Neuroscience 136 (2005) 907–925
THE INFERIOR COLLICULUS OF THE RAT: QUANTITATIVE
IMMUNOCYTOCHEMICAL STUDY OF GABA AND GLYCINE
M. MERCHÁN,a,b L. A. AGUILAR,a,b1
E. A. LOPEZ-POVEDAb AND M. S. MALMIERCAa,b*
a
Laboratory for the Neurobiology of Hearing, Department of Cell Biol-
ogy and Pathology, Faculty of Medicine, University of Salamanca,
Salamanca, Spain
b
Institute for Neuroscience of Castilla y León, University of Salamanca,
Salamanca, Spain
Abstract—Both GABA and glycine (Gly) containing neurons
send inhibitory projections to the inferior colliculus (IC),
whereas inhibitory neurons within the IC are primarily
GABAergic. To date, however, a quantitative description of
the topographic distribution of GABAergic neurons in the
rat’s IC and their GABAergic or glycinergic inputs is lacking.
Accordingly, here we present detailed maps of GABAergic
and glycinergic neurons and terminals in the rat’s IC. Semi-
thin serial sections of the IC were obtained and stained for
GABA and Gly. Images of the tissue were digitized and used
for a quantitative densitometric analysis of GABA immuno-
staining. The optical density, perimeter, and number of
GABA- and Gly immunoreactive boutons apposed to the so-
mata were measured. Data analysis included comparisons
across IC subdivisions and across frequency regions within
the central nucleus of the IC.
The results show that: 1) 25% of the IC neurons are GABAergic;
2) there are more GABAergic neurons in the central nucleus of
the IC than previously estimated; 3) GABAergic neurons are
larger than non-GABAergic; 4) GABAergic neurons receive
less GABA and glycine puncta than non-GABAergic; 5) dif-
ferences across frequency regions are minor, except that the
non-GABAergic neurons from high frequency regions are
larger than their counterparts in low frequency regions;
6) differences within the laminae are greater along the dor-
somedial–ventrolateral axis than along the rostrocaudal axis;
7) GABA and non-GABAergic neurons receive different num-
bers of puncta in different IC subdivisions; and 8) GABAergic
puncta are both apposed to the somata and in the neuropil,
glycinergic puncta are mostly confined to the neuropil.
© 2005 Published by Elsevier Ltd on behalf of IBRO.
1
Present address: University Cayetano Heredia, Division of Neuro-
science and Behavior, Lima, Peru.
*Correspondence to: M. S. Malmierca, Laboratory for the Neurobiol-
ogy of Hearing, Department of Cell Biology and Pathology, Faculty of
Medicine, University of Salamanca, and Institute for Neuroscience of
Castilla y León, Campus Miguel de Unamuno, s/n, 37007 Salamanca,
Spain. Tel: ⫹34-923-294500x1861; fax: ⫹34-923-294549.
E-mail address: msm@usal.es (M. S. Malmierca).
Abbreviations: CNIC, central nucleus of the inferior colliculus; DCIC,
dorsal cortex of the inferior colliculus; DNLL, dorsal nucleus of the
lateral lemniscus; F, flat neuron; GABA-IN, immunonegative neurons
for GABA; GABA-IR, immunopositive neurons for GABA; Gly, glycine;
Gly-IR, immunopositive neurons for glycine; GP, glutaraldehyde/
paraformaldehyde; IC, inferior colliculus; LCIC, lateral part of the
external cortex; LF, less-flat neuron; LSO, lateral superior olive;
MGB, medial geniculate body; NSS, normal swine serum; OD, optical
densitometry; PB, sodium phosphate buffer; TPBS, Tris-phosphate-
buffered saline; VCLL, ventral complex of the lateral lemniscus.
0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2004.12.030
907
Key words: amino acids, auditory pathway, inhibitory neuro-
transmitters, axosomatic endings, inferior colliculus, optical
densitometry.
Most ascending auditory tracts converge on the inferior
colliculus (IC), which is a major relay en route to the medial
geniculate body (MGB; Malmierca et al., 2002; Malmierca
and Merchán, 2004). Afferent projections to the IC are both
excitatory and inhibitory (Oliver, 1984a, 1987; Shneiderman
and Henkel, 1987; Saint Marie et al., 1989; Saint Marie and
Baker, 1990; Li and Kelly, 1992; Riquelme et al., 2001).
Likewise, projections from the IC to the MGB are also
excitatory and inhibitory (Winer et al., 1996; Peruzzi et al.,
1997; Bartlett et al., 2000).
Fast excitatory neurotransmission in the auditory sys-
tem is mainly mediated by the action of an excitatory amino
acid such as glutamate on AMPA receptors (Lerma et al.,
2001; Zhang and Kelly, 2001, 2003), whereas inhibition
depends largely on two neurotransmitters: GABA and gly-
cine (Gly). The functional roles played by GABA and Gly
have been reported in studies of the physiology (Rose
et al., 1963; Nelson and Erulkar, 1963; Kuwada et al.,
1997; Rees et al., 1997) and pharmacology (Faingold et al.,
1989, 1991; Roberts and Ribak, 1987; Oliver et al., 1994;
LeBeau et al., 1995, 1996, 2001; Zhang and Kelly, 2001,
2003; Malmierca et al., 2003) of IC neurons.
By contrast, anatomical studies on the distribution of
GABA and Gly in the IC are scarce. A number of them
have focused on the distribution of GABA, Gly and gluta-
mate receptors (Sanes et al., 1987; Glendenning and
Baker, 1988; Suneja et al., 1998; Marianowski et al., 2000;
LeBeau et al., 1995, 1996, 2001; Shiraishi et al., 2001;
Zhang and Kelly, 2001, 2003; Ma et al., 2002; reviewed in
Malmierca, 2003), but few have investigated the distribu-
tion of inhibitory neurons and their inputs in the IC (Roberts
and Ribak, 1987; Oliver et al., 1994; Winer et al., 1995). Of
these, the most detailed study is that of Oliver et al. (1994)
in the cat, which showed that up to 20% of the neurons are
GABAergic and GABA immunoreactive (GABA-IR) neu-
rons differ from GABA immunonegative neurons in their
soma size, orientation, and axosomatic endings. Studies of
gerbil (Roberts et al., 1985), guinea-pig (Thompson et al.,
1985) and bat (Winer et al., 1995) also described GABAer-
gic cells in the IC, but only a few brief reports are available
for rat (Vetter and Mugnaini, 1984, 1985; Roberts and
Ribak, 1987).
The main goal of this study is to determine the
morphological types of GABAergic and non-GABAergic
neurons as well as their axosomatic GABAergic and gly-
cinergic inputs. Quantitative anatomical data are needed to
908 M. Merchán et al. / Neuroscience 136 (2005) 907–925

Fig. 1. Photomicrograph of case R-283 showing the immunostaining for GABA in a panoramic view of the IC seen in a transverse semithin section
(0.5 ␮m thick) at the “rostral” level indicated in the inset. Inset in the bottom left part of the panel illustrates a schematic drawing of the IC in the sagittal
plane with the location of the rostral and caudal transverse sections used for the quantitative analysis. Large arrows indicate GABA-IR fibers from the
lateral lemniscus; small arrowheads points to a large GABA-IR cell and a cluster of small GABA-IN neurons. Arrow with open arrowheads indicate
GABA-IR and GABA-IN fibers through the commissure of the IC, and arrowheads show GABA-IR and GABA-IN fibers through the brachium of the
IC. D, dorsal; L, lateral, SC, superior colliculus. Scale bar⫽500 ␮m.

