Download Neurochemistry of identified motoneurons of the tensor tympani

Hearing Research 248 (2009) 69–79
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Research papers
Neurochemistry of identified motoneurons of the tensor tympani muscle
in rat middle ear
Stefan Reuss a,*, Inna Kühn a, Reinhard Windoffer a, Randolf Riemann b
a
b
Department of Anatomy and Cell Biology, School of Medicine, Johannes Gutenberg-University, Becherweg 13, 55099 Mainz, Germany
Department of Otorhinolaryngology, City Hospital Frankfurt-Hoechst, Germany
a r t i c l e
i n f o
Article history:
Received 11 September 2008
Received in revised form 26 November 2008
Accepted 6 December 2008
Available online 24 December 2008
Keywords:
Nitric oxide synthase
Neuropeptides
Fluoro-Gold
Retrograde tracing
a b s t r a c t
The objective of the present study was to identify efferent and afferent transmitters of motoneurons of
the tensor tympani muscle (MoTTM) to gain more insight into the neuronal regulation of the muscle.
To identify MoTTM, we injected the fluorescent neuronal tracer Fluoro-Gold (FG) into the muscle after
preparation of the middle ear in adult rats. Upon terminal uptake and retrograde neuronal transport,
we observed FG in neurons located lateral and ventrolateral to the motor trigeminal nucleus ipsilateral
to the injection site. Immunohistochemical studies of these motoneurons showed that apparently all contained choline acetyltransferase, demonstrating their motoneuronal character. Different portions of these
cell bodies were immunoreactive to bombesin (33%), cholecystokinin (37%), endorphin (100%), leuenkephalin (25%) or neuronal nitric oxide synthase (32%). MoTTM containing calcitonin gene-related
peptide, tyrosine hydroxylase, substance P, neuropeptide Y or serotonin were not found. While calcitonin
gene-related peptide was not detected in the region under study, nerve fibers immunoreactive to tyrosine
hydroxylase, substance P, neuropeptide Y or serotonin were observed in close spatial relationship to
MoTTM, suggesting that these neurons are under aminergic and neuropeptidergic influence. Our results
demonstrating the neurochemistry of motoneuron input and output of the rat tensor tympany muscle
may prove useful also for the general understanding of motoneuron function and regulation.
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
There is evidence that the middle ear muscles, i.e., the tensor
tympani muscle (TTM) and the stapedius muscle, modulate acoustic input by influencing the ossicular chain in non-primate mammals, such as rabbit and cat (Borg and Counter, 1989; Carmel
and Starr, 1963; Kevanishvili and Gvacharia, 1972). While in primates, including man, the TTM does not appear to play a major role
for the acoustic reflex (Feldman, 1967; Klockhoff, 1961), it may act
synergistically to the tensor veli palatini muscle in facilitating middle ear aeration via the Eustachian tube (Kamerer and Rood, 1978).
The TTM of rats that demonstrates several atypical features, such
as the lack of muscle spindles, was found to be part of the acoustic
Abbreviations: ACh, acetylcholine; BBS, bombesin; CCK, cholecystokinin; CGRP,
calcitonin gene-related peptide; ChAT, choline acetyltransferase; CN, cochlear
nucleus; END, endorphin; FG, Fluoro-Gold; HRP, horseradish peroxidase; ir,
immunoreactive; leu-ENK, leu-enkephalin; LSO, lateral superior olivary nucleus;
MoTTM, motoneurons of the tensor tympani muscle; nNOS, neuronal nitric oxide
synthase; NO, nitric oxide; NPY, neuropeptide Y; PBS, phosphate-buffered 0.9%
saline; SER, serotonin; SP, substance P; TH, tyrosine hydroxylase; TMN, trigeminal
motor nucleus; TTM, tensor tympani muscle; VCN, ventral cochlear nucleus
* Corresponding author. Tel.: +49 6131 3923207; fax: +49 6131 3923719.
E-mail address: [email protected] (S. Reuss).
0378-5955/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.heares.2008.12.003
reflex (van den Berge and Wirtz, 1989; van den Berge and van der
Wal, 1990; van den Berge et al., 1990).
The location of motoneurons of the TTM (MoTTM) was studied
previously in mammals. In cats, rabbits, guinea pigs and rats, they
were localized by neuronal retrograde tracing upon injection into
the muscle (Lyon, 1975; Mizuno et al., 1982; Shaw and Baker,
1983; Spangler et al., 1982; Strutz et al., 1988; Takahashi et al.,
1984). Although there are some differences in the number and
location of MoTTM between species, a general feature of these neurons is that they are found in a region lateral and ventrolateral to
the motor nucleus of the trigeminal nerve. This region corresponds
to ‘‘cell group k”, described first by Meessen and Olszewski (1949).
It is thought to contain also motoneurons supplying masseter,
digastric and Eustachian tube muscles (Donga et al., 1992; Saad
et al., 1997).
