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Immunohistochemical Evaluation of Cholinergic Neurons
in the Rat Superior Olivary Complex
Department of Otolaryngology, Medical College of Ohio, Toledo, Ohio 43699-0008
acetylcholinesterase; choline acetyltransferase; vesicular acetylcholine transporter
The cholinergic system in the rat superior olivary complex (SOC) was evaluated by
immunohistochemistry for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter
(VAChT) and histochemistry for acetylcholinesterase (AChE). ChAT-positive somata were found
mostly in the lateral superior olive (LSO) and ventral nucleus of the trapezoid body (VNTB). In the
LSO, there were both rostral-caudal and medial-lateral gradients in concentration of ChAT-positive
somata; the highest concentration was in the middle of the rostral-caudal extent and the most
medial part. The estimated total number of ChAT-positive neurons in the LSO was similar to
previous estimates of the total number of lateral olivocochlear neurons. Two groups of ChAT-positive
somata were found in the VNTB: a dorsolateral group of larger, multipolar, and more darkly labeled
neurons and a ventromedial group of smaller, oval, and more lightly labeled neurons, which was
about 5 times as numerous. There was a caudal-to-rostral increase in number of neurons in each
group. VAChT immunoreactivity, predominantly localized in puncta, was seen in LSO, VNTB, and
LNTB, and, to a lesser extent, in other parts of the SOC. VAChT-positive somata were also found in
the VNTB and medial LSO. This distribution pattern of VAChT was generally similar to that of
ChAT. AChE labeling had a similar appearance to ChAT labeling in the VNTB but differed in the
LSO, where AChE labeling was lighter and associated more with neuropil than with somata.
Microsc. Res. Tech. 41:270–283, 1998. r 1998 Wiley-Liss, Inc.
The superior olivary complex (SOC) is a major auditory center in the lower brain stem, which not only
relays ascending auditory information bilaterally from
the cochlear nucleus (CN) to higher levels but also
provides important descending pathways to the cochlea
and CN (Spangler and Warr, 1991). The projections
from the SOC to the cochlea are described as the lateral
and medial olivocochlear bundle (OCB) systems. The
lateral OCB of the rat originates almost entirely from
neurons in the ipsilateral lateral superior olive (LSO)
and terminates on the peripheral terminals of auditory
nerve fibers synapsing with inner hair cells. The medial
OCB originates bilaterally, mostly contralaterally, from
the ventral nucleus of the trapezoid body (VNTB) and
terminates on the outer hair cells (White and Warr,
1983). Projections from the SOC to the CN include some
collaterals of the OCB as well as other fibers which
project via the trapezoid body (Benson and Brown,
1990; Osen et al., 1984; Sherriff and Henderson, 1994;
Spangler and Warr, 1991; Warr, 1992; Warr and Beck,
1996; White and Warr, 1983). Although many neurotransmitters may be involved in descending auditory
pathways (Godfrey et al., 1988b; Potashner et al., 1993;
Spangler and Warr, 1991; Wenthold, 1991), ample
evidence has established that acetylcholine (ACh) is
important in the OCB systems and other projections
from the SOC to the CN (Altschuler et al., 1985;
Godfrey and Ross, 1985; Godfrey et al., 1987a,b; Jasser
and Guth, 1973; Osen et al., 1984; Sherriff and Henderson, 1994; Vetter et al., 1991). The exact roles of the
cholinergic pathways in hearing mechanisms are still
unclear. It has been proposed that the OCB system may
be important for modulating cochlear sensitivity to
sound, that its branches to the CN may be important for
resetting neuronal intensity coding to compensate for
the adjustments of cochlear sensitivity (Benson and
Brown, 1990), and that during cochlear pathology this
feedback system might be involved in a mechanism of
tinnitus (Levine and Kiang, 1995). Moreover, there is
evidence that the OCB system has important influences
on the early development of cochlear function (Walsh
and McGee, 1997) and that neurons of the LSO, including lateral OCB neurons, show plasticity after cochlear
lesions (Illing et al., 1997a,b).