understand the functional role of inhibition in the rat’s IC
and to provide a comparative basis for studies concerned
with pathologies of GABA- and Gly-mediated inhibitory
transmission such as age-related hearing loss, tinnitus or
audiogenic seizures (Caspary et al., 1999; Bauer et al.,
2000; Faingold, 1999, 2002; Suneja et al., 1998). To
achieve these specific goals, we have performed an optical
densitometry (OD) analysis of IC neurons after postem-
bedding immunocytochemistry for GABA and Gly, and
compared the results within and across IC subdivisions.
EXPERIMENTAL PROCEDURES
Immunocytochemistry for GABA and Gly
Six adult Wistar rats of either sex (B.W., 250 –300 g) were anesthe-
tized with sodium pentobarbital (60 mg/kg, i.p.) and perfused tran-
 M. Merchán et al. / Neuroscience 136 (2005) 907–925 909

Fig. 2. Images of neurons in the CNIC immunostained alternately for GABA (left) and Gly (right). Each neuron is shown in two micrographs, taken
from a pair of adjacent semithin sections stained respectively for GABA (left panel, A–C) and Gly (right panel, D–F). Areas highlighted in frames (A,
D) are shown at higher magnification in B, C and E, F, respectively. Neurons with strong immunostaining for GABA (A) and with weak immunosignal
for Gly (D) and with numerous GABA-positive (B, C) and few Gly-positive perisomatic puncta (E, F). Arrowheads in A indicate a row of four to five
neurons that are GABA-IN. Note that there are GABA-IN and immunonegative neurons for Gly fibers (e.g. arrows in D). Scale bars⫽50 ␮m in A and
D; B, C, E and F⫽10 ␮m.
scardially with a cold (6 °C) wash solution (40 ml) composed of 2% IC were rinsed in PB, postfixed with osmium tetroxide (0.5% in PB)
dextran (MW 70,000) in 0.1 M sodium phosphate buffer, pH 7.4 (PB), for 45 min, dehydrated in ascending ethanols to propylene oxide, and
followed by 750 ml of a fixative containing 1% paraformaldehyde and embedded in epoxy resin (Durcupan ACM; Fluka, Milwaukee, WI,
2.5% glutaraldehyde in the same buffer at room temperature. The USA). Three consecutive 0.5-␮m-thick sections of each slice (cut on
specimens were kept at 4 °C overnight, and the next day the brains an ultramicrotome) were mounted on different gelatinized slides. The
were removed. The brainstems were cut into 200 – 400-␮m-thick first two sections were immunocytochemically stained for GABA and
slices in the transverse plane on a Vibratome. Slices containing the Gly, respectively, and the third section was stained with Toluidine
910 M. Merchán et al. / Neuroscience 136 (2005) 907–925

A 2
R283: All neurons
2

1 1

0 0
Normalized density

Threshold
-1 -1

-2 -2

-3 -3
Inferior colliculus (N=932)
Granule cells (N=35)
-4 Golgi neurons (N=35)
-4
Purkinje neurons (N=36)
Blood vessels (N=16)
-5 -5
0 200 400 600 800 1000 0 20 40 60 80 100
Neuron ID number Number of neurons

R1119: All neurons

B 3 3

2 2

1 1
Normalized density

0 0 Threshold

-1 -1

-2 -2
Inferior colliculus (N=330)
Granule cells (N=32)
-3 Golgi neurons (N=19)
-3
Purkinje neurons (N=20)
Blood vessels (N=25)
-4 -4
0 100 200 300 400 0 10 20 30 40
Neuron ID number Number of neurons
Fig. 3. Scatter plots showing the distribution of the gray values (normalized density) obtained for all IC neurons in cases R-283 (A) and R-1119 (B;
open circles) used in the densitometric analysis together with the control neurons (granule-, Golgi-, and Purkinje cells from the cerebellum and blood
vessels). Horizontal stipple line shows the threshold calculated as the mean plus two times the S.D. of the optical density of the granule cells. The
vertically oriented histogram shows a bimodal distribution of the same values seen in the scatter plot for the IC neurons. Note that the valley that
separates the two peaks in the histograms approximates the threshold of OD that separates GABA-IR from GABA-IN neurons.
 M. Merchán et al. / Neuroscience 136 (2005) 907–925 911