Despite the functional importance of the TTM, there is little
information about the efferent and afferent transmitter substances
of its motoneurons. One report on cat MoTTM demonstrated their
cholinergic nature (Godfrey et al., 1990). In addition, one report described the innervation of MoTTM by serotonergic fibers in bush
babies (Otolemur garnettii) (Thompson et al., 1998). We therefore
injected the neuronal tracer Fluoro-Gold (FG) into the muscle in
rats to identify motoneurons upon retrograde axonal transport
and combined that with the immunofluorescent detection of
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S. Reuss et al. / Hearing Research 248 (2009) 69–79
putative transmitter antigens. We first sought to confirm the motoneuronal nature of the traced neurons by demonstrating immunohistochemically that they contain the acetylcholine-synthesizing
enzyme, choline acetyltransferase (ChAT).
We then studied the distribution of transmitter candidates or
their marker enzymes found in the lower brain stem, in particular
in the trigeminal motor nucleus (TMN) (Briski, 1999; Cortes et al.,
1990; Covenas et al., 1990; Fodor et al., 1992; Fukuoka et al.,
1999; Khachaturian et al., 1983; Marcos et al., 1994; Simmons
et al., 1999; Sutin and Jacobowitz, 1990). To investigate whether
identified MoTTM or fiber systems in their vicinity exhibit respective immunoreactivity, we studied the distribution of bombesin
(BBS), cholecystokinin (CCK), calcitonin gene-related peptide
(CGRP), endorphin (END), leu-enkephalin (leu-ENK), neuropeptide
Y (NPY), neuronal nitric oxide synthase (nNOS), serotonin (SER),
substance P (SP) and tyrosine hydroxylase (TH) by the immunofluorescence detection method with simultaneous detection of
FG. This study is part of a larger program to explore the neurochemistry of middle ear muscles. Within that scope we recently
presented data on the stapedius muscle motoneurons (Reuss
et al., 2008).
2. Materials and methods
2.1. Animals and treatment
The procedures concerning animals reported in this study
complied with German laws for the protection of animals and
were approved by the county-government office (Bezirksregierung
Rheinhessen-Pfalz). Fifteen adult female Sprague–Dawley rats,
200–220 g b.wt., were maintained under constant conditions
(light:dark 12:12 h, room temperature 21 ± 1 °C) with food and
water ad libitum. For application of the tracer, rats were deeply
anesthetized with tribromoethanol (0.3 g/kg b.wt., i.p.). The tensor tympani muscle of the left middle ear was exposed by an
endaural microsurgical approach. After creating a tympanomeatal flap and partially removing the posterior wall of the outer
ear canal, the incus and parts of the malleus were removed.
The tensor tympany muscle was exposed and its tendon identified. A total of approximately 0.3 ll of a 5% Fluoro-Gold solution
(FG; Fluorochrome, Englewood, Colorado, USA; dissolved in distilled water) was, in three portions, pressure-injected into the
muscle using a glass micropipette. After five days, animals were
reanesthetized at the middle of the light period and perfused
transcardially with phosphate-buffered 0.9% saline (PBS), to
which 15000 IU heparin/l were added, at room temperature
(RT), followed by an ice-cold fixative (4% paraformaldehyde,
1.37% L-lysine, 0.21% sodium-periodate in phosphate buffer;
according to McLean and Nakane (1974). The right atrium was
opened to enable venous outflow.
2.2. Tissue processing
The brain was removed and postfixed for one hour in the same
fixative, stored overnight at 4 °C in phosphate-buffered 30% sucrose and sectioned serially at 40 lm thickness on a freezing
microtome in the frontal plane. Sections were collected in PBS
and, for immunohistochemistry, incubated free-floating in PBS-diluted primary antibodies (see Table 1), to which 1% normal swine
serum and 0.1% Triton-X 100 were added. After three rinses in PBS,
sections were incubated in IgG directed against the host species of
the respective primary antibody, coupled to Cy3 (1:400 in PBS,
Amersham, Hannover, Germany). Sections were mounted onto
gelatin-coated slides, shortly dehydrated, coverslipped with
Merckoglas (Merck, Darmstadt, Germany) and analyzed using a
Leitz Orthoplan microscope with a Ploemopak epifluorescence unit
through filter sets A (Fluoro-Gold) and N2 (Cy3).
Fluoro-Gold-labelled cell bodies were quantified from complete
sections from seven animals. All sections containing FG-neurons
(from a total of fifteen rats) were processed for immunohistochemistry. For each antibody, at least fifteen sections from at least three
animals were incubated. Some sections were incubated with two
antibodies (with additional secondary antibody coupled to Cy2),
thus increasing the number of sections tested for a specific transmitter. Neurons exhibiting immunoreactivity to the antisera tested
were counted when immunoreactivity was clearly over the background level. Counts were corrected according to Abercrombie
(1946) to prevent double counting of cells. Single- and double-labeled neurons were quantified separately and the respective percentages were calculated. Brain regions were identified using the
stereotaxic atlas of Paxinos and Watson (1998). Photomicrographs
were taken on Ilford HP 5 plus films. Some sections were counterstained with hematoxilin-eosin after removing the coverslips to
facilitate localization of labelled neurons. Due to the pretreatment,
however, staining quality has proved less satisfactory in these
cases.