There is limited information about the organization
of the cholinergic neurons in the SOC and their relation
to the OCB. A delineation of this organization is essential to an understanding of the roles of these cholinergic
Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; ANOVA,
analysis of variance; ChAT, choline acetyltransferase; CN, cochlear nucleus;
LNTB, lateral nucleus of the trapezoid body; LSO, lateral superior olive; LSO-b,
border of lateral superior olive; LSO-i, intermediate limb of lateral superior olive;
LSO-l, lateral limb of lateral superior olive; LSO-m, medial limb of lateral
superior olive; MNTB, medial nucleus of the trapezoid body; MSO, medial
superior olive; OCB, olivocochlear bundle; SOC, superior olivary complex; SPN,
superior paraolivary nucleus; VAChT, vesicular acetylcholine transporter; VNTB,
ventral nucleus of the trapezoid body; VNTB-l, group of more lateral and larger
VNTB ChAT-positive neurons; VNTB-m, group of more medial and smaller
VNTB ChAT-positive neurons.
Contract grant sponsor: NIH; Contract grant number: DC 00172.
*Correspondence to: Weiping Yao, Department of Otolaryngology, Medical
College of Ohio, P.O. Box 10008, Toledo, OH 43699-0008. E-mail:
Received 15 May 1997; Accepted in revised form 12 September 1997
neurons in the plasticity of the auditory system. The
present report represents a step toward this delineation in the rat. We used, as markers of cholinergic
structures, antibodies against choline acetyltransferase (ChAT), the synthetase for ACh, and vesicular
acetylcholine transporter (VAChT), which facilitates
packaging of ACh into neurotransmitter vesicles in
cholinergic terminals and has been used recently as a
marker for localizing especially cholinergic terminals
(Gilmor et al., 1996). Because of the consistency and
clarity of the soma labeling with the ChAT antibody, we
were able to quantitatively estimate the numbers,
sizes, and labeling densities of ChAT-immunopositive
somata in rat SOC regions. We also compared the
results of ChAT and VAChT immunohistochemistry
with our preparations of histochemistry for acetylcholinesterase (AChE), the degradative enzyme for ACh,
which has long been used as a marker in studies of
cholinergic systems.
Young adult male rats weighing 250–350 g were used
in this study. Their care and use were approved by the
National Institutes of Health (NIDCD, DC 00172) and
by the Medical College of Ohio Institutional Animal
Care and Use Committee.
The immunohistochemical procedures basically followed the antibody providers’ recommendations and
were similar to those previously reported (Yao and
Godfrey, 1995). The animals were, under sodium pentobarbital anesthesia, intracardially perfused with 3–4%
paraformaldehyde in phosphate-buffered saline (PBS,
pH 7.4). Brain blocks were isolated and cryoprotected
with 30% sucrose in PBS. Transverse sections 30-µm
thick were cut and collected in cold PBS. Two sets of
every fourth section were immunostained, one for ChAT
and an adjacent set for VAChT (the other two sets were
used for other experiments). Antibody dilutions with
PBS containing 5% normal rabbit serum and 0.2%
Triton X-100 were 1:4–6 for anti-ChAT antibody (monoclonal, Boehringer Mannheim, Inc., Indianapolis, IN)
and 1:1,000 for anti-VAChT antibody (monoclonal,
INCSTAR, Stillwater, MN). After one overnight incubation at 4°C with primary antibody, the sections were
processed with a Vectastain ABC kit and diaminobenzidine as chromogen. Sections were mounted on slides for
light microscopic studies. Controls in which primary
antibodies were replaced with normal serum showed no
labeling above background. For ChAT immunohistochemistry, we did not include 15% picric acid in the
fixative, as recommended by the antibody provider
(Boehringer Mannheim, Inc.) for optimal staining of
terminals (Yao and Godfrey, 1993). This was because we
were using the same animals for VAChT immunohistochemistry as well as ChAT and were not primarily concerned with ChAT-labeled terminals, but rather somata. Nevertheless, there was no substantial difference
in terminal labeling for ChAT in these sections from
what we have seen with the inclusion of picric acid.
Histochemistry for AChE involved preparations in
which frozen 10-µm-thick brain sections were incubated for 1–2 hours in a buffered (pH 5.6) medium
containing acetylthiocholine iodide, cupric sulfate, potassium ferricyanide, and a blocker of pseudocholinesterase (El-Badawi and Schenk, 1967; Karnovsky and
Roots, 1964; Yao and Godfrey, 1993).