Table 1. Summary of the two cases (all neurons)a means of a Leica optical microscope equipped with a black-and-
white video camera (Cohu CCD Mod. 4912–5000). Pixel values of
TOTAL GABA-IR GABA-IN 0 and 255 correspond to white and black colors, respectively. The
camera was connected to a Macintosh computer via a video
R283 digitizing card (Scion Corporation). Images were digitized and
IC 932 240 692 analyzed using Scion NIH Image software.
25.8% 74.2% In an attempt to preserve identical illumination conditions for
CNIC 415 138 277 different image capturing sessions, the settings of all components
33.3% 66.7% were kept unchanged and the intensity of the microscope lamp
DCIC 140 34 106 was always set at saturation. As a further control, the microscope
24.3% 75.7% illumination was adjusted (if necessary) using neutral density fil-
LCIC 322 58 264 ters so that similar gray level distributions (mean and standard
18.0% 82.0% deviation) were obtained at the beginning of each session for the
R1119 digital image of an empty slide.
IC 330 80 250 Camera lucida drawings of the IC outline were made using a
24.2% 75.8% dry 40⫻ objective (PL Fluotar, N.A.: 0.70). For each slice, rectan-
CNIC 185 47 138 gular digital images were obtained for portions of the IC (hereafter
25.4% 74.6% referred to as “fields”) viewed with the 40⫻ objective. For animal
DCIC 60 13 47 R-283, images for nearly-adjacent fields were obtained sequen-
21.7% 78.3% tially until the whole IC was covered; for case R-1119 fields were
LCIC 85 20 65 more sparse but still evenly distributed over the IC slice. Each field
23.5% 76.5% was numbered and assigned approximate (x, y) coordinates ac-
cording to a calibrated Cartesian space with an arbitrary origin.
a
Indicated are the absolute number (and the percentage, %) of neu- For each field, the mean gray level (MF) and the standard devia-
rons. tion of gray values (S.D.F) were measured.
From each field (768⫻512 pixels), five to seven neurons
Blue. To assess the selectivity of the immunoreaction, a 0.5-␮m- among those that showed a nucleus were selected at random by
section of a multi-layered resin-embedded “sandwich” of amino acid– the experimenter. Typically, this number amounted to 70 – 80% of
glutaraldehyde–paraformaldehyde–rat brain protein conjugates (Ot- the total number of neurons that met our criterion in any given
tersen, 1987) was also mounted on each slide to be immunostained. field. This allowed obtaining a random sample of neurons distrib-
In addition, a 0.5-␮m section of the cerebellar cortex (referred to uted over the IC slice. Neuronal somata were outlined manually on
below as control nucleus) from the same animal was mounted on the digital image and tagged uniquely. The initial dendritic seg-
each slide to compare IC tissue with structures of known GABA ment was ignored for analysis whenever it was stained (which
immunostaining patterns (Ottersen and Storm-Mathisen, 1984; So-
happened very rarely due to the section thickness; cf. Fig. 2).
mogyi et al., 1986; Ottersen, 1987; Wenthold et al., 1987; Saint Marie
Nevertheless, the initial part of the dendritic segments was never
et al., 1989; Ottersen et al., 1995; Spirou and Berrebi, 1997).
longer than 2–3 ␮m and negligible compared with the length of a
Postembedding immunocytochemistry followed the modification
whole dendritic arbor in rat (range: 490 –1812 ␮m long; Table 3 in
of Ottersen (1987) from the method of Somogyi et al. (1984). The
sections were etched in sodium ethanolate and then immersed in 1% Malmierca et al., 1993). All neurons in a given field were assigned
sodium metaperiodate. They were subsequently preincubated in the same (x, y) coordinates of its corresponding field. These
20% normal swine serum (NSS) in Tris-phosphate-buffered saline, coordinates allowed producing gray-coded maps like those shown
pH 7.6 (TPBS), for 20 min. Sections were then incubated (overnight, in Figs. 9 and 10 using DeltaGraph.
at 4 °C) in the rabbit primary antiserum in TPBS containing 1% NSS, For each selected neuron, the mean cytoplasmic gray level
followed by 40 min in sheep anti-rabbit IgG and 1 h in rabbit peroxi- (MN), the standard deviation (S.D.N), and the perimeter of the soma
dase–antiperoxidase complex and, finally, diaminobenzidine/H2O2. were measured using image processing techniques. The number
Sections were thoroughly rinsed between steps. of GABA and Gly immunoreactive (Gly-IR) puncta for the neuron
The primary antisera used were GABA antiserum 990 and Gly were also counted. The counts were performed by viewing the
antiserum 290 (e.g. Kolston et al., 1992; Moore et al., 1996; neuron with 100⫻ oil immersion objective. GABA and Gly puncta
Riquelme et al., 2001). Prior to use, these antisera were preincu- for the same neuron were counted on adjacent IC slices pro-
bated for 18 –24 h with glutaraldehyde/paraformaldehyde (GP) cessed separately for reactivity against antibodies to GABA or
conjugates of possible cross-reacting amino acids. The final work- Gly. Puncta counts given below are expressed as per 100 ␮m of
ing solutions for the antibodies were as follows: anti-GABA 1:200 perimeter.
with 300 ␮M ␤-alanine-GP and 300 ␮M Gly-GP; and anti-Gly The neuron’s mean gray level (a value between 0 and 255)
1:600 with 400 ␮M ␤-alanine-GP, 200 ␮M GABA-GP, and 100 ␮M was used as a measure of the neuron’s immunoreactivity to
Glu-GP. Negative controls comprised omission of the primary GABA. The neuron’s gray level reflects the neuron’s true optical
antiserum or adsorption of the primary antiserum with the antigen density, but may also depend on the illumination conditions. De-
conjugate, carried out by adding 300 ␮M of GABA-GP to the spite the precautions taken (see above), illumination conditions
anti-GABA working solution and 400 ␮M of Gly-GP to the anti-Gly may have varied slightly across sections. To minimize the risk of
working solution. A positive control was provided by the “sand-
influencing the results by fluctuations in illumination, normalized
wich” sections described above. Adsorption resulted in the com-
gray levels (ZN) were used instead of direct gray level measures.
plete suppression of immunostaining in the tissue and “sandwich”
The normalized gray level for each neuron was obtained by sub-
sections.
tracting the field’s mean gray level from the neuron’s mean gray
Densitometry analysis level, and dividing the result by the field’s standard deviation; in
mathematical terms: ZN⫽(MN⫺MF)/S.D.F (Riquelme et al., 2001).
Image processing techniques were employed to perform an OD Therefore, positive/negative normalized gray values indicate that
analysis on the immunostained tissue from two animals (R-283 the neuron is darker/lighter, respectively, than its corresponding
and R-1119). Eight-bit digital images of the IC were obtained by field.
912 M. Merchán et al. / Neuroscience 136 (2005) 907–925

Table 2. Summary of immunoreactivity of IC neurons (case: R283)a

GABA-IR GABA-IN
Mean⫾SD (Range) Mean⫾SD (Range)

Whole IC sample (N⫽932) 240 692

(*) Normalized OD ⫺0.18⫾0.32 (⫺0.76, 0.61) ⫺1.60⫾0.44 (⫺3.18, ⫺0.77)
(*) Perimeter 55.06⫾15.25 (26.21, 121.87) 49.04⫾11.20 (19.24, 98.98)
(*) GABA punctae 13.87⫾5.25 (3.64, 35.38) 17.75⫾5.10 (4.42, 39.54)
Glycine punctae 1.85⫾3.00 (0.00, 18.78) 1.74⫾3.11 (0.00, 21.91)
CNIC sample (N⫽415) 138 277
(*) Normalized OD ⫺0.11⫾0.31 (⫺0.76, 0.59) ⫺1.55⫾0.38 (⫺2.85, ⫺0.77)
(*) Perimeter 57.06⫾14.33 (29.42, 102.64) 49.27⫾10.41 (27.58, 93.91)
(*) GABA punctae 12.47⫾4.49 (3.90, 31.18) 16.90⫾4.48 (6.88, 30.07)
Glycine punctae 2.23⫾3.18 (0.00, 18.78) 1.93⫾3.35 (0.00, 21.91)
Low freq sample (CNIC: N⫽115) 37 78
(*) Normalized OD ⫺0.08⫾0.31 ⫺0.65, 0.58) ⫺1.58⫾0.41 (⫺2.85, ⫺0.81)
Perimeter 54.80⫾13.96 (31.95, 86.03) 50.50⫾10.85 (27.87, 74.51)
(*) GABA punctae 12.62⫾5.17 (4.32, 29.20) 16.92⫾4.23 (8.50, 28.71)
Glycine punctae 2.10⫾4.01 (0.00, 18.78) 1.22⫾2.65 (0.00, 14.87)
Middle freq sample (CNIC; N⫽133) 53 88
(*) Normalized OD ⫺0.09⫾0.31 (⫺0.75, 0.59) ⫺1.47⫾0.33 (⫺2.15, ⫺0.77)
(*) Perimeter 58.46⫾14.03 (29.42, 97.19) 50.42⫾10.92 (30.03, 93.91)
(*) GABA punctae 12.39⫾4.44 (6.16, 31.18) 16.41⫾4.38 (8.47, 29.61)
Glycine punctae 2.81⫾3.16 (0.00, 12.29) 2.34⫾3.36 (0.00, 16.65)
High freq sample (CNIC; N⫽129) 37 92
(*) Normalized OD ⫺0.17⫾0.31 (⫺0.75, 0.59) ⫺1.62⫾0.38 (⫺2.61, ⫺0.81)
(*) Perimeter 57.57⫾15.47 (32.61, 102.64) 47.45⫾8.92 (27.58, 77.24)
(*) GABA punctae 12.52⫾3.56 (3.90, 21.46) 16.87⫾4.69 (6.88, 30.07)
Glycine punctae 1.78⫾2.44 (0.00, 8.35) 2.28⫾3.96 (0.00, 21.91)
DCIC sample (N⫽140) 34 106
(*) Normalized OD ⫺0.26⫾0.32 (⫺0.75, 0.58) ⫺1.55⫾0.47 (⫺3.17, ⫺0.82)
Perimeter 49.58⫾10.43 (26.21, 78.71) 44.35⫾10.14 (22.98, 88.21)
(*) GABA punctae 13.02⫾4.62 (3.64, 22.89) 18.39⫾5.01 (4.79, 30.44)
Glycine punctae 0.82⫾1.68 (0.00, 7.10) 0.91⫾2.27 (0.00, 13.11)
LCIC sample (N⫽322) 58 264
(*) Normalized OD ⫺0.30⫾0.28 (⫺0.75, 0.34) ⫺1.54⫾0.47 (⫺3.18, ⫺0.77)
Perimeter 54.07⫾18.91 (30.13, 121.87) 50.25⫾11.96 (19.24, 98.98)
GABA punctae 17.49⫾5.53 (8.20, 35.38) 18.45⫾5.53 (4.67, 39.54)
Glycine punctae 1.79⫾3.21 (0.00, 12.19) 1.83⫾3.11 (0.00, 15.41)
a
Asterisks denote statistically significant differences between GABA-IR and GABA-IN neurons.