For dual color demonstration of backfilled neurons and neighboring fibers (Fig. 6), selected sections were studied using a TLS
NT (Leica, Wetzlar, Germany) laser scanning microscope. Digital
images were taken and overlayed using the Adobe Photoshop program that was also used to adjust image contrast and brightness
and to add labels.
2.3. Control studies
Immunohistochemical specificity studies, carried out by omitting primary or secondary antibodies or by absorbing the primary
antibody with the immunogen, showed the absence of the immunofluorescent signal. The antisera had been used earlier in our laboratory and were characterized previously. Further details are
given in the references of Table 1.
Table 1
Antibodies used for the characterization of identified tensor tympani muscle motoneurons. The references refer to characterizations given by the producer and/or to previous use
of the antibodies in our laboratory (mc, monoclonal; pc, ployclonal).
Primary antibody
Abbreviation
Host
Dilution
Source
References
Bombesin
Calcitonin gene-related peptide
Cholecystokinin 26–33
Choline acetyltransferase
b-Endorphin
Leu-enkephalin
Neuronal nitric oxide synthase
Neuropeptide Y
Serotonin
Substance P
Tyrosine hydroxylase
BBS
CGRP
CCK
ChAT
END
ENK
nNOS
NPY
SER
SP
TH
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rabbit, pc
Rat, mc
Rabbit, pc
1:500
1:1000
1:500
1:100
1:100
1:100
1:1500
1:500
1:2000
1:200
1:1000
Peninsula, Belmont, CA, USA
Amersham, Hannover, Germany
Peninsula
Chemicon, Temecula, CA, USA
ICN Biomedicals, Costa Mesa, CA, USA
Amersham
Laboserv Giessen, Germany
Amersham
Eugene Tech Int., Ridgefield Park, NJ, USA
Boehringer, Ingelheim, Germany
Eugene Tech Int., Ramsey, NJ, USA
Reuss (1991)
Reuss et al. (1999)
Reuss (1991)
Shiromani et al. (1990)
Bullock and Petrusz (1982)
Reuss et al. (1999)
Alm et al. (1993), Reuss and Reuss (2001)
Reuss and Olcese (1995)
Kawano et al. (1996)
Cuello et al. (1979), Reuss and Reuss (2001)
Reuss et al. (1999)
S. Reuss et al. / Hearing Research 248 (2009) 69–79
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To control for unspecific tracing due to possible spread of the
tracer into the middle ear, three animals received an additional
application of 1 ll cholera toxin subunit B (CTB, 1:1000 in PBS,
List, Campbell, CA, USA) into the middle ear. One of three sets
of sections from each of these rats was incubated in goat antiCTB (1:2000 in PBS, List), and the immunoreaction was visualized with biotinylated anti-goat IgG coupled to Cy2 (1:100 in
PBS, Rockland, Gilbertsville, PA, USA). In these cases, perikarya
labelled by CTB were found in a region medially and caudally
to the MoTTM group. However, MoTTM were not double-labelled
by both FG and CTB.
tion approximately is at the level of 9.7 mm posterior to bregma,
corresponding to fig. 58 of the rat stereotaxic atlas (Paxinos and
Watson, 1998).
Labelled cells, mostly exhibiting strong FG deposition, measured 24.6 and 11.2 lm in average in the longitudinal and vertical
axis, respectively. Neurons were counted from sections of seven
animals, and the numbers were corrected for double counting. A
mean of 162.8 ± 17.7 (ranging from 140 to 187) cells per animal
were labelled.
3. Results
Selected sections were immunohistochemically incubated in
antibodies directed against BBS, CCK, CGRP, ChAT, END, leu-ENK,
nNOS, NPY, SER, SP and TH. From the eleven antisera tested, six
resulted in immunohistochemical labelling of identified TTM motoneurons. Apparently all MoTTM were ChAT-positive, and different
portions of FG-labelled cell bodies exhibited immunoreactivity to
BBS, CCK, END, leu-ENK or nNOS (see below). Immunoreactivity to
CGRP, TH, SP, NPY or SER was not found in MoTTM.
The light-microscopical evaluation of incubated sections
showed that apparently all FG-neurons exhibited strong to moderate ChAT-staining (Fig. 1c–f). In the vicinity of double-labelled neurons, we also observed ChAT neurons that were not retrogradely
labelled by FG (asterisks in Fig. 1f).