Imaging and Profile Counts
The SOC regions, corresponding to the terminology of
White and Warr (1983), including LSO, VNTB, medial
superior olive (MSO), medial nucleus of the trapezoid
body (MNTB), lateral nucleus of the trapezoid body
(LNTB), and superior paraolivary nucleus (SPN), were
recognizable in our preparations, after consulting Nisslstained sections from other rats. The boundaries of the
structures were traced using a computer program,
Neurolucida 2.1 (MicroBrightField, Inc., Colechester,
VT), and the locations, sizes (areas in square micrometers), and labeling densities of ChAT-positive soma
profiles were recorded on maps. Each staining density,
measured as a grey value within the range of 0 to 255
(lightest to darkest), was corrected by subtraction of the
average density of the spinal trigeminal tract, an
adjacent non-cholinergic structure, as a background
control, and compared with those of the facial nucleus,
a nearby cholinergic structure. Although these relative
staining density estimates do not provide direct quantitation of amounts of cholinergic elements, high correlations have been found between such estimates and
their respective assayed enzyme activities for ChAT
and AChE in the rat CN (Yao and Godfrey, 1995), and
between such measurements and antigen concentrations (Huang et al., 1996).
Labeled neurons were grouped, based on their locations and morphological features. The total number of
ChAT-positive neurons in each group was estimated by
counting labeled profiles. The criteria for counting
positively labeled profiles included elimination of small
fractions (,5–8 µm in diameter, proportional to soma
sizes for the various groups) of labeled structures
without somatic features and very lightly labeled profiles (occasional uncertainties, one or two per SOC
section, were resolved by comparing measured densities to the background; the required minimal density
for counting was the mean background density 1 2
standard deviations). The total count for all sections of
each SOC in each rat was multiplied by 4 since every
fourth section was counted. The mean from each side of
3 rats (n 5 6) was calculated. Our method of profile
counting is the most commonly used method in the
literature for estimating numbers of neurons (Coggeshall and Lekan, 1996), and the results should thus be
comparable to those of others using similar counting
methods in the rat SOC (Aschoff and Ostwald, 1987,
1988; Robertson et al., 1989; Vetter and Mugnaini,
1990, 1992; Vetter et al., 1991; White and Warr, 1983).
Although this method can only provide estimates, it
was the only reasonable choice for our series of every
fourth section in which intracellular details were usually obscured by the immunoreaction. Konigsmark’s
equation (4) (Konigsmark, 1970) was applied to correct
for overestimation of the total number of neurons
because of double-counting of somata cut by the sectioning (Coggeshall and Lekan, 1996). Both total and
corrected counts were recorded. The corrected ones
should be more accurate representations of the actual
numbers of neurons, although they may still be overes-
Fig. 1. Photomicrographs of ChAT immunoreactivity in SOC regions and the facial nucleus (FN). The
approximate locations of the SOC regions are indicated in the center drawing. Scale bar (50 µm) applies to
all photographs.
timates (Coggeshall et al., 1990). However, the uncorrected total numbers are also reported because they
should be more comparable to previous counts of OCB
neurons, for which correction factors were not applied
(Vetter and Mugnaini, 1992). Statistical comparisons
were made across groups using analysis of variance
(ANOVA, one-sided) and t-tests.
Appearance of Histochemistry
ChAT-positive (presumably cholinergic) somata were
mostly found in the LSO, the VNTB, and, to a much
lesser extent, in the LNTB. No ChAT-positive somata
were found in the MSO, SPN, or MNTB. ChAT-positive
somata varied in size, shape, and labeling density in
different parts of the SOC. Locations of ChAT-positive
somata were grouped under lateral, intermediate, and
medial limbs of LSO (LSO-l, LSO-i, LSO-m), the border
of LSO (LSO-b), which represents the capsular structure surrounding the LSO proper, the LNTB, the
lateral, larger-cell group of VNTB (VNTB-l), and the
medial, smaller-cell group of VNTB (VNTB-m). Figure
1 shows examples of ChAT-positive somata from these
designated regions or groups. Examples of ChAT-
Fig. 2. Photomicrographs of VAChT immunoreactivity in SOC regions and the FN. The approximate
locations of the SOC regions are indicated in the center drawing. Scale bar (50 µm) applies to all
positive somata of the facial nucleus (FN), a well-known
group of cholinergic neurons close to the SOC in the
brain stem, are included for comparison. From similar