GABA controls RESULTS

Sections of the cerebellum were obtained for the same animals
and were processed using identical immunocytochemical and OD
methods as for the IC sections. The granule cells of the cerebel-
lum were regarded as a GABA immunonegative control. The
optical density threshold for GABA immunoreactivity was defined
as the mean plus two standard deviations (95% confidence inter-
val) of the normalized gray values for the granule cells (see open
triangles in Fig. 3 and below).
Statistical analyses
When required (v.i.), two-tailed Student’s t-tests were employed to
compare the mean values of the variables under study. Statistical
significance was set at P⬍0.01.
All experimental animals used in this study were handled and
cared for according to the NIH Guidelines and the Society for
Neuroscience Policy on the Use of Animals in Neuroscience Re-
search under the supervision of the Institutional Animal Care and
Use Committee. All procedures were vetted and approved by The
University of Salamanca Animal Care Committee. In accordance
with these guidelines, efforts were made to minimize the numbers
of animals used and the suffering experienced by those animals.
Standard microscopic analysis of GABA and Gly
immunoreactivity in the IC
Visual inspection of the immunostained tissue under the
light microscope revealed that all parts of the IC show (1)
dense punctate immunostaining of the neuropil to both
GABA (Figs. 1 and 2) and Gly (Fig. 2), (2) some IC neurons
are GABA-IR, and (3) no IC neurons are Gly-IR.
GABA-IR neurons show a variable degree of immuno-
staining regardless of their topographical location. They
are found in all subdivisions of the IC in slightly different
proportions, and have cell bodies of variable sizes and
shapes (e.g. multipolar, triangular, round and fusiform;
Figs. 1 and 2).
Neurons immunonegative for GABA (GABA-IN) have
somata that appear to be smaller than those of GABA-IR
cells, but show the same variability as their GABA-IR coun-
terparts with regard to their shape (Figs. 1 and 2).
GABA-IN neurons tend to form rows or groups of three to
 M. Merchán et al. / Neuroscience 136 (2005) 907–925 913

Fig. 4. Histograms showing the distribution of optical density, perimeter, number of GABA puncta/100 ␮m perimeter and number of Gly puncta/
100 ␮m perimeter in all neurons (GABA-IR, thick line; GABA-IN, thin line) of the IC (top row), and separately in the CNIC, DCIC and LCIC for cases
R-283 (N⫽932) and R-1119 (N⫽330), respectively.
914 M. Merchán et al. / Neuroscience 136 (2005) 907–925

Whole IC sample Gly-IR neuropil puncta are diverse in size, shape and their
80 targets (Fig. 2). They are likely to represent cross-sections
[*]
of small dendrites and axons or terminal boutons. These
puncta were excluded from the subsequent quantitative
60
Perimeter (micra)

analysis, which pertains only to perisomatic puncta.

Quantitative densitometric analysis of GABA and Gly

40 immunoreactivity in the IC
The OD, the perimeter, and the number of perisomatic
20 GABA-IR and Gly-IR puncta were measured for a total of
1262 IC neurons from two cases (R-283 and R-1119;
Tables 1 and 2; Figs. 3– 8). These neurons were selected
0 from transverse sections as illustrated in the inset of Fig. 1.
GABA-IR GABA-IN Numerical data were virtually identical for the two
30 cases (Figs. 3– 8; Table 1). Statistical tests were run to
[*]
compare the proportions of GABA-IR and GABA-IN neu-
GABA punctae (/100 micra)

rons in the two cases (Table 1) and to compare the mean

20
values for all variables measured, for all IC subdivisions
and frequency regions (Figs. 4 – 8). No significant differ-
ences were found. On these grounds, and for the sake of
conciseness, the numerical data discussed in the text below
10 corresponds to case R-283 (the one for which more neurons
were measured; Table 2). Nevertheless, Figs. 3– 8 illustrate
results for both cases.
In the following sections, we will describe the data for
0 the IC as a whole; then we will focus on the central nucleus
GABA-IR GABA-IN of the inferior colliculus (CNIC); and last, we shall compare
10 the data for CNIC with that of the dorsal cortex (DCIC) and
Glycine punctae (/100 micra)

R283 the lateral part of the external cortex, hereafter referred to as

8
R1119 the lateral cortex (LCIC; Malmierca, 1991, 2003). Data are
summarized in Tables 1 and 2 and illustrated in Figs. 3– 8).
6 Densitometric analysis of the IC as a whole