3.1. Retrograde tracing of tensor tympani motoneurons
Following unilateral FG injection into the TTM, the tracer was
found in cell bodies and their processes in a distinct region restricted ipsilateral to the injection site. The labelled neurons were
located ventrally and ventrolaterally to the trigeminal motor nucleus. They were often seen in vertical rows or in clusters but
loosely arranged neurons were also observed (in some cases in
more caudal aspects of their location). A section in which labelled
neuronal somata are typically located ventrally to the TMN and
medially to the trigeminal motor root is shown in Fig. 1. The sec-
3.2. Immunoreactivity of FG-labelled cell bodies
Fig. 1. Location and characterization of neurons innervating the tensor tympani muscle (TTM) of the rat middle ear. (a) Hematoxiline-eosine (HE) stained section (medial is to
the right). (b) Higher magnification from the boxed area in (a). (c) Identified TTM motoneurons are labelled by FG upon injection of the substance into the muscle. (d) Choline
acetyltransferase (ChAT)-immunoreactivity in the same section. Higher magnifications from the top region are shown in (e,f). The arrows in (b–f) depict the same neuron. The
asterisks in (f) depict ChAT-positive neurons not labelled by FG. Scale bars = 300 lm (a), 50 lm (b–d), 20 lm (e,f). Abbreviations: TMN, trigeminal motor nucleus; m5,
trigeminal motor root; 7, facial nerve; py, pyramidal tract.
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S. Reuss et al. / Hearing Research 248 (2009) 69–79
We also found that a considerable portions exhibited immunoreactivity to BBS (33.2 ± 2.1% of FG cells, mean ± standard deviation) or CCK (36.8 ± 3.2%). Some of these neurons exhibited
relatively strong immunoreactivity while others were faintly labelled and were only hardly distinguished from background
(Fig. 2). They were included in the immunopositive neurons when
their staining was clearly above background as judged from higher
magnifications (see Fig. 2 c,d and g,h). The immunofluorescence
was not restricted to FG-labelled neurons but was observed also
in neighboring neurons and in other brain regions. For example,
the CCK-antiserum brightly stained neurons of the lateral olivary
nucleus (LSO) in the same sections.
Immunoreactivity to END was observed in many neurons in the
region under investigation (Fig. 3a–d). The reaction was relatively
weak and similar to that observed with BBS and CCK and often
stained only parts of the perikaryon. The immunostained neurons
apparently included all cell bodies labelled retrogradely by FG
(Fig. 3 c,d).
A relatively low level of leu-ENK-immunoreactivity was observed in many neurons of the studied region. It was found in
25.2 ± 2.3% of the FG-labelled neurons (Fig. 3e–h).
Bright staining of the cell soma, in contrast, was found when
immunoreactivity to nNOS was investigated. We observed that
32.5 ± 1.2% of the FG-labelled neurons were nNOS-ir. A section
exhibiting a high level of double-labelling is shown in Fig. 4. Neurons
that showed only nNOS-ir (asterisk in Fig. 4d) were also seen in the
region. While the incubations with BBS-, CCK-, END- and leu-ENKantibodies labelled mainly the neuronal perikaryon with leaving
processes rather unstained, we observed that nNOS-ir structures included cell bodies and fibers. Some appeared to be processes of ir
perikarya that were also FG-labelled. Since more nNOS-ir fibers than
FG-labelled fibers were seen in the region (Fig. 4c,d), some nNOS-ir
processes may be axons originating elsewhere.
Immunoreactivity to CGRP, NPY, SER, SP or TH was not found in
identified MoTTM or in other cell bodies of the region. While NPY,
SER, SP and TH were found in fibers and terminals in the region
Fig. 2. Localization of retrogradely labelled motoneurons following application of Fluoro-Gold (FG) into the tensor tympani muscle and neuropeptide immunoreactivity in
the same sections. (a,b) FG and bombesin (BBS). Higher magnifications are given in (c,d). The neuron depicted by an arrow in (a) and (c) clearly is BBS-ir, while the other two
FG-labelled cells exhibit only faint BBS-labelling. FG-containing neurons in another section and cholecystokinin (CCK)-immunoreactivity is shown in (e,f), demonstrated in
higher magnification in (g,h) where the neuron depicted by arrow is double-labelled. Scale bars = 50 lm (a,b,e,f), 20 lm (c,d,g,h).
S. Reuss et al. / Hearing Research 248 (2009) 69–79
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Fig. 3. Localization of retrogradely labelled motoneurons following application of Fluoro-Gold (FG) into the tensor tympani muscle and neuropeptide immunoreactivity in
the same sections. (a,b) FG and endorphin (END). Higher magnifications are given in (c,d), where it is seen that most FG-labelled neurons exhibit faint BBS-labelling. The same
neuron is depicted by an arrow in (a,c,d), for comparison. (e,f) Retrogradely labelled motoneurons and immunoreactivity to enkephalin (leu-ENK) in the same section. The
neurons to the right of the asterisk are shown in (g,h) in higher magnification where the one depicted by arrow exhibits leu-ENK-immunolabelling. Scale bars = 50 lm
(a,b,e,f), 20 lm (c,d,g,h).
that included the location of FG-neurons, CGRP-ir was not seen
here but was clearly present in neuronal somata of the lateral
superior olive.
3.3. Immunoreactive structures in the vicinity of MoTTM
In the region where identified MoTTM somata were located, we
observed distinct immunoreactivity to TH, SP, NPY and SER. The
overlapping zones of immunofluorescence and FG-neurons were,
however, different between the substances tested. Figs. 5 and 6
demonstrate the location of ir fiber systems in relation to FG-labelled motoneurons.
