Download the inferior colliculus of the rat: quantitative

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*
Key words: amino acids, auditory pathway, inhibitory neurotransmitters, axosomatic endings, inferior colliculus, optical
densitometry.
a
Laboratory for the Neurobiology of Hearing, Department of Cell Biology and Pathology, Faculty of Medicine, University of Salamanca,
Salamanca, Spain
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 system 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 glycine (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 glutamate 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 distribution 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) neurons 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 GABAergic 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 glycinergic inputs. Quantitative anatomical data are needed to
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. Semithin 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 immunostaining. The optical density, perimeter, and number of
GABA- and Gly immunoreactive boutons apposed to the somata 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) differences 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 dorsomedial–ventrolateral axis than along the rostrocaudal axis;
7) GABA and non-GABAergic neurons receive different numbers 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 Neuroscience and Behavior, Lima, Peru.
*Correspondence to: M. S. Malmierca, Laboratory for the Neurobiology 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: [email protected] (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-phosphatebuffered 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
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 anesthetized 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%
dextran (MW 70,000) in 0.1 M sodium phosphate buffer, pH 7.4 (PB),
followed by 750 ml of a fixative containing 1% paraformaldehyde and
2.5% glutaraldehyde in the same buffer at room temperature. The
specimens were kept at 4 °C overnight, and the next day the brains
were removed. The brainstems were cut into 200 – 400-␮m-thick
slices in the transverse plane on a Vibratome. Slices containing the
IC were rinsed in PB, postfixed with osmium tetroxide (0.5% in PB)
for 45 min, dehydrated in ascending ethanols to propylene oxide, and
embedded in epoxy resin (Durcupan ACM; Fluka, Milwaukee, WI,
USA). Three consecutive 0.5-␮m-thick sections of each slice (cut on
an ultramicrotome) were mounted on different gelatinized slides. The
first two sections were immunocytochemically stained for GABA and
Gly, respectively, and the third section was stained with Toluidine
910
M. Merchán et al. / Neuroscience 136 (2005) 907–925
Normalized density
A
R283: All neurons
2
2
1
1
0
0
-1
-1
-2
-2
-3
-3
Inferior colliculus (N=932)
Granule cells
(N=35)
Golgi neurons
(N=35)
Purkinje neurons (N=36)
Blood vessels
(N=16)
-4
-5
0
200
400
Threshold
-4
-5
600
800
0
1000
Normalized density
R1119: All neurons
3
2
1
1
0
0
-1
-1
-2
-2
Inferior colliculus (N=330)
Granule cells
(N=32)
Golgi neurons
(N=19)
Purkinje neurons (N=20)
Blood vessels
(N=25)
-4
0
100
200
60
80 100
3
2
-3
40
Number of neurons
Neuron ID number
B
20
Threshold
-3
-4
300
Neuron ID number
400
0
10
20
30
40
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
Table 1. Summary of the two cases (all neurons)a
R283
IC
TOTAL
GABA-IR
GABA-IN
932
240
25.8%
138
33.3%
34
24.3%
58
18.0%
692
74.2%
277
66.7%
106
75.7%
264
82.0%
80
24.2%
47
25.4%
13
21.7%
20
23.5%
250
75.8%
138
74.6%
47
78.3%
65
76.5%
CNIC
415
DCIC
140
LCIC
322
R1119
IC
330
CNIC
185
DCIC
60
LCIC
85
a
Indicated are the absolute number (and the percentage, %) of neurons.
Blue. To assess the selectivity of the immunoreaction, a 0.5-␮msection of a multi-layered resin-embedded “sandwich” of amino acid–
glutaraldehyde–paraformaldehyde–rat brain protein conjugates (Ottersen, 1987) was also mounted on each slide to be immunostained.
In addition, a 0.5-␮m section of the cerebellar cortex (referred to
below as control nucleus) from the same animal was mounted on
each slide to compare IC tissue with structures of known GABA
immunostaining patterns (Ottersen and Storm-Mathisen, 1984; Somogyi et al., 1986; Ottersen, 1987; Wenthold et al., 1987; Saint Marie
et al., 1989; Ottersen et al., 1995; Spirou and Berrebi, 1997).