4 Fig. 3 illustrates the normalized OD values for all 1262 IC

2
0
GABA-IR GABA-IN
Neuron type
Fig. 5. Mean values and SDs for the perimeter, number of GABA
puncta/100 ␮m perimeter and number of Gly puncta/100 ␮m perimeter
for GABA-IR and GABA-IN in the whole IC. Asterisks indicate statis-
tically significant differences.
seven cells that are oriented parallel to the axonal fascicles of
the fibrodendritic laminae (Fig. 2). Many of these fascicles
contain GABA-IR and/or Gly-IR fibers. Their preterminal
trunks could be easily traced down to the lateral lemniscus
(Fig. 1). Both GABA-IR and GABA-IN, but no Gly-IR, fibers
could be traced to the commissure of the IC and the
laterally placed brachium of the IC (Fig. 1).
Examination of perisomatic puncta (which presumably
correspond to axosomatic synapses) also reveals that
GABA-IR puncta are more noticeable than Gly-IR puncta;
however, the two are equally abundant in the adjacent
neuropil, with the exception of the dorsal region of the IC
where Gly-IR puncta are less numerous. GABA-IR and
neurons (circles) in the two analyzed cases (A, R-283;
B, R-1119). The OD values for Golgi cells (known to be
GABA-IR; filled squares), Purkinje cells (known to be
GABA-IR; filled triangles) and granule cells (known to be
GABA-IN; open triangles) from the cerebellum are also
shown for comparison. In addition, OD values for cross-
sectioned blood vessels (crosses) are included to act as
indicators of background illumination. The dashed horizontal
line indicates the numerical OD threshold that permits clas-
sifying the neurons as GABA-IR or GABA-IN (see Experi-
mental Procedures). The vertically oriented histograms in Fig.
3 show that the OD follows a bimodal distribution with a valley
that matches approximately the numerical threshold used to
separate the two distinct populations. The group of GABA-IR
neurons comprises 25% of all IC neurons (Table 1).
Means and ranges for the perimeter and the number of
perisomatic GABA-IR and Gly-IR puncta for GABA-IR and
GABA-IN neurons are shown in Table 2. GABA-IR cells
have a mean perimeter of 55.06 ␮m with 13.87 perisomatic
GABA-IR puncta/100 ␮m and 1.85 perisomatic Gly-IR
puncta/100 ␮m. The GABA-IN cells have a mean perime-
ter of 49.04 ␮m with 17.75 perisomatic GABAergic puncta/
100 ␮m and 1.74 perisomatic Gly-IR puncta/100 ␮m
(Table 2). These data show that, on average, GABAergic
IC neurons are significantly larger and contain fewer peri-
 M. Merchán et al. / Neuroscience 136 (2005) 907–925
80 25
GABA puncta (/100 micra)
20
Perimeter (micra)
60
15
40
10
20
5 2
0 0
LF HF
80 25
GABA puncta (/100 micra)
20
Perimeter (micra)
60
15
40
10
20
5 2
0 0
LF HF
Fig. 6. Mean values and SDs for the perimeter, number of GABA puncta/100 ␮m and number of Gly puncta/100 ␮m for GABA-IR and GABA-IN in
the low- (LF) vs the high-frequency (HF) regions of the CNIC. Asterisks indicate statistically significant differences.
somatic GABA puncta (per 100 ␮m of perimeter) than
non-GABAergic neurons (Table 2; Fig. 5). In the subse-
quent sections we will analyze the numerical data for the
CNIC, DCIC and LCIC, separately (Table 2; Figs. 4, 6 – 8).
For this analysis, 55 neurons of the original sample of 932
IC neurons (Case R-283) were excluded because they
were located on the CNIC borders and could not be clas-
sified as part of any one subdivision with certainty. Accord-
ingly, only 877 neurons were studied. Of these, 415 were
from the CNIC, 140 from the DCIC, and 322 from the LCIC
(Table 2).
Densitometric analysis of CNIC neurons
For the analysis, CNIC neurons were either pooled, irre-
spective of their location within the CNIC, or examined in
groups established according their presumed frequency
representation (low vs. high) within the CNIC (v.i.) as de-
termined by separate electrophysiological studies of isofre-
quency laminae in the IC (e.g. Kelly et al., 1991; Malmierca
et al., 2003). Table 2 summarizes data on the perimeter and
the number of perisomatic GABA puncta and perisomatic
Gly puncta terminating on these neurons (Figs. 4, 6 – 8).
GABA-IR neurons comprise one third of all CNIC neurons
(Table 1). The somata of GABA-IR cells are significantly
larger than those of GABA-IN cells (perimeter values of
57.06 vs. 49.27 ␮m, respectively). Likewise, the number of
perisomatic GABA-IR puncta/100 ␮m is significantly
smaller for GABA-IR cells than for GABA-IN neurons
(12.47 vs. 16.9, respectively). Both cell types show very
few perisomatic Gly puncta (2.23 vs. 1.93, respectively).
915
GABA-IR
10
Glycine puncta (/100 micra)
R283
8 R1119
6
4
0
LF HF LF HF
GABA-IN
10
Glycine puncta (/100 micra)
8 [*]
6
4
0
LF HF LF HF
Frequency region
A fundamental property of the auditory system is its
tonotopic organization (von Bèkèsy, 1960; for reviews see
Irvine, 1992; Malmierca, 2003). It is now well established
that the morphological substrate for such tonotopy in the
CNIC is its laminar organization (Oliver and Morest, 1984;
Malmierca et al., 1993). Thus, we investigated the distri-
bution of GABA-IR and GABA-IN neurons across and
within the frequency-band laminae (Table 2, Figs. 6, 7) to
check whether or not there are neurochemical differences
with regard to the tonotopic organization of the CNIC.
Comparisons across laminae (Fig. 6). Of the 415
neurons (case R-283) sampled in the CNIC, 115 neurons
were located in the dorsolateral, low frequency region
(corresponding approximately to 1– 4 kHz frequency
bands; Ryan et al., 1988) and 129 were in the ventrome-
dial, high frequency region (corresponding approximately
to 30 – 60 kHz; Ryan et al., 1988). The remaining neurons
were excluded from the analysis to avoid overlapping
between these frequency specific samples. Differences
between GABA-IR and GABA-IN neurons in frequency
specific regions are similar to those described for the IC as
a whole, except for the fact that GABA-IN and GABA-IR
neurons in the low frequency region have similar perime-
ters regardless of their immunoreactivity (Table 2).
The comparison between the low- and high-frequency
regions shows that there is a slightly larger proportion of
GABA-IR neurons in the low- than in the high-frequency
region (32% vs. 28%; Table 2). This is also appreciated by
visual inspection (Fig. 1). GABA-IR neurons in the dorso-
916 M. Merchán et al. / Neuroscience 136 (2005) 907–925

A GABA-IR
80 30 10

Glycine puncta (/100 micra)

R283

GABA puncta (/100 micra)

8 R1119
[*]
Perimeter (micra)

60
20
6
40
4
10
20
2

0 0 0
Dorsomedial Ventrolateral Dorsomedial Ventrolateral Dorsomedial Ventrolateral

GABA-IN
80 30 10
[*]

Glycine puncta (/100 micra)

GABA puncta (/100 micra)

[*] [*]
8
Perimeter (micra)

60
20
6
40
4
10
20
2

0 0 0
Dorsomedial Ventrolateral Dorsomedial Ventrolateral Dorsomedial Ventrolateral
Portion of the CNIC lamina

B GABA-IR
80 30 10
[*]
Glycine puncta (/100 micra)
GABA puncta (/100 micra)

8
Perimeter (micra)

60
20
6
40
4
10
20
2

0 0 0
Rostral Caudal Rostral Caudal Rostral Caudal

GABA-IN
80 30 10
[*]
Glycine puncta (/100 micra)
GABA puncta (/100 micra)

8
Perimeter (micra)

60
20
6
40
4
10
20
2

0 0 0
Rostral Caudal Rostral Caudal Rostral Caudal
Portion of the CNIC lamina

Fig. 7. Mean values and SDs for the perimeter, number of GABA puncta/100 ␮m and number of Gly puncta/100 ␮m for GABA-IR and GABA-IN within
the CNIC lamina. (A) Dorsomedial vs ventrolateral; (B) rostral vs. caudal. Asterisks indicate statistically significant differences.
 M. Merchán et al. / Neuroscience 136 (2005) 907–925 917

GABA-IR
80 30 10
[*] [*]

Glycine puncta (/100 micra)

R283

GABA puncta (/100 micra)

8 R1119
[*]
Perimeter (micra)

60 [*]
20
6
40
4
10
20
2

0 0 0
CNIC DCIC LCIC CNIC DCIC LCIC CNIC DCIC LCIC

GABA-IN
80 30 10
[*]

Glycine puncta (/100 micra)

GABA puncta (/100 micra)

[*] [*] [*]

8
Perimeter (micra)

60 [*] [*]
20
6
40
4
10
20
2

0 0 0
CNIC DCIC LCIC CNIC DCIC LCIC CNIC DCIC LCIC

IC subdivision

Fig. 8. Mean values and SDs for the perimeter, number of GABA puncta/100 ␮m and number of Gly puncta/100 ␮m for GABA-IR and GABA-IN in
the CNIC, DCIC and LCIC. Asterisks indicate statistically significant differences.