locations, examples of VAChT immunohistochemistry
and AChE histochemistry are shown in Figures 2 and 3,
respectively. ChAT immunoreactivity was primarily
localized in the cytoplasm of somata and major proximal dendrites. There was also diffuse neuropil labeling
for ChAT in the SOC, more prominent in the lateral
aspect of the LSO and in the VNTB-m. The VAChT
immunoreactivity was predominantly localized in
puncta (presumably axon terminals) throughout the
SOC, more in the LSO, VNTB, and LNTB than in the
MNTB, MSO, and SPN. The VAChT-positive puncta
were mostly found near somatic profiles, in the LSO,
LNTB, VNTB-l, and MNTB, and sparsely along processes, in the LSO, MSO, LNTB, and VNTB. VChAT
immunoreactivity was also found in the cytoplasm of
some of the somata in the SOC, especially in the
VNTB-l, LSO-m, and LSO-i (Fig. 2). Darkly labeled
VAChT-positive puncta and somata were both seen in
the FN. Histochemical labeling for AChE was more
Fig. 3. Photomicrographs of AChE histochemistry in SOC regions
and the FN. The approximate locations of the SOC regions are
indicated in the center drawing. The association of AChE with
membranes and extracellular spaces (Yao and Godfrey, 1996) probably
contributes to the less clearly defined somatic profiles than in the
immunohistochemical preparations. Scale bar (50 µm) applies to all
Fig. 4. Soma profiles of ChAT-positive neurons in the rat SOC. The approximate locations of the SOC regions are indicated in the center
drawing. Scale bar in VNTB-l applies to all soma profile tracings. Bottom left: Comparison of soma profile areas. Analysis of variance (ANOVA)
indicates significant differences among SOC regions. Group vs. group comparisons (t-tests) indicate significant differences between LSO-b and
each LSO limb and VNTB-l, and between each LSO limb and VNTB-l, VNTB-m, and LNTB. Bottom right: Comparison of labeling densities
with those of facial motoneurons; ANOVA indicates significant differences among them. The average density of ChAT-negative somata in LSO,
MNTB, MSO, and SPN was 11% of that of facial motoneurons, less than half the values in SOC ChAT-positive somata. Comparisons (t-tests)
between groups indicate significant differences (P , 0.01), except between LSO-l and LSO-b, LSO-l and VNTB-m, and LSO-b and VNTB-m. The
lower densities in smaller somata may partly reflect the smaller proportions of the section thickness which they occupy, and darker neuropil
labeling in LSO may contribute to the measured densities of somata there. The data were measured on sections from four rats. Numbers of
profiles measured were 55 for LSO-l, 79 for LSO-i, 152 for LSO-m, 17 for LSO-b, 20 for LNTB, 81 for VNTB-l, 140 for VNTB-m, and 30 for FN.
Fig. 4.
Fig. 5.
Legend on page 278.
Fig. 5.
diffuse and primarily found in the neuropil of the SOC.
Relatively dark labeling for AChE activity was seen in
somata in the VNTB-l and the FN, closely associated
with the somatic membrane. Thus, as expected, ChAT
appeared to be a more definitive marker than VAChT or
AChE for identifying cholinergic somata. Therefore, the
results from ChAT immunohistochemistry were used
for quantitative analysis of cholinergic somata in the
To provide direct visual comparison of the shapes and
sizes of ChAT-positive neuronal somata in the LSO,
LNTB, VNTB, and FN, soma profiles were traced
(Schofield and Cant, 1996) using Neurolucida and
shown in Figure 4. These tracings provide an impression of the amounts of heterogeneity in soma appearance within and among groups. Within all limbs of the
LSO, ChAT-positive somata were small, oval to elongated. In LSO-b, labeled somata were more variable in
shape and size, but generally larger than those within
the LSO. The labeled somata in the LNTB and VNTB-l
were generally relatively large and multiangular in
shape, whereas those in VNTB-m were more similar to
those within the LSO. The areas and labeling densities
of ChAT-positive soma profiles, compared to the values
for facial motoneurons as an internal standard, are
shown in Figure 4. ANOVA indicated significant differences among the regions for both soma profile area and
ChAT labeling density.
The distributions of ChAT-positive somata in the
LSO and VNTB showed gradients along both lateralmedial and caudal-rostral axes (Fig. 5). The distributional pattern of VAChT immunoreactivity in the SOC
generally resembled that of ChAT, but the apparent
labeling of somata was less certain when observed at
higher magnification (Fig. 2). Histochemical labeling
for AChE activity generally corresponded to the ChAT
and VAChT immunoreactivities in the VNTB but not in
the LSO, where AChE labeling was less prominent.
Quantitative Analysis
The LSO is the origin of the lateral OCB in the rat
(White and Warr, 1983) and is tonotopically organized
(Friauf, 1992; Schwartz, 1992; Warr and Beck, 1996).