Postembedding immunocytochemistry followed the modification
of Ottersen (1987) from the method of Somogyi et al. (1984). The
sections were etched in sodium ethanolate and then immersed in 1%
sodium metaperiodate. They were subsequently preincubated in
20% normal swine serum (NSS) in Tris-phosphate-buffered saline,
pH 7.6 (TPBS), for 20 min. Sections were then incubated (overnight,
at 4 °C) in the rabbit primary antiserum in TPBS containing 1% NSS,
followed by 40 min in sheep anti-rabbit IgG and 1 h in rabbit peroxidase–antiperoxidase complex and, finally, diaminobenzidine/H2O2.
Sections were thoroughly rinsed between steps.
The primary antisera used were GABA antiserum 990 and Gly
antiserum 290 (e.g. Kolston et al., 1992; Moore et al., 1996;
Riquelme et al., 2001). Prior to use, these antisera were preincubated for 18 –24 h with glutaraldehyde/paraformaldehyde (GP)
conjugates of possible cross-reacting amino acids. The final working solutions for the antibodies were as follows: anti-GABA 1:200
with 300 ␮M ␤-alanine-GP and 300 ␮M Gly-GP; and anti-Gly
1:600 with 400 ␮M ␤-alanine-GP, 200 ␮M GABA-GP, and 100 ␮M
Glu-GP. Negative controls comprised omission of the primary
antiserum or adsorption of the primary antiserum with the antigen
conjugate, carried out by adding 300 ␮M of GABA-GP to the
anti-GABA working solution and 400 ␮M of Gly-GP to the anti-Gly
working solution. A positive control was provided by the “sandwich” sections described above. Adsorption resulted in the complete suppression of immunostaining in the tissue and “sandwich”
sections.
Densitometry analysis
Image processing techniques were employed to perform an OD
analysis on the immunostained tissue from two animals (R-283
and R-1119). Eight-bit digital images of the IC were obtained by
911
means of a Leica optical microscope equipped with a black-andwhite video camera (Cohu CCD Mod. 4912–5000). Pixel values of
0 and 255 correspond to white and black colors, respectively. The
camera was connected to a Macintosh computer via a video
digitizing card (Scion Corporation). Images were digitized and
analyzed using Scion NIH Image software.
In an attempt to preserve identical illumination conditions for
different image capturing sessions, the settings of all components
were kept unchanged and the intensity of the microscope lamp
was always set at saturation. As a further control, the microscope
illumination was adjusted (if necessary) using neutral density filters so that similar gray level distributions (mean and standard
deviation) were obtained at the beginning of each session for the
digital image of an empty slide.
Camera lucida drawings of the IC outline were made using a
dry 40⫻ objective (PL Fluotar, N.A.: 0.70). For each slice, rectangular digital images were obtained for portions of the IC (hereafter
referred to as “fields”) viewed with the 40⫻ objective. For animal
R-283, images for nearly-adjacent fields were obtained sequentially until the whole IC was covered; for case R-1119 fields were
more sparse but still evenly distributed over the IC slice. Each field
was numbered and assigned approximate (x, y) coordinates according to a calibrated Cartesian space with an arbitrary origin.
For each field, the mean gray level (MF) and the standard deviation of gray values (S.D.F) were measured.
From each field (768⫻512 pixels), five to seven neurons
among those that showed a nucleus were selected at random by
the experimenter. Typically, this number amounted to 70 – 80% of
the total number of neurons that met our criterion in any given
field. This allowed obtaining a random sample of neurons distributed over the IC slice. Neuronal somata were outlined manually on
the digital image and tagged uniquely. The initial dendritic segment was ignored for analysis whenever it was stained (which
happened very rarely due to the section thickness; cf. Fig. 2).
Nevertheless, the initial part of the dendritic segments was never
longer than 2–3 ␮m and negligible compared with the length of a
whole dendritic arbor in rat (range: 490 –1812 ␮m long; Table 3 in
Malmierca et al., 1993). All neurons in a given field were assigned
the same (x, y) coordinates of its corresponding field. These
coordinates allowed producing gray-coded maps like those shown
in Figs. 9 and 10 using DeltaGraph.
For each selected neuron, the mean cytoplasmic gray level
(MN), the standard deviation (S.D.N), and the perimeter of the soma
were measured using image processing techniques. The number
of GABA and Gly immunoreactive (Gly-IR) puncta for the neuron
were also counted. The counts were performed by viewing the
neuron with 100⫻ oil immersion objective. GABA and Gly puncta
for the same neuron were counted on adjacent IC slices processed separately for reactivity against antibodies to GABA or
Gly. Puncta counts given below are expressed as per 100 ␮m of
perimeter.