lateral, low- and ventromedial, high-frequency IC regions input, and rostrally located GABA-IN neurons are larger
are similar in all respects (Fig. 6). The same holds true for (Fig. 7B).
the GABA-IN neurons except that these neurons have In summary, these data demonstrate that GABA-IR and
more perisomatic Gly puncta in the high frequency region GABA-IN neurons tend to be alike regardless of the fre-
(Fig. 6). quency region to which they belong. However, the data do
show some diversity of GABA-IR and GABA-IN neurons
Comparisons within laminae (Fig. 7). When seen en
within the CNIC laminae. For the same neural type, differ-
face, each frequency-band lamina can be depicted as a
ences are more evident along the ventrolateral-to-dorsomedial
two-dimensional plane that adds a rostrocaudal dimension
axis of the IC.
to the lateromedial axis more commonly portrayed in coro-
nal sections of the CNIC (cf., Fig. 17 in Malmierca et al.,
Densitometric analysis for the DCIC and LCIC
1993). Comparisons along these two axes allow us to
analyze the topographical distribution of GABA-IR and The R-283 sample from the collicular cortical regions in-
GABA-IN neurons in all locations within a lamina. First we cludes 462 neurons (322 from the LCIC and 140 from the
compared GABA-IN and GABA-IR neurons from the ven- DCIC; Tables 1 and 2; Figs. 4 and 8). In the analysis, we
trolateral region of the CNIC with their corresponding coun- excluded neurons from the rostral part of the external
terparts located in dorsomedial region of the CNIC (Fig. 7A). cortex because this area is difficult to delineate and the
These comparisons show that GABA-IR neurons are sim- border with the CNIC and adjacent tegmentum is not clear
ilar along the main ventrolateral-dorsomedial axis of the IC, (Faye-Lund and Osen, 1985; Malmierca et al., 1993,
except for the fact that neurons in the ventral region re- 1995a,b).
ceive more axosomatic glycinergic input (Fig. 7A). In ad- On average, around 23% of the DCIC neurons are
dition, GABA-IN neurons in the ventral CNIC are larger, GABA-IR. The OD distribution for the DCIC sample is
receive more axosomatic glycinergic input and less axo- shown in Fig. 4. Similar to our findings for the CNIC, this
somatic GABAergic input than those located in the dorso- distribution is bimodal and reveals two distinct groups of
medial portion of the CNIC (Fig. 7A). immunoreactive neurons. GABA-IR cells in the DCIC have
Next, we compared the samples from the rostral and a mean perimeter of 49.58 ␮m with 13.02 perisomatic
caudal sections of the CNIC (Fig. 7B). The results show GABA-IR puncta/100 ␮m, and 0.82 perisomatic Gly-IR
that neurons located rostrally and caudally are very similar puncta/100 ␮m. The GABA-IN cells have a mean perime-
in most respects with two exceptions: rostrally located ter of 44.35 ␮m with 18.39 perisomatic GABA-IR puncta/
GABA-IR neurons receive more GABAergic axosomatic 100 ␮m, and 0.91 perisomatic Gly-IR puncta/100 ␮m
918 M. Merchán et al. / Neuroscience 136 (2005) 907–925

%
&# (Table 2, Fig. 8). These data imply that GABAergic and
% non-GABAergic cells in the DCIC are similar except for the
 "%

"$
%#
%'
number of perisomatic GABA-IR puncta that they receive
$# (Table 2; Fig. 4 and 8).
# %'
!
About 21% of the LCIC neurons are GABA-IR. The OD
$
distribution for this group is shown in Figs. 4. Like their
$ &
counterparts in the CNIC and DCIC, the distribution is
#'
bimodal. The GABAergic and non-GABAergic cells in the
!"
'
LCIC are similar in most respects with regard to the vari-
#
" !!
'  !
ables under study (Table 2, Figs. 4 and 8).
& ' %
#
Topographic distribution of GABA and Gly elements

in the IC
! " "

In the preceding sections, we have described the results


 regarding OD, perimeter, and perisomatic GABA-IR and
Gly-IR puncta for GABA-IR and GABA-IN neurons within
  each subdivision of the IC, independently. To facilitate the
comparison of the four parameters across subdivision
 boundaries and to illustrate graphically their spatial distri-
bution, we have produced gray-coded density (contour)
maps that illustrate the topographical distribution of immu-

noreactive elements in the IC of case R-283 (Figs. 9, 10).
The most remarkable finding after inspection of these

 gray-coded maps is the occurrence of patches (or clusters)
 of different densities of labeling (Fig. 10). This is particu-

 larly noticeable for the perimeter and the perisomatic
GABA puncta (Fig. 10). Patches of these two variables
 
   


with different degrees of labeling occur within and across
the frequency-band laminae. The patches are more evi-
 
dent for perisomatic GABA puncta in both GABA-IR and
GABA-IN neurons (Fig. 10). Altogether, these findings sug-
gest that neurons of different sizes are mixed up and
  evenly distributed throughout the IC, i.e. small, medium
and large GABA-IR and GABA-IN neurons intermingle.
  The same holds true for the number of perisomatic
GABA-IR puncta. However, clear differences are also ev-
ident. There are larger GABA-IR neurons in the ventral and

 
rostral portions of the LCIC. Additionally, it seems that
neurons in the ventral portions of the CNIC and in the LCIC

  possess more perisomatic GABA-IR puncta. The results
   
 
 regarding the perisomatic Gly-IR puncta are rather differ-
  ent (Fig. 10). Although, this is reminiscent of a cluster-like
organization, it is obvious that most puncta are concen-
 
trated in the CNIC, with a minor component in the cortices.
Interestingly, a single and distinct cluster of perisomatic
Gly-IR puncta is seen in the DCIC close to the commissure
 that interconnects the two ICs (Fig. 10). The cluster is more
dense for the GABA-IN neurons than for the GABA-IR
 cells. Another interesting feature related to the number of


calibrated Cartesian plane. The scales of the x and y axes are ex-
pressed in microns. Five neurons were typically chosen from each field

 and were assigned the same (x, y) coordinates. (C) An example
    

  gray-coded map. Each field was assigned a gray value between 0 and
255 according to the average value of the investigated variable for the
Fig. 9. (A) Outline of the IC. Rectangles illustrate the location of the IC five neurones in the field. Linear interpolation between gray values for
fields considered in the study. The numbers by the rectangles corre- adjacent fields was done automatically by the plotting application
spond to the fields’ ID numbers (c.f. Fig. 3). (B) Dots illustrate the DeltaGraph. (D) Resulting gray-coded map for the three regions of
approximate (x, y) coordinates assigned to each field in an arbitrary the IC.
 M. Merchán et al. / Neuroscience 136 (2005) 907–925 919

Fig. 10. Gray-coded maps that illustrate the topographical distribution for different immunoreactive elements in the IC. Left panel compares the
distribution of the somata, number of perisomatic GABA and Gly puncta in the rostral and caudal sections though the CNIC for the GABA-IN neurons.
Right panel, similar comparison for GABA-IN neurons. Note the patchy distribution for most parameters assessed.
920 M. Merchán et al. / Neuroscience 136 (2005) 907–925

A Lamina on edge B Lamina en face

DL
DCIC

LF DM
LCIC
DM
CNIC
HF

VL

90º
R
VM LL VL
C
LL

Non-GABAergic neurons Glycinergic puncta

GABAergic neurons GABAergic puncta

Fig. 11. Tentative scheme of the arrangement of the GABAergic and non-GABAergic neurons in the IC subdivisions with the CNIC lamina seen on
edge (A) and en face (B). Neuronal soma sizes and number of punta are artificial, but depict relative sizes and number of puncta for comparative
purposes (cf. Table 2). In general, GABAergic neurons are larger than non-GABAergic. GABAergic neurons receive less GABA- and Gly puncta than
non-GABAergic. GABAergic and non-GABAergic neurons receive different number of puncta as a function of their topographical location into IC
subdivisions. Differences across frequency regions (A) are minor, except that in the high-frequency (HF) region, non-GABAergic neurons receive more
Gly puncta than in the low-frequency region (LF). Differences within the laminae are more profound along the dorsomedial–ventrolateral (DM-VL) axis
than along the rostro-caudal (R-C) axis. GABAergic puncta are found both on somata and in neuropil, whereas glycinergic puncta are mostly confined
to the neuropil. LL, lateral lemniscus; VM, ventromedial; DL, dorsolateral.

