the DRG neurons is positively regulated by an autocrine mechanism
that is mediated by muscarinic receptors.
Chick DRG neurons release ACh not only at the nerve terminals
but also along the fibers and possibly from the soma. The presence
of both VAChT and mediatophore transcripts in the DRG neurons
Fig. 4. Analysis of m2, m3 and m4 transcripts in chick DRG at different developmental
stages by real time PCR. The levels of the transcripts were normalized to those of the
housekeeping gene GAPDH and compared with expression levels at day E12, which
were considered as reference samples.
V. Corsetti et al. / Life Sciences 91 (2012) 783–788
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suggests that two release mechanisms coexist at different develop-
mental stages. The elevated mediatophore expression compared to
VAChT suggests that the “non-vesicular mechanism” might be the
main release system in non-cholinergic neurons, although one can-
not exclude the possibility that the two modes of release might dif-
ferentially support neurite outgrowth and to contribute to the cross-
talk between the neurons and glial cells within the DRG. In fact, the
time-dependent expression of the muscarinic receptor subtypes in
both DRG neurons and glial cells suggests a differential ability of
the two cell populations to respond to ACh stimuli during DRG de-
velopment and in adults (Bernardini et al., 1999 and 2001a; Loreti
et al., 2006).
Conflict of interest statement
The authors declare that there are no conflicts of interest.
This work was supported by the funds of The University of Rome
“La Sapienza”. The authors are grateful to Dr. Fiona Leckie for the
English revision of this manuscript.
Abreu- Villaça Y, Filgueiras CC, Manhães AC. Developmental aspects of cholinergic sys-
tem. Behav Brain Res 2011;221:367–78.
Antonov I, Chang S, Zakharenko S, Popov SV. Distribution of neurotransmitter secretion
in growing axons. Neuroscience 1999;90:975–84.
Bernardini N, De Stefano ME, Tata AM, Biagioni S, Augusti-Tocco G. Neuronal and
non-neuronal cell populations of the avian dorsal root ganglia express muscarinic
acetylcholine receptors. Int J Dev Neurosci 1998;16:365–77.
Bernardini N, Levey AI, Augusti-Tocco G. Rat dorsal root ganglia express m1–m4 mus-
carinic receptor proteins. J Peripher Nerv Syst 1999;4:222–32.
Bernardini N, Sauer SK, Haberberger R, Fischer MJ, Reeh P. Excitatory nicotinic and
desensitizing muscarinic (M2) effects on C-nociceptors in isolated rat skin. J Neurosci
Bernardini N, Reeh P, Sauer SK. Muscarinic M2 receptors inhibit heat-induced CGRP
release. Neuroreport 2001b;12:2457–60.
Bernardini N, Srubek Tomassy G, Tata AM, Augusti-Tocco G, Biagioni S. Detection of
basal and potassium evoked acetylcholine release fron embryonic DRG explants.
J Neurochem 2004;88:1533–9.
Biagioni S, Tata AM, Augusti-Tocco G. Expression of cholinergic system components in
dorsal root ganglion (DRG) neurons: its possible dual role in development and
nociception. Recent Res Dev Neurochem 1999;2:443–61.
Biagioni S, Tata AM, De Jaco A, Augusti-Tocco G. Acetylcholine and regulation of gene
expression in nerve system development. Curr Top Neurochem 2002;3:177–88.
Bovetti S, Gribaudo S, Puche AC, De Marchis S, Fasolo A. From progenitors to integrated
neurons: role of neurotransmitters in adult olfactory neurogenesis. J Chem
Neuroanat 2011. http://dx.doi.org/10.1016/j.jchemneu.2011.05.006.
Buznikov GA, Shmukler YB, Lauder JM. From oocyte to neuron neurotransmitter func-
tion in the same way throughout development. Cell Mol Neurobiol 1996;16:
Campenot RB. Local control of neurite development by nerve growth factor. Proc Natl
Acad Sci 1977;74:4516–9.
Cervini R, Houhou L, Pradat PF, Béjanin S, Mallet J, Berrard S. Specific vesicular acetyl-
choline transporter promoters lie within the first intron of the rat choline
acetyltrasnferase gene. J Biol Chem 1995;270:24654–7.
Chow I, Poo MM. Release of acetylcholine from embryonic neurons upon contact with
muscle cell. J Neurosci 1985;5:1076–82.
Evans PD, Reale V, Merzon RM, Villegas J. A comparison of the release of a vasoactive-
intestinal-peptide-like peptide and acetylcholine in the giant axon-Schwann cell
preparation of the tropical squid Sepioteuthis Sepioidea. J Exp Biol 1999;202:417–28.
Fujii T, Takada-Takatori Y, Horiguchi K, Kawashima K. Mediatophore regulates acetyl-
choline release from T cells. J Neuroimmunol 2012. [Jan. 13 Epub ahead of print].