Tyrosine hydroxylase-ir fibers were seen in a dense plexus in
the brainstem. While most TH-ir fibers were found medially to
the location of FG-labelled perikarya, a region of overlapping was
observed in which both ir fibers and retrogradely labelled cell
bodies were present (Fig. 5a,b). In some instances, ir thickenings
were observed that appear in close contact to a FG-neuron
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S. Reuss et al. / Hearing Research 248 (2009) 69–79
Fig. 4. (a,b) Localization of retrogradely labelled motoneurons following application of Fluoro-Gold (FG) into the tensor tympani muscle and immunoreactivity to neuronal
nitric oxide synthase (nNOS) in the same section. The higher magnification is given in (c,d). While most FG-labelled neurons are nNOS-immunoreactive, some are not (arrows
in (c)) and NOS-neurons not retrogradely labelled by FG were also seen (asterisk in (d)). Scale bars = 50 lm (a,b), 20 lm (c,d).
(Fig. 6a), while this was rarely seen at neighboring unlabelled neurons. On the whole, however, it appeared that most TH-fibers pass
through the region of MoTTM rather than contacting them.
Substance P was observed in a dense plexus the location of
which partly matched the groups of FG-labelled neurons. As seen
in Fig. 5d, the highest amounts of SP-ir structures were situated
more dorsally than the FG-cells (Fig. 5c). In the more ventral parts,
from which the higher magnifications in Fig. 5e,f were taken, we
observed dense accumulations of SP-ir structures surrounding
FG-neurons. In these cases, demonstrated also in Fig. 6b, the
light-microscopical study using high magnification suggested that
FG-labelled neurons were contacted by SP-ir structures. In the
same sections, we observed a group of strongly stained SP-ir perikarya in the motor root of the trigeminal nerve from which fibers
ran in a dorsomedial direction aiming at the FG-labelled cell group.
A relatively weak concentration of neuropeptide Y-immunofluorescence was observed. However, we found in few cases that
NPY-ir thickenings were in close vicinity to retrogradely labelled
neurons (Fig. 6c). These were concentrated in the dorsal parts of
the MoTTM-containing regions.
Finally, we saw SER-ir fibers in a dense plexus that was located
mainly dorsally to the MoTTM region but extended ventrally to
clearly cover the region of identified MoTTM. Fibers of fine caliber
with small varicosities were located distinctly close to FG-cell
bodies (Fig. 6d).
4. Discussion
4.1. Location and number of identified motoneurons
The present data are the first to demonstrate efferent transmitters in motoneurons of the middle ear tensor tympani muscle in
rats. We also provide some evidence for the character of their afferent innervation. We identified MoTTM by Fluoro-Gold injection
into the muscle and found retrogradely labeled neurons in a region
ventral and ventrolateral to the trigeminal motor nucleus ipsilateral to the injection site.
In an early study (Szentagothai, 1949), the representation of the
TTM was determined within the posteroventral part of the trigeminal motor nucleus (TMN). However, tracing studies using horseradish peroxidase (HRP) or pseudorabies virus injections into the
TTM unanimously resulted in retrograde staining of a neuronal
group outside the TMN in several mammalian species such as rabbits, cats and guinea pigs (Mizuno et al., 1982; Shaw and Baker,
1983; Strutz et al., 1988; Takahashi et al., 1984). In particular in
rats, MoTTM were found in a parvocellular column or cluster ventrolateral to the magnocellular division of the ipsilateral TMN,
extending rostrally toward the medial aspect of the lateral lemniscus (Billig et al., 2007; Rouiller et al., 1986; Spangler et al., 1982).
Our findings are thus in accordance to the literature, and also
confirm the approximate number of motoneurons labelled. With
a mean of 163, our results are in agreement with previous rat studies (Billig et al., 2007; Rouiller et al., 1986; Spangler et al., 1982).
Evidence that all motoneurons were labelled in the present study
comes from earlier experiments in our laboratory (not included
in the present study) showed that decreasing the amount of tracer
to approximately 100 nl reduced the number of labelled TTM
motoneurons. Increasing the amount to up to 1 ll did not significantly increase the number but augmented unspecific labelling,
possibly due to tracer spilling into the middle ear (see also Section
2).
In guinea pigs, 300–400 HRP-filled neurons were described
(Mizuno et al., 1982; Strutz et al., 1988). In cats, up to 700–800
motoneurons were labelled (Ito et al., 1987; Mizuno et al., 1982;
S. Reuss et al. / Hearing Research 248 (2009) 69–79
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Fig. 5. Demonstration of the localization of retrogradely labelled motoneurons following application of Fluoro-Gold (FG) into the tensor tympani muscle and of ir fiber
systems in the same sections. (a,b) FG and tyrosine hydroxylase (TH). The region containing FG-neurons contains less TH-ir fibers than the region medial to it. Retrogradely
labelled motoneurons and immunoreactivity to substance P (SP) in the same section are seen in (c,d) and in (e,f) in higher magnification. Immunoreactive fibers and terminals
are seen in close vicinity to some retrogradely labelled neurons (asterisks). Scale bars = 50 lm (a–d), 20 lm (e,f).