We arbitrarily divided the S-shaped LSO into 9 lateralto-medial sectors in order to quantitate the distribution
of ChAT-positive somata along its tonotopic axis. Figure
6 (top) shows increasing concentrations (number of
Fig. 5. Photomicrographs of ChAT immunohistochemistry, VAChT
immunohistochemistry, and AChE histochemistry in the rat SOC
across a caudal-rostral range of 480 µm, along with Neurolucida
tracings illustrating the locations of ChAT-positive somata. For each
column (series), top is caudal, bottom is rostral, and sections are at
120-µm intervals. Sections beyond each end, caudally and rostrally,
contained no recognizable LSO segments. Symbols in the tracing
indicate different sizes, shapes, and labeling densities of ChATpositive somata: solid circles are darkly labeled somata in LSO and
LSO-b; open diamonds are less darkly labeled somata in LNTB; solid
triangles are dorsolaterally located, larger, multipolar, and more
darkly labeled somata (VNTB-l) in VNTB; open circles are ventromedially located, smaller, round or oval, and less darkly labeled somata
(VNTB-m) in VNTB. Two cholinergic fiber tracts are included in the
sections: the bundle of ChAT-positive fibers lateral to the LSO is the
facial motor root, and the medial ChAT-positive bundles of fibers
traversing the MNTB are abducens nerve rootlets. For each figure, top
is dorsal and left is lateral, as indicated in top tracing. Scale bar (500
µm) applies to all photographs.
Fig. 6. Distribution of ChAT-positive somata in rat LSO. For
lateral-medial comparisons (top), the LSO is divided into nine sectors
by straight lines perpendicular to its S-shaped axis, and the numbers
1–9 in the inset correspond to the abscissa labels, from lateral to
medial. Sectors 1–3, 4–6, and 7–9 approximately correspond to the
lateral, intermediate, and medial limbs, respectively. Sector areas
ranged from 0.017–0.030 mm2 with a mode close to 0.025 mm2/sector.
Therefore, the spatial densities of ChAT-positive somata are expressed
as number per 0.025 mm2. The means and standard errors were
obtained from 30 LSO sections in 3 animals. ANOVA indicates
significant differences across the sectors (P , 0.001). For caudalrostral comparisons (bottom), the numbers of ChAT-positive somata
are plotted at five levels 120 µm apart. ANOVA indicates significant
differences across levels (P , 0.001).
somata/area) of ChAT-positive somata from lateral to
intermediate to medial LSO limbs. There is also a
lateral-to-medial increase in concentrations of ChATpositive somata within the intermediate and medial
limbs (4 to 6, and 7 to 9). In the caudal-to-rostral
direction, a symmetric distribution of cholinergic somata in the LSO was found (Fig. 6, bottom). ANOVA
indicated that the variations in number of ChATpositive somata along both lateral-medial (P , 0.001)
and caudal-rostral (P , 0.001) axes were statistically
significant. ChAT-positive somata within the LSO, the
TABLE 1. Estimated numbers of ChAT-positive somata in rat SOC
regions (mean 6 SEM for 6 SOCs of 3 rats)
Total counted
soma profiles
440 6 33
54 6 9
461 6 14 2235 6 85
360 6 27
42 6 7
346 6 10 1744 6 64
were corrected by applying equation (4) of Konigsmark (1970) to somata.
The average soma radius was calculated from the average soma profile area (Fig.
4), and the diameter of uncounted fragments was estimated as 5–8 µm,
proportional to soma sizes of the different groups.
ergic neurons; (4) Within the VNTB, there are two
subpopulations of cholinergic neurons: a more dorsolaterally located group with larger, multipolar somata,
more darkly labeled for ChAT, and a more numerous
ventromedial group with smaller, round/oval somata,
more lightly labeled for ChAT; and (5) Cholinergic
terminals, as revealed by VAChT-positive puncta, are
most concentrated in the LSO and VNTB, similar to the
distribution of ChAT-positive somata. Whether these
terminals derive from the ChAT-positive neurons in
these nuclei or from elsewhere is not known.
region immediately surrounding it (LSO-b), and the
LNTB ventral to it were counted (Table 1). Of those
within LSO, 16% were in the lateral, 33% in the
intermediate, and 51% in the medial limb. Of those in
LSO-b, 50% were next to the lateral limb of the LSO,
17% next to the intermediate limb, and 33% next to the
medial limb.
Compared to the LSO, the VNTB extends for a much
longer distance along the caudal-rostral axis: 1.8 mm as
measured in our preparations. The distribution of
ChAT-positive somata along a 1.4-mm length of the
VNTB is shown in Figure 7. The larger ChAT-positive
somata were clustered more laterally (VNTB-l) and
dorsally, while the smaller ChAT-positive somata were
spread out more medially (VNTB-m) and ventrally. The
numbers of both groups of ChAT-positive somata were
counted (Table 1). From caudal to rostral in the VNTB,
the ChAT-positive somata in both groups showed significant (P , 0.01), although nonlinear, increases in number (Fig. 8).