perisomatic Gly-IR puncta is that they seem to form dense
bands that alternate with less dense bands, similar to those
described in the studies of afferent fibers to the IC using
tritiated amino acids (Oliver 1984a, 1987; Shneiderman and
Henkel, 1987; Shneiderman et al., 1988).
These topographical results are supported by the con-
clusions drawn from the statistical comparisons of the OD,
perimeter, perisomatic GABA-IR and Gly-IR puncta of
GABA-IR and GABA-IN neurons across IC subdivisions
(Fig. 8). Table 2 shows the mean values for these four
parameters for the CNIC, DCIC and LCIC. Finally, Fig. 8
illustrates also significant differences (asterisks) across
nuclear divisions in the IC for GABA-IR and GABA-IN,
respectively, showing the more notorious differences when
the CNIC and the cortices are compared.
DISCUSSION
The present account demonstrates that all neurons in the
IC are under the influence of GABAergic and/or glycinergic
inhibitory input. Furthermore, a quarter of the IC neurons in
the rat are GABAergic and, in contrast to lower auditory
centers, the IC lacks glycinergic cells.
Fig. 11 illustrates our main findings in schematic rep-
resentations of the IC subdivisions (A) and of the CNIC
lamina seen en face (B). The results may be summarized
as follows: 1) GABAergic neurons are larger than non-
GABAergic; 2) GABAergic neurons receive fewer GABA
and Gly puncta than non-GABAergic (this is not related to
the somata size, since the larger GABAergic neurons have
less puncta than the smaller non-GABAergic neurons);
3) GABAergic and non-GABA neurons from different IC
subdivisions receive different proportions of GABA and Gly
puncta; 4) differences across frequency regions are minor,
except that in the high-frequency region, non-GABAergic
neurons are larger than GABAergic neurons; 5) differ-
ences within the laminae are greater along the dorsomedial–
ventrolateral axis than along the rostro-caudal axis;
6) GABAergic puncta are found both on somata and in the
neuropil, whereas glycinergic puncta are mostly confined
to the neuropil.
These main results are in general agreement with the
previous immunocytochemical studies on the IC in the cat
(Oliver et al., 1994) and the bat (Vater et al., 1992; Winer
et al., 1995). Our results further extend previous studies
 M. Merchán et al. / Neuroscience 136 (2005) 907–925
because they are the first to show inhibitory inputs and
neurons across IC subdivisions as well as within and
across the tonotopic axis of the CNIC. We have found a
larger proportion of inhibitory neurons in the rat IC than in
the cat or bat (Oliver et al., 1994; Winer et al., 1995). This
is particularly apparent in the CNIC (approximately 30% of
GABAergic neurons in the rat vs. approximately 20% in the
cat or bat; ratio of 1:1.5).
Technical considerations and limitations
Although the current study constitutes a comprehensive
qualitative and quantitative analysis of the GABAergic im-
munoreactive somata and GABAergic and glycinergic im-
munoreactive puncta in the IC, several technical problems
should be considered before discussing the functional im-
plications of the results. First, only quantitative data related
to puncta apposed to the somata are provided. These
puncta may represent small cross-sections of dendrites or
axons rather than terminal boutons. Consequently, it is
appropriate to note that without verification at the ultra-
structural level, immunoreactive puncta can only be iden-
tified as synaptic terminals with any degree of certainty
when they are apposed to cell bodies or unambiguously
identified dendrites arising from the soma (Fig. 2). There-
fore, we have limited our study to the quantitative analysis
of perisomatic puncta following the example of other stud-
ies of the auditory system (e.g. Osen et al., 1990; Kolston
et al., 1992; Moore et al., 1996; Riquelme et al., 2001). The
present material is based on thin (0.5 ␮m thick) sections
and could not be used to analyze puncta apposed to the
proximal dendrites of the neurons. This is a significant
limitation, as the main distinguishing feature of IC neurons,
particularly of those in the CNIC, is the orientation and
thickness of their dendritic arbors (Morest, 1964; Oliver
and Morest, 1984; Faye-Lund and Osen, 1985; Malmierca
et al., 1993, 1995a). Furthermore, the counting of puncta in
the neuropil was difficult. This is an important constraint
because it made it virtually impossible to evaluate the
co-localization of GABA and Gly puncta in the neuropil.
A second technical problem is that neurons with iden-
tical concentrations of GABA or Gly could show different
degrees of immunostaining. Indeed, some GABA-IR neu-
rons were more darkly stained (Figs. 1 and 2) than others,
a fact reflected in the wide range of ODs seen in Figs. 3
and 4. This difference may be due to the variability of the
immunopenetration, although one important advantage of
the postembedding immunocytochemistry technique is
that it results in relatively uniform thickness of sections and
penetration of antibodies and reagents, which ensures
evenness of immunoreactivity. The different immunostain-
ing could also be due to other variables difficult to control
such as depth of anesthesia or vascular dilation in the
perfusion procedure. Alternatively, the different degree of
immunostaining may reflect a truly metabolic phenome-
non. Despite all these caveats, our methodology is power-
ful and reliable. It has proved useful in similar studies in
other brain regions (e.g. Brodal et al., 1988; Walberg and
Ottersen,1989, 1992; Osen et al., 1990; Ornung et al.,
1996; Reichenberger et al., 1997) including the auditory
921
system (Kolston et al., 1992; Ottersen et al., 1995; Moore
et al., 1996; Riquelme et al., 2001). Our study is the first to
use an objective measure such as the OD to distinguish
between GABAergic and non-GABAergic neurons in the
IC. Previous quantitative studies regarding GABA in IC
neurons have been based solely on visual ratings (Oliver
et al., 1994), which could be the reason for the large
differences that are reported across cases (e.g. data
shown in Oliver et al., 1994; their Table 3). Our data from
two cases are very robust because they show highly con-
sistent and statistically comparable results (Figs. 3– 8).
GABAergic cells in the CNIC
Our results suggest a possible correlation between the
immunocytochemical neuronal types described here (i.e.
GABAergic and non-GABAergic classes) and previously
described morphological cell types in the rat IC, namely flat
(F) and less-flat (LF) neurons (Malmierca et al., 1993,
1995a). We propose that a majority of F neurons are
excitatory and non-GABAergic and a majority of LF neu-
rons are GABAergic. Several reasons support this pro-
posal: 1) LF neurons have larger cell body diameters
(Malmierca et al., 1993) and constitute about 25–30% of
the CNIC neurons (Table 1 in Malmierca et al., 1993).
Similarly, the GABAergic neurons in the CNIC, on average,
are larger than the non-GABAergic cells and represent
about 30% of CNIC neurons (Table 2 in the present ma-
terial); 2) recent studies based on track-tracing studies
combined with immunocytochemistry in bats (Fremouw
et al., 1999) and rats (Yang et al., 2000) have shown that
a majority of the intrinsic laminar inputs originate from F
neurons (Oliver et al., 1991) and are non-GABAergic; 3) in
the present material, many GABA-IN neurons form rows or
groups of three to five neurons oriented along the fre-
quency band-laminae (Fig. 2), a distribution that resembles
the location and orientation of the F neurons, which form
the fibrodendritic laminae of the CNIC (Morest, 1964;
Oliver and Morest, 1984; Malmierca et al., 1993).
Intrinsic (Fremouw et al., 1999; Yang et al., 2000) and
descending (Mulders and Robertson, 2000) projections
emerging from the IC are mostly excitatory; while ascend-
ing projections to the MGB are both excitatory and inhibi-