Shaw and Baker, 1983), while in the monkey Macaca fascicularis
only 150 MoTTM per animal were observed (Gannon and Eden,
1987). This may accompany differences in TTM function between
mammalian non-primate and primate species, what was indeed
postulated previously (see Section 1).
4.2. Transmitter immunoreactivities of identified motoneurons
The application of different antibodies then showed that apparently all traced neurons exhibited ChAT-ir, supporting the assumption that they all were motoneurons. Thus, our study demonstrates
by combined retrograde tracing and immunohistochemistry that
MoTTM are cholinergic. ChAT was preferentially found in caudal
brain regions, including the TMN in the present and in previous
studies (Cortes et al., 1990; Saad et al., 1999; Tatehata et al.,
1987). It is likely that ChAT-positive, FG-negative neurons
observed in the present study are not motoneurons but rather project to neural sites such as cerebellum and oculomotor complex, as
was shown previously for neurons of this region (Graybiel and
Hartwieg, 1974; Grottel et al., 1986).
The next question was whether MoTTM may belong to different
subpopulations with regard to their co-localized neurotransmitter
or neuromodulator substances. Our incubation studies testing the
possible presence of ten additional antigens known to have neuromodulatory effects in the central nervous system showed that distinct portions of the FG-labelled neurons were ir to BBS, CCK, END,
leu-ENK or nNOS, while other substances studied were not found
in motoneuron perikarya. However, we observed that fibers and
terminals ir to TH, SP, NPY or SER were found in close spatial relations to MoTTM (see below).
Muscles that are involved in the masticatory system are often
modulated by the ‘‘satiety peptides” CCK and BBS. This seems to
be similar for some non-masticatory muscles such as the tensor
tympani muscle since approximately one-third of the FG-labelled
neurons of the present study was ir to CCK and/or BBS. Since we
did not accomplish double immunofluorescence detection of these
peptides, we cannot tell whether there is overlap between both
motoneuron pools. The staining generally was rather faint and
not restricted to MoTTM. In the TMN, BBS-immunoreactivity did
not appear to exceed background level, whereas we observed
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S. Reuss et al. / Hearing Research 248 (2009) 69–79
Fig. 6. Dual color imaging of perikarya showing FG-fluorescence (in green false color) upon injection of the substance into the TTM and immunofluorescence related to
neuroactive substances (in red). (a) Tyrosine hydroxylase (TH), (b) substance P (SP), (c) neuropeptide Y (NPY), (d) serotonin (SER). Scale bars = 5 lm.
CCK-ir staining of respective neurons. This is in accordance to previous studies reporting that many cells in the rat TMN express
CCK-mRNA (Cortes et al., 1990; Sutin and Jacobowitz, 1990). In
the trigeminal motor nucleus, CCK coexists with acetylcholine
(ACh), and this may similarly be the case in MoTTM. Although
the concrete role of CCK produced by MoTTM is at present unclear,
it is likely that it modulates the release of ACh at the neuromuscular junction (Cortes et al., 1990). The presence of CCK and BBS in
both, MoTTM and masticatory motoneurons again points to the
hypothesis that activities of both systems are related (Ramirez
et al., 2007).
A similarly weak cytoplasmic expression was found for endorphin that was however observed in every retrogradely labelled
neuron. It was also described previously to be moderately present
in the TMN (Zamir et al., 1984).
Leu-enkephalin-immunoreactivity was found in neurons of the
region. As seen for BBS, CCK and END, the staining was weak in
most cases. The comparison with the results of the FG-tracing
showed that approximately one quarter of MoTTM contained
leu-ENK, although mainly at low levels. Previously, some leuENK-ir perikarya were found in the rat TMN (Khachaturian et al.,
1983) but only upon colchicine treatment. The alternative splicing
form, methionine-enkephalin (met-ENK) was observed in various
auditory structures, including the cochlear nuclei (CN) and
superior olivary complex (Aguilar et al., 2004; Reuss et al., 1999;
Robertson and Mulders, 2000).
Another neuroactive substance produced by MoTTM is the gas,
nitric oxide. We identified the neuronal isoform of the NO-synthesizing enzyme (nNOS) in approximately one-third of retrogradely
labelled neurons. The enzyme was found also in other neuronal cell
bodies of the region and, furthermore, in cells of the TMN in the
present and in a previous study (Briski, 1999). A colocalization of
ACh and nNOS was observed in other parts of the central nervous
system such as the spinal autonomic regions (Wetts and Vaughn,
1994). Interestingly, NO-producing neurons were also detected in
other parts of the auditory system such as the cochlear spiral ganglion and the superior olivary complex (Reuss, 1998; Riemann and
Reuss, 1999; cf. Reuss and Riemann, 2000). The functions of NO, in
general, include the long-term depression or potentiation of synaptic transmission. Its actions are mediated via the stimulation of
cytosolic soluble guanylyl cyclase that is often located in adjacent
cells and whose activation results in increased production of the
second messenger cGMP (cf. Garthwaite, 2000). Although it is open
what the specific role of nitric oxide for MoTTM or neighboring
cells is, it is conceivable that the gas influences the excitability of
neurons involved in the tensor tympani reflex pathway.