The neuron’s mean gray level (a value between 0 and 255)
was used as a measure of the neuron’s immunoreactivity to
GABA. The neuron’s gray level reflects the neuron’s true optical
density, but may also depend on the illumination conditions. Despite the precautions taken (see above), illumination conditions
may have varied slightly across sections. To minimize the risk of
influencing the results by fluctuations in illumination, normalized
gray levels (ZN) were used instead of direct gray level measures.
The normalized gray level for each neuron was obtained by subtracting the field’s mean gray level from the neuron’s mean gray
level, and dividing the result by the field’s standard deviation; in
mathematical terms: ZN⫽(MN⫺MF)/S.D.F (Riquelme et al., 2001).
Therefore, positive/negative normalized gray values indicate that
the neuron is darker/lighter, respectively, than its corresponding
field.
912
M. Merchán et al. / Neuroscience 136 (2005) 907–925
Table 2. Summary of immunoreactivity of IC neurons (case: R283)a
Whole IC sample (N⫽932)
(*) Normalized OD
(*) Perimeter
(*) GABA punctae
Glycine punctae
CNIC sample (N⫽415)
(*) Normalized OD
(*) Perimeter
(*) GABA punctae
Glycine punctae
Low freq sample (CNIC: N⫽115)
(*) Normalized OD
Perimeter
(*) GABA punctae
Glycine punctae
Middle freq sample (CNIC; N⫽133)
(*) Normalized OD
(*) Perimeter
(*) GABA punctae
Glycine punctae
High freq sample (CNIC; N⫽129)
(*) Normalized OD
(*) Perimeter
(*) GABA punctae
Glycine punctae
DCIC sample (N⫽140)
(*) Normalized OD
Perimeter
(*) GABA punctae
Glycine punctae
LCIC sample (N⫽322)
(*) Normalized OD
Perimeter
GABA punctae
Glycine punctae
a
GABA-IR
Mean⫾SD (Range)
GABA-IN
Mean⫾SD (Range)
240
⫺0.18⫾0.32 (⫺0.76, 0.61)
55.06⫾15.25 (26.21, 121.87)
13.87⫾5.25 (3.64, 35.38)
1.85⫾3.00 (0.00, 18.78)
138
⫺0.11⫾0.31 (⫺0.76, 0.59)
57.06⫾14.33 (29.42, 102.64)
12.47⫾4.49 (3.90, 31.18)
2.23⫾3.18 (0.00, 18.78)
37
⫺0.08⫾0.31 ⫺0.65, 0.58)
54.80⫾13.96 (31.95, 86.03)
12.62⫾5.17 (4.32, 29.20)
2.10⫾4.01 (0.00, 18.78)
53
⫺0.09⫾0.31 (⫺0.75, 0.59)
58.46⫾14.03 (29.42, 97.19)
12.39⫾4.44 (6.16, 31.18)
2.81⫾3.16 (0.00, 12.29)
37
⫺0.17⫾0.31 (⫺0.75, 0.59)
57.57⫾15.47 (32.61, 102.64)
12.52⫾3.56 (3.90, 21.46)
1.78⫾2.44 (0.00, 8.35)
34
⫺0.26⫾0.32 (⫺0.75, 0.58)
49.58⫾10.43 (26.21, 78.71)
13.02⫾4.62 (3.64, 22.89)
0.82⫾1.68 (0.00, 7.10)
58
⫺0.30⫾0.28 (⫺0.75, 0.34)
54.07⫾18.91 (30.13, 121.87)
17.49⫾5.53 (8.20, 35.38)
1.79⫾3.21 (0.00, 12.19)
692
⫺1.60⫾0.44 (⫺3.18, ⫺0.77)
49.04⫾11.20 (19.24, 98.98)
17.75⫾5.10 (4.42, 39.54)
1.74⫾3.11 (0.00, 21.91)
277
⫺1.55⫾0.38 (⫺2.85, ⫺0.77)
49.27⫾10.41 (27.58, 93.91)
16.90⫾4.48 (6.88, 30.07)
1.93⫾3.35 (0.00, 21.91)
78
⫺1.58⫾0.41 (⫺2.85, ⫺0.81)
50.50⫾10.85 (27.87, 74.51)
16.92⫾4.23 (8.50, 28.71)
1.22⫾2.65 (0.00, 14.87)
88
⫺1.47⫾0.33 (⫺2.15, ⫺0.77)
50.42⫾10.92 (30.03, 93.91)
16.41⫾4.38 (8.47, 29.61)
2.34⫾3.36 (0.00, 16.65)
92
⫺1.62⫾0.38 (⫺2.61, ⫺0.81)
47.45⫾8.92 (27.58, 77.24)
16.87⫾4.69 (6.88, 30.07)
2.28⫾3.96 (0.00, 21.91)
106
⫺1.55⫾0.47 (⫺3.17, ⫺0.82)
44.35⫾10.14 (22.98, 88.21)
18.39⫾5.01 (4.79, 30.44)
0.91⫾2.27 (0.00, 13.11)
264
⫺1.54⫾0.47 (⫺3.18, ⫺0.77)
50.25⫾11.96 (19.24, 98.98)
18.45⫾5.53 (4.67, 39.54)
1.83⫾3.11 (0.00, 15.41)
Asterisks denote statistically significant differences between GABA-IR and GABA-IN neurons.