tory (Winer et al., 1996; Peruzzi et al., 1997; Saint Marie
et al., 1997; Coomes et al., 2002). The lack of GABAergic
neurons in the rat auditory thalamus is well known (Winer
and Larue, 1988); thus, the GABAergic input to the rat
MGB must originate from neurons outside the MGB
(Peruzzi et al., 1997; Coomes et al., 2002). The CNIC
projections to the MGB are from both F and LF neurons
(Oliver, 1984b; Malmierca et al., 1997; Peruzzi et al., 1997;
Oliver et al., 1999) and it is known that a population of
GABAergic neurons in the IC projects to the MGB. Fur-
thermore, the inhibitory projection from the IC to the MGB
is more prominent in rat (about 40% of the cells projecting
to the MGB are GABAergic) than in other species (Peruzzi
et al., 1997). The present data show that the CNIC in the
rat possesses up to 30% of GABAergic neurons. This
contrasts with a comparatively lower proportion (about
20%) of GABAergic neurons in the cat and bat CNIC
922 M. Merchán et al. / Neuroscience 136 (2005) 907–925
(Oliver et al., 1994; Winer et al., 1995). Perhaps the large
number of GABAergic axons in the colliculo-geniculate
pathway compensates for the lack of GABAergic interneu-
rons in rats (Bartlett et al., 2000; Coomes et al., 2002).
However, the interneuron content of the thalamic nuclei in
the somatosensory system also varies among different
species (Arcelli et al., 1997) and there does not appear to
be an ascending GABAergic projection in the somatosen-
sory system that could compensate for lack of interneurons
(Coomes et al., 2002).
GABAergic and glycinergic inputs to the CNIC
Several previous studies have demonstrated the different
projection patterns of inputs to the IC in different species
(Adams, 1979, 1983; Brunsø-Bechtold et al., 1981; Ryugo
et al., 1981; Aitkin and Phillips, 1984a,b; Oliver, 1984a,
1987; Shneiderman and Henkel, 1987; Shneiderman et al.,
1988; Malmierca et al., 1998; reviewed in Oliver and
Shneiderman, 1991; Oliver and Huerta, 1992; Casseday
et al., 2002) including rat (Beyerl, 1978; Coleman and
Clerici, 1987; Bajo et al., 1993; Merchán et al., 1994;
González-Hernández et al., 1996; Merchán and Berbel,
1996; Kelly et al., 1998; Malmierca et al., 1999a,b, 2003;
Oliver et al., 1999; reviewed in Malmierca, 2003; Malmierca and
Merchán, 2004). Only the projections to the IC that originate
in the cochlear nuclei are exclusively excitatory, while
those from the superior olive (medial and lateral, LSO) and
lateral lemniscus nuclei (dorsal, DNLL, and ventral com-
plex, VCLL) are purely inhibitory or a mixture of excitatory
and inhibitory. Of these, the terminals from the DNLL and
the superior paraolivary nucleus are mostly GABAergic
(Shneiderman et al., 1988, 1998; Kulesza and Berrebi,
2000). The terminals arising from the LSO and the VCLL
can be excitatory, GABAergic and glycinergic or even co-
localize GABA and Gly (Saint Marie et al., 1989; Saint
Marie and Baker, 1990; Riquelme et al., 2001). Our quan-
titative analysis demonstrates that IC neurons possess
very few axosomata Gly puncta (Table 2; Fig. 11) and
further suggests that most glycinergic inputs contact only
the dendrites. Electron-microscopy studies have shown
that the DNLL terminals are consistent with inhibitory syn-
apses and contact both the somata and the dendrites of IC
neurons, while only a very small proportion (3%) of LSO
terminals (some of which must be glycinergic) contacts the
somata of the IC neurons (Oliver et al., 1995). Although
similar studies are pending for the VCLL, these previous
studies are in harmony with our immunocytochemical re-
sults. It is known that some neurons in the VCLL co-
localize GABA and Gly, and that many of these neurons
must project to the IC (Riquelme et al., 2001). We have
looked for evidence of co-localization of GABA and Gly in
the puncta of the IC neuropil, but can neither confirm nor
rule out this possibility. Cell bodies in the IC are sufficiently
large (thus can be used as reference marks themselves) to
observe co-localization of GABA and Gly in two consecu-
tive sections, but terminal boutons in the neuropil are too
small for such analysis in our material. The fact that the IC
neurons receive most of their glycinergic input on the
dendrites rather than on the somata suggests some that
some projections from the brainstem nuclei could remain
segregated at the neuronal level. However, assigning a
functional role for such segregation must await future stud-
ies.
The present results also suggest that the neuronal
circuitry is likely to be similar across frequency regions of
the IC, although different synaptic domains occur within a
given frequency band lamina. Differences in the number of
GABA and Gly puncta seem to occur only within the same
frequency lamina but not across tonotopic laminae. These
observations are consistent with the hypothesis that syn-
aptic domains vary within frequency lamina in CNIC
reflecting different combinations of inputs (Oliver, 2000;
Oliver and Huerta, 1992; Oliver et al., 1997). Previous
physiological studies also support this notion and suggest
that there is an orderly variation of responses within the
lamina for different parameters of sounds such as the peri-
odicity of amplitude modulation (Schreiner and Langner,
1988; Langner et al., 2002), latency (Langner et al., 2002),
frequency response maps, and frequency sweeps (Hage
and Ehret, 2003). In a recent report, Hage and Ehret
(2003) have suggested that inhibition decreases from the
center to the periphery within each laminae. This model
would explain the mappings of the distribution of frequency
response maps and representation of sweep direction that
they have found in the mouse IC. Our density maps of
inhibition shown in Fig. 10 support their notion regarding
Gly puncta. It seems that there are more puncta apposed
to neurons in the center of the lamina rather than in the
more ventrolateral and dorsomedial parts (i.e. more pe-
ripheral). However, our data seem to follow an inverse
pattern regarding GABA puncta, although the distribution
of GABA puncta in the CNIC is very patchy as described
(Fig. 10). Our anatomical data in conjunction with the
conclusions from emerging physiological studies, support
the notion that there might be diverse maps within a single
IC frequency lamina and that such maps depend, at least
in part, on different patterns of inhibitory inputs.
Summary and conclusions
In this study we have used a quantitative analysis to dis-
tinguish GABAergic from non-GABAergic cells in the rat
IC. We have demonstrated that there are more GABAergic
neurons in the rat IC than previously estimated for other
species. GABAergic neurons differ from non-GABAergic
neurons in their proportion (ratio 1:4 for the IC and 1:3 for
the CNIC), their soma size and in the number of GABAer-
gic and glycinergic inputs apposed to their somata.
GABAergic puncta are found both in the neuropil and
neuronal somata whereas very few glycinergic terminals
contact the somata. The glycinergic terminals are found
mostly in the neuropil. These results strongly suggest that
GABA-mediated inhibition spreads over dendrites and cell
bodies, while Gly-mediated inhibition is mostly associated
with the dendritic domain of the IC neurons.
Acknowledgments—Dr. Ole P. Ottersen kindly provided GABA
and glycine antibodies. We thank Ignacio Plaza for his excellent
technical assistance, and Jack Kelly, Douglas Oliver, Kirsten
 M. Merchán et al. / Neuroscience 136 (2005) 907–925
Osen and Bruce Warr for their critical reading and comments on a
previous version of the manuscript. This study was supported by
the Spanish DGES; grant number: BFI-2000-1396, Spanish
DGES; grant number BFI-2003-09147-02-01 to M.A.M. and
M.S.M.; the Spanish JCYL-UE grant number: SA040/04 to M.S.M.
and M.A.M.), and the Spanish FIS; grant numbers PI020343 and
G03/203 to E.A.L.-P. L.A.A. was supported by the Fundación
Carolina (Spain).
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