Our findings of particularly rich nNOS-ir fiber networks among
MoTTM indicate that not all of them belong to these neurons but
rather may originate in other brain regions such as the ventral
CN and periolivary cell groups. In both regions, ir neuronal perikarya had been detected (Riemann and Reuss, 1999; Zheng et al.,
2006; present study), and both regions provide MoTTM innervation (see below).
We did not detect CGRP-immunoreactivity in identified MoTTM. In the same sections, however, the substance was observed
in the LSO and in some TMN neurons, where low levels of CGRPmRNA were previously detected in few neurons (Cortes et al.,
1990; Fukuoka et al., 1999). Whether CGRP-mRNA is present also
in MoTTM and whether the protein may be produced in very low
amounts (below the immunohistochemical detection limit) is unknown. Our results, however, suggest that the substance does
not serve as a transmitter of these motoneurons.
Notably, animals were not treated with the unspecific axonal
transport blocker colchicine in the present study. This may have
led to an underestimation of the true number of neurons that produce a substance under investigation in cases where the amount is
below the detection limit. For example, the use of relatively high
doses of colchicine was required to demonstrate leu-ENK perikarya
in the rat brainstem (Khachaturian et al., 1983). The underestimation may be the case with BBS and CCK immunoreactivity. We
counted these neurons only when immunoreactivity was clearly
above background fluorescence. Doubling the incubation time or
the concentration of antibodies in these cases did not significantly
improve the strength of specific immunoreactivity (but in some
cases increased background staining). It is thus not probable that
low immunoreactivity is due to insufficient antiserum concentration since neurons in other brain regions were heavily stained
(e.g., CCK in the LSO).
Our results suggest that MoTTM use ACh as the classical neurotransmitter, several neuropeptides such as BBS, CCK, END and leuENK as cotransmitters, and NO as a gaseous neuroactive substance.
The functions of coexisting neuropeptides include modulation of
S. Reuss et al. / Hearing Research 248 (2009) 69–79
the effects of classical neurotransmitters; however, the functional
significance of these coexistences for MoTTM remains to be
elucidated.
4.3. Possible inputs to identified MoTTM
Our immunofluorescence investigations demonstrated that four
out of eleven tested antigens were found in structures in close
vicinity to FG-backfilled neurons, i.e., fibers and putative terminals
ir to serotonin, substance P, tyrosine hydroxylase and neuropeptide Y. The serotonergic projection to MoTTM in O. garnettii was
demonstrated previously in the only report available so far on
afferent contacts to these neurons (Thompson et al., 1998). Our
findings in rats of SER-ir fibers and terminals in close spatial relation to identified motoneurons support the view that MoTTM are
contacted by serotonergic afferents. We observed a distinct concentration of fibers over and close to FG-labelled neurons while
there was a relatively homogenous distribution of ir fibers in the
TMN. The function of SER for MoTTM regulation is supported also
by the presence of serotonin receptors in rabbit group k neurons
(Kolta et al., 1993). (Thompson et al., 1998) suggested that serotonergic fibers, originating in the midline raphe, may present an integral component of the tensor tympani reflex pathway.
Furthermore, Manaker and Zucchi (1998) reported that substance P binding sites are expressed in rat trigeminal motoneurons.
A dense plexus of SP-ir fibers was seen in the MoTTM area. The
evaluation of neighboring regions showed that respective immunoreactivity was clearly reduced outside the MoTTM area. Our data
reveal that the high concentration of SP-ir structures close to MoTTM may be interpreted as a strong afferent pattern. Although the
neuropeptide is distributed over many regions of the mammalian
brain and body and is known to be involved in many neural mechanisms, the role of SP in the regulation of MoTTm is unknown. It
has been suggested that SP excites MoTTm similar to hypoglossal
motoneurons (Manaker and Zucchi, 1998). Respective fibers may
originate in the VCN (see below) where some immunoreactive
neurons were seen in the present study. Alternatively, the motor
root of the trigeminal nerve that stained positive for SP in the present study may send fibers to MoTTM.
In contrast to the rather dense occurrence of SER and SP close to
MoTTM, the distribution of NPY-ir structures was rather low in the
region under investigation, including the TMN. Relatively few
MoTTM appeared to be neighbored by NPY-ir structures, but these
putative contacts were relatively obvious. We did not observe NPYir perikarya in the MoTTM region, while moderate numbers of
NPY-ir cell bodies were found in the TMN of cat and man (Covenas
et al., 1990; Fodor et al., 1992).
The light-microscopical evaluation of tyrosine hydroxylaseimmunoreactivity further revealed that most ir fibers pass through
rather than terminate in the MoTTM region, and that most of these
fibers were located medially to MoTTM. In this region, bundles of
TH fibers were seen while rather singular structures were observed
close to MoTTM. Immunoreactive thickenings were clearly seen at
motoneurons suggesting a (sparse) catecholaminergic innervation.