The larger, more lateral ChAT-positive VNTB neurons are considered to give rise to the medial OCB
(Sherriff and Henderson, 1994; Vetter et al., 1991),
which provides a nonuniform innervation along the
basal-to-apical tonotopic axis in the outer hair cell
region of the rat cochlea (Dannhof and Bruns, 1993).
Since the VNTB has been reported to have a lateral
(low frequency) to medial (high frequency) tonotopic
organization (Friauf, 1992; Warr and Beck, 1996), we
wanted to examine the medial-lateral distribution of
the larger, darker ChAT-positive somata. To do this, we
measured their distances from the midline of the brain
stem (Fig. 9). The group of these neurons gradually
shifted laterally at increasingly rostral locations. As
would be predicted from the increasing number of
larger, darker ChAT-positive somata at successively
more rostral levels (Fig. 8), there were more located
laterally than medially within their medial-lateral
Comparisons to Previous Studies
Our observations of ChAT immunohistochemistry in
the rat SOC are generally in agreement with those in
the literature. For example, the results of Vetter et al.
(1991) suggested that ChAT-positive neurons were not
homogeneously distributed in rat LSO, with 14, 33, and
53% in the lateral, intermediate, and medial limbs,
respectively. Our data are similar (16, 33, 51%). Our
results further revealed a trend for a lateral-to-medial
increase in concentration of cholinergic neurons within
the intermediate and medial limbs of the LSO (Fig. 6),
and for a higher density of ChAT immunolabeling in
more medially located somata (Fig. 4). In addition, the
larger ChAT-positive somata at the border of the LSO
may correspond, at least in part, to the shell neurons of
Vetter and Mugnaini (1992) and are likely to represent
a group of neurons distinct from those inside the LSO
(Warr et al., 1997). Nevertheless, since these neurons
appear to project with the lateral OCB (Warr et al.,
1997), we included them in our total counts of LSO
neurons. In the VNTB, which corresponds to the combination of medioventral (MVPO) and rostral (RPO)
periolivary regions of Vetter et al. (1991), our results
agree with those of Vetter et al. (1991) and Sherriff and
Henderson (1994) in finding lateral ChAT-positive neurons that are larger and more darkly labeled than those
more medially located. Our results reveal, moreover,
that the lateral group is actually located dorsolaterally
and the medial group ventromedially. It can be noted
from Figure 4 that the smaller, medial somata (VNTBm), although they appear in sections to have lighter
ChAT labeling, do not have much lower measured
densities than the larger, lateral somata (VNTB-l). The
lighter appearance may merely reflect the smaller
proportion of the section thickness that they occupy
because of their smaller size. The caudal-to-rostral
variations in numbers of ChAT-positive neurons in the
LSO and VNTB have not been reported previously,
although a greater dorsal-ventral size of the VNTB
rostrally was noted by Vetter et al. (1991).
Cholinergic Neurons in the SOC
Based on our immunohistochemical results, the characteristics of cholinergic neurons in the rat SOC can be
proposed to be as follows: (1) Almost all cholinergic
somata are in the LSO and VNTB; (2) Within the LSO,
small cholinergic somata are distributed across all
limbs, but there is a distinct lateral-to-medial increase
in number and labeling density for ChAT; (3) At the
border of the LSO, a small group of cholinergic somata,
larger and more variable in shape than within the LSO
proper, may represent a distinct subpopulation of cholin-
ChAT-Positive LSO Neurons
It is difficult to compare our ChAT-positive neuron
counts with counts of OCB neurons to conclude definitely whether or not all lateral or medial OCB neurons
may be cholinergic. Differences in tracers, which are
probably not all taken up equally well (Aschoff and
Ostwald, 1988; Schofield and Cant, 1996), as well as
differences in section thicknesses and in assumptions
used for counting soma profiles, may lead to significant
differences in total counts (Coggeshall and Lekan,
1996). The estimated numbers of lateral and medial
OCB neurons for adult rats vary considerably across
studies: lateral 238–720; medial 213–583 (Aschoff and
Ostwald, 1987, 1988; Robertson et al., 1989; Vetter and
Mugnaini, 1990, 1992; Vetter et al., 1991; White and
Warr, 1983). Since these counts were not corrected for
double counting, our uncorrected counts should be more
comparable to them than our corrected counts. Some
previous studies suggested that as few as half the
lateral OCB neurons may be cholinergic (Vetter et al.,
1991), while others suggested that all are (Godfrey et
al., 1984). Our uncorrected count of LSO-region ChATpositive neurons (within LSO 1 LSO-b: 494) is similar
to those of Aschoff and Ostwald (1988, 540) and Robertson et al. (1989; 573) for lateral OCB neurons. This
similarity suggests that at least most, if not all, lateral
OCB neurons are cholinergic. The study of Vetter et al.