Hamburger V, Hamilton HL. A series of normal stages in the development of chick em-
bryo. J Morphol 1951;88:85-100.
Hamburger V, Levi-Montalcini R. Proliferation, differentiation and degeneration in
the spinal ganglia of the chick embryo under normal and experimental condi-
tions. J Exp Zool 1949;111:457–501.
Hanani M. Satellite glial cells in sensory ganglia: from form to function. Brain Res Rev
Hanani M. Satellite glial cells in sympathetic and parasympathetic ganglia: in search of
function. Brain Res Rev 2010;64:304–27.
Israel M, Dunant Y. Mediatophore, a protein supporting quantal acetylcholine release.
Can J Physiol Pharmacol 1999;77:689–98.
Israel M, Lesbats B. Continuous determination by a chemiluminescent method of ace-
tylcholine release and compartmentation in Torpedo electric organ synaptosomes.
J Neurochem 1981;37:1475–83.
Karczmar GA. Cholinergic aspects of growth and development. In: Karczmar GA, editor.
Exploring the vertebrate central cholinergic system. New York: Spinger-Verlag;
2007. p. 311–409.
non-neuronal cholinergic systems and their biological significance. J Pharmacol Sci
Loreti S, Vilaró MT, Visentin S, Rees H, Levey AI, Tata AM. Rat Schwann cells express
M1–M4 muscarinic receptor subtypes. J Neurosci Res 2006;84:97–105.
Loreti S, Ricordy R, De Stefano ME, Augusti-Tocco G, Tata AM. Acetylcholine inhibits cell
cycle progression in rat Schwann cells by activation of the M2 receptor subtype.
Neuron Glia Biol 2007;4:269–79.
Malo M, Israel M. Expression of the acetylcholine release mechanism in various cells
and reconstruction of the release mechanism in non-releasing cells. Life Sci
Ohara Pt, Vit JP, Bhargava A, Romero M, Sundberg C, Charles AC, et al. Gliopathic pain:
when satellite glial cells go bad. Neuroscientist 2009;15:450–63.
Pannese E. The histogenesis of spinal ganglia. Adv Anat Embryol Cell Biol 1974;47(5).
Pannese E. Neurocytology. New York: George Thieme Verlag, Stuttgart, Thieme Medical
Reiser G, Miledi R. Characteristic of Schwann-cell miniature end-plate currents in de-
nervated frog muscle. Pflugers Arch 1988;412:22–8.
Salani M, Anelli T, Augusti Tocco G, Lucarini E, Mozzetta C, Poiana G, et al.
Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regu-
lates EGR-1 and REST expression in neuroblastoma cells. J Neurochem 2009;108:
Salt TE, Hill RG. Neurotransmitter candidates of somatosensory primary afferent fibers.
Schaeffer V, Mayer L, Patte-Mensah C, Mensah- Nyagan AG. Progress in dorsal root
ganglia neurosteroidogenic activity: basic evidence and pathophysiological corre-
lation. Prog Neurobiol 2010;92:33–41.
Tata AM, Plateroti M, Cibati M, Biagioni S, Augusti-Tocco G. Cholinergic markers are
expressed in developing and mature chick dorsal root ganglia. J Neurosci Res
Tata AM, Vilarò MT, Agrati C, Biagioni S, Mengod G, Agusti Tocco G. Expression of musca-
rinic m2 receptor mRNA in dorsal root ganglia of neonatal rat. Brain Res 1999;824:
Tata AM, Vilaró MT, Mengod G. Muscarinic receptors subtypes expression in rat and
chick dorsal root ganglia. Mol Brain Res 2000;82:1-10.
Tata AM, Cursi S, Biagioni S, Augusti-Tocco G. Cholinergic modulation of neurofilament
expression and neurite outgrowth in chick sensory neurons. J Neurosci Res
Tata AM, De Stefano ME, Srubek Tomassy G, Vilarò MT, Levey A, Biagioni S.
Suppopulation of rat dorsal root ganglion neurons express active vesicular acetyl-
choline transporter. J Neurosci Res 2004;75:194–202.
Whittaker VP. The cellular basis of synaptic transmission: an overview. In:
Zimmerman A, editor. Cellular and Molecular basis of synaptic transmission.
NATO ASI SeriesSpringer Verlag; 1988. p. 1-23.
Whittaker VP. Some currently neglected aspects of cholinergic function. J Mol Neurosci
Yao WD, Rusch J, Poo M, Wu CF. Spontaneous acetylcholine secretion from developing
growth cone of Drosophila central neurons in culture: effects of cAMP-pathway
mutations. J Neurosci 2000;20:2626–37.
Young SZ, Taylor MM, Bordey A. Neurotransmitters couple brain activity to subventricular
zone neurogenesis. Eur J Neurosci 2011;33:1123–32.
V. Corsetti et al. / Life Sciences 91 (2012) 783–788