Since TH, the first enzyme in catecholamine synthesis, marks all
three types of catecholaminergic neurons (i.e., dopaminergic, norepinephrineric and epinephrinergic), it is presently open to which
of these types the observed fibers may belong. It is further unknown where the fibers originate but from the evaluation of our
material we gained the impression that A5 noradrenergic cells
(cf. Hökfelt et al., 1984) may send fibers into the MoTTM region.
This area, however, is not among those yet known to innervate
TTM motoneurons (see below), but it provides noradrenergic input
to trigeminal motoneurons (Min et al., 2007).
The present results suggest that MoTTM are innervated by
structures containing serotonin and substance P and, to a lesser ex-
77
tent, tyrosine hydroxylase and neuropeptide Y. It should be noted,
however, that our study concentrated on the demonstration of
putative contacts at the neuronal soma or at proximal processes.
For technical reasons, possible contacts at more distal parts of axons or dendrites were difficult to demonstrate by light microscopy.
An intracellular HRP study of cat MoTTM demonstrated large dendritic trees extending far beyond the distribution of trigeminal nuclear boundaries (Friauf and Baker, 1985). It is believed that TTM
motoneurons receive multiple sensory inputs what is supported
also by the location of its dendrites close to the auditory SOC and
to trigeminal somatosensory structures.
4.4. Putative sources of MoTTM innervation
The question arises as to where the ir structures observed close
to MoTTM may originate. Upon lesioning, tract-tracing and electrophysiological experiments, some brain regions were candidates to
provide the innervation of these neurons, viz. the cochlear nuclei, a
group of neurons located near the superior olivary complex, and
the pontine reticular formation.
Data on the cochlear nucleus stemming from the last thirty-five
years gave inconsistent results ranging from ipsilateral ventral cochlear nucleus (Borg, 1973), bilateral ventral and dorsal CN (Ito and
Honjo, 1988; Itoh et al., 1986) to bilateral VCN (Billig et al., 2007).
As part of acoustic middle ear reflex pathways, however, the VCN
at least sends fibers to motoneurons innervating the TTM. CN neurons are glycinergic or GABA-ergic but little is known about which
other neuroactive substances they may contain. In our study and in
a previous report (Wynne and Robertson, 1997), some neurons in
the CN were faintly stained for SP, and this may well be the origin
of respective MoTTM innervation.
The transneuronal viral tracing technique demonstrated that
MoTTM are further innervated by periolivary neurons located
bilaterally near the nuclei of the trapezoid body (Rouiller et al.,
1986). The authors, however, did not detect labelled neurons in
the CN that was the major target in a recent study using virus tracing to identify MoTTM innervation (Billig et al., 2007). Periolivary
neurons were not fully characterized yet by immunohistochemistry although some are known to contain, e.g., neuronal NOS.
Last, the dorsolateral part of the pontine reticular formation
sends many fibers to the region where MoTTM are located in rats
(Li et al., 1995). In humans, this region contains both SP- and THir neuronal perikarya (Halliday et al., 1990; Kitahama et al.,
1996). If this holds true also for rats, these neurons may represent
the source of respective afferent or through-passing fiber systems
as seen in this study.
An interesting point is the comparative view at the motoneuron
neurochemistry of both middle ear muscles. Our recent study on
the stapedius muscle motoneurons (Reuss et al., 2008) was, for
technical reasons, not as comprehensive as the present study.
However, we observed some discrepancies and similarities between both motoneuronal pools. Stapedius motoneurons use CGRP
as a cotransmitter to ACh but do not produce nitric oxide. Vice versa, tensor tympani motoneurons do not express CGRP but are nitrergic. Similarities include that both motoneuron groups do not
express serotonin or substance P but exhibit close spacial relationship to structures ir to these neuroactive substances. It remains unknown what this may imply for the function of these motoneurons
and their muscles and from where the respective input may originate. Transsynaptic tracing studies were conducted only for the
TTM (see above), so that we may only speculate about the location
of stapedius premotor neurons (see Reuss et al., 2008).
In conclusion, our present results suggest that motoneurons of
the rat tensor tympani muscle are cholinergic and use - to different
extents - bombesin, cholecystokinin, endorphin and leu-enkephalin as well as nitric oxide as neurotransmitter or -modulator sub-
78
S. Reuss et al. / Hearing Research 248 (2009) 69–79
stances. We also provide morphological evidence that these motoneurons receive input by structures immunoreactive to serotonin,
substance P, tyrosine hydroxylase or neuropeptide Y.
Acknowledgments
The authors thank U. Disque-Kaiser for excellent technical
assistance. Data in this study are part of a thesis presented by I.
Kühn in partial fulfillment of her M.D. degree at the Johannes
Gutenberg-University, Mainz. This work was supported by the
Deutsche Forschungsgemeinschaft (Re 644/3-1), the University
Medical Faculty Science Program (MAIFOR) and the SchleicherStiftung (Baden-Baden).
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