(1991), which estimated that about half the lateral OCB
neurons are ChAT-positive, found a much smaller number of ChAT-positive LSO neurons than we found. This
could have resulted from poor penetration of antibody
(Altschuler et al., 1984; Vetter et al., 1991), particularly
after the sections had been reacted for HRP activity,
and/or use of a polyclonal antibody, which, in our
preparations, gives less reliable labeling of LSO somata
than that used here. Additional work will be needed to
more confidently determine what proportion of lateral
OCB neurons is cholinergic.
The LSO is known to be tonotopically organized
(Friauf, 1992; Schwartz, 1992). From lateral-to-medial,
the characteristic frequencies of LSO neurons are arranged from low to high (Friauf, 1992; Guinan et al.,
1972; Tsuchitani and Boudreau, 1966). The lateral-tomedial increasing gradient of ChAT-positive neurons in
the LSO is consistent with a stronger cholinergic
influence on higher frequency auditory processing in
more basal parts of the cochlea. Cholinergic innervation of the cochlea by the lateral OCB has been documented as ChAT-positive puncta under the inner hair
cells (Altschuler et al., 1985), as measured ChAT and
AChE enzyme activities in the inner hair cell region
(Godfrey and Ross, 1985; Godfrey et al., 1986), and as
size of the AChE-positive inner spiral bundle (Firbas,
1978). The latter two measures correlate well with each
other and with the estimated distribution of lateral
OCB fibers to the inner hair cell region (Dannhof and
Bruns, 1993). In the rat inner hair cell region, measured ChAT enzyme activity and size of the inner spiral
bundle were much lower in the apical turn than in the
middle and basal turns. This is consistent with the
lower number of ChAT-positive neurons in the lateral
limb of the LSO. However, the finding that the highest
density of ChAT-positive somata is in the most medial
part of the LSO does not agree with the decreased size
of the inner spiral bundle in the most basal part of the
cochlea. Also, the distribution of CGRP-positive, presumed cholinergic, terminals in the inner hair cell
region does not correspond well with the medial-lateral
Fig. 7. Neurolucida tracing of ChAT-positive somata in a series of
sections spaced at 120-µm intervals through the rat VNTB, from a
different rat than in Figure 5. Top-to-bottom represents caudal-torostral. From top downward, the third through seventh sections
contain S-shaped LSO. Solid triangles represent larger and more
darkly labeled somata; open circles represent smaller and more lightly
labeled somata.
Fig. 8. Distribution of ChAT-positive somata along the caudalrostral axis of the VNTB. The S-shaped LSO is present at 480–960 µm.
VNTB-l represents larger, more darkly labeled, and more dorsolaterally located ChAT-positive somata (solid triangles in Figs. 5 and 7).
VNTB-m represents smaller, more lightly labeled, and more ventrome-
dially located somata (open circles in Figs. 5 and 7). The means and
standard errors were calculated from 6 series of VNTB sections in 3
animals. The increasing trend from caudal to rostral is statistically
significant by analysis of variance (P , 0.01) for each group of somata.
distribution of ChAT-positive somata in the LSO (Vetter
et al., 1991).
frey et al., 1987a,b). To the extent that these fibers
derive from the SOC, our results suggest that there are
few potential sources available besides the smaller
ChAT-positive VNTB neurons. Their large number is
sufficient to account for the amount of ChAT activity
that disappears from the CN when the trapezoid body is
completely cut, and their light staining for AChE
activity may correlate with its smaller decline in the
CN (Godfrey et al., 1983; Osen et al., 1984) than in the
cochlear inner hair cell region (Godfrey and Ross, 1985)
after transection of their centrifugal innervations. The
larger number rostrally of the smaller ChAT-positive
VNTB neurons could be consistent with the relatively
larger effects on CN ChAT activity of lesions including
more rostral damage (Godfrey et al., 1987b) and correlates with evidence for higher ChAT activities rostrally
than caudally in the trapezoid body (Godfrey et al.,
The rat VNTB has been reported to be tonotopically
organized in a lateral-to-medial low-to-high frequency
organization (Friauf, 1992; Warr and Beck, 1996). A
lateral-to-medial increasing gradient in number of
larger, darker ChAT-positive neurons (VNTB-l) might,
therefore, be expected to correlate with cochlear outer
hair cell region gradients of ChAT enzyme activities
(Godfrey and Ross, 1985; Godfrey et al., 1986) and
ChAT immunoreactivities (Altschuler et al., 1985; Dannhof et al., 1991; Eybalin, 1993), more prominent
basally than apically. Since only a few larger, darker
ChAT-positive somata are clustered in each transverse
section (Figs. 5,7), there is only a small range of
medial-lateral locations at any given rostral-caudal
level. Therefore, medial-to-lateral variations cannot be
easily assessed at one coronal plane. However, a wider
ChAT-Positive VNTB Neurons
In the VNTB, the counts of ChAT-positive neurons
are more involved because the somata are spread over a
larger volume and exist as two groups. In our preparations, the uncorrected number of darkly ChAT-labeled
large somata (461) is within the range of the counts of
medial OCB neurons, to which they correspond (Sherriff and Henderson, 1994; Vetter et al., 1991), similar to
those of Aschoff and Ostwald (1988; 556) and Robertson
et al. (1989; 474), the studies for which the LSO count
was also similar. This similarity is consistent with a
conclusion that most or all medial OCB neurons are
cholinergic (Dannhof et al., 1991; Vetter et al., 1991).
The medial OCB neurons are considered to send some
cholinergic collaterals to the CN (Benson and Brown,
1990; Godfrey et al., 1987a; Osen et al., 1984; Sherriff
and Henderson, 1994). The smaller, more lightly labeled ChAT-positive neurons in the VNTB (VNTB-m,
Figs. 7,8) are about five times as numerous as the larger
neurons. It is believed that they are, at least in part,
responsible for the cholinergic projections to the CN via
the trapezoid body route (Godfrey et al., 1987b) and do
not project to the cochlea (Sherriff and Henderson,
1994). These neurons do not stain darkly for AChE (Fig.
3) and may correspond to the more lightly AChElabeled neurons noted in cat by Adams (1989), who also
suggested that they contribute cholinergic descending
inputs to the CN. In the rat, approximately 65% of the
ChAT activity in the CN appears to be related to
centrifugal fibers entering from the trapezoid body, as
compared to only 20% related to OCB branches (God-
VNTB-m neurons, which are much more numerous,
may be important in cochlear nucleus plasticity. The
VNTB neuronal groups, with their widespread connections, may have particularly significant influences on
plasticity in the auditory system. Further, the VAChTpositive puncta in SOC regions suggest some intimate
cholinergic connections among SOC neurons, which
may be important for coordinating their activities.
We thank Dr. William Gunning and Mr. Edward
Calomeni of the Department of Pathology, Medical
College of Ohio, for their generous provision of photographic facilities and expert assistance. We also thank
Dr. W. Bruce Warr, Boys Town National Research
Hospital, for valuable consultations on olivocochlear
neurons. This work was supported by NIH grant DC
Fig. 9. Medial-lateral distribution of large ChAT-pozsitive VNTB
somata. Top: The mean 6 SD of soma locations at each rostral-caudal
distance. Bottom: The mean (6 SEM) numbers of somata (per series
of 12 transverse sections spaced at 120-µm intervals) located at
different distances from the midline are plotted. Each value on the
abscissa represents a 100-µm range centered at that distance. Data
are from 6 series of VNTB sections in 3 animals. ANOVA indicates
significant variance across distances (P , 0.001). By t-test, the number of ChAT-positive somata at 1,800–1,899 µm from the midline is
larger than that at 1,600–1,699 µm (P , 0.05).
range exists across the entire rostral-caudal extent of
the VNTB because of the lateral shift of the group of
these somata at increasingly rostral levels. In our
analysis, the distribution of the larger, darker ChATpositive somata showed more of them laterally than
medially, corresponding to lower and higher frequencies, respectively (Friauf, 1992; Warr and Beck, 1996),
and opposite to the expected distribution. This discrepancy suggests that our current knowledge of the tonotopic mapping in the VNTB is probably inadequate,
especially considering the several neuronal groups present there (Warr and Beck, 1996) and the differences
among studies regarding the definition of the VNTB.
Our results suggest that, if the cholinergic OCB
neurons are important in cochlear plasticity, the
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