GABA controls
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 cerebellum 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 interval) 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 Research 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.
RESULTS
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 immunostaining 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 counterparts 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
Perimeter (micra)
80
Gly-IR neuropil puncta are diverse in size, shape and their
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
analysis, which pertains only to perisomatic puncta.
Whole IC sample
[*]
60
Quantitative densitometric analysis of GABA and Gly
immunoreactivity in the IC
40
20
0
GABA-IR
GABA-IN
GABA punctae (/100 micra)
30
[*]
20
10
0
Glycine punctae (/100 micra)
GABA-IR
GABA-IN
10
R283
R1119
8
The OD, the perimeter, and the number of perisomatic
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
from transverse sections as illustrated in the inset of Fig. 1.
Numerical data were virtually identical for the two
cases (Figs. 3– 8; Table 1). Statistical tests were run to
compare the proportions of GABA-IR and GABA-IN neurons in the two cases (Table 1) and to compare the mean
values for all variables measured, for all IC subdivisions
and frequency regions (Figs. 4 – 8). No significant differences were found. On these grounds, and for the sake of
conciseness, the numerical data discussed in the text below
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
the IC as a whole; then we will focus on the central nucleus
of the inferior colliculus (CNIC); and last, we shall compare
the data for CNIC with that of the dorsal cortex (DCIC) and
the lateral part of the external cortex, hereafter referred to as
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
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 crosssectioned blood vessels (crosses) are included to act as
indicators of background illumination. The dashed horizontal
line indicates the numerical OD threshold that permits classifying the neurons as GABA-IR or GABA-IN (see Experimental 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 perimeter 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-
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 statistically 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
M. Merchán et al. / Neuroscience 136 (2005) 907–925
915
GABA-IR
60
40
20
Glycine puncta (/100 micra)
GABA puncta (/100 micra)
Perimeter (micra)
10
25
80
20
15
10
5
LF
LF
HF
R1119
6
4
2
0
0
0
R283
8
LF
HF
HF
GABA-IN
60
40
20
Glycine puncta (/100 micra)
GABA puncta (/100 micra)
Perimeter (micra)
10
25
80
20
15
10
5
LF
HF
6
4
2
0
0
0
[*]
8
LF
HF
LF
HF
Frequency region
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 subsequent 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 classified as part of any one subdivision with certainty. Accordingly, 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, irrespective 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 determined by separate electrophysiological studies of isofrequency 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).
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 distribution 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 ventromedial, 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 perimeters 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
30
60
40
20
0
20
10
0
Ventrolateral
80
GABA puncta (/100 micra)
60
40
20
0
2
Ventrolateral
10
[*]
20
10
Ventrolateral
R1119
4
Dorsomedial
0
Dorsomedial
[*]
6
Ventrolateral
GABA-IN
30
[*]
R283
8
0
Dorsomedial
Glycine puncta (/100 micra)
Dorsomedial
Perimeter (micra)
10
Glycine puncta (/100 micra)
GABA puncta (/100 micra)
Perimeter (micra)
80
[*]
8
6
4
2
0
Dorsomedial
Ventrolateral
Dorsomedial
Ventrolateral
Rostral
Caudal
Rostral
Caudal
Portion of the CNIC lamina
B
GABA-IR
30
60
40
20
0
20
10
0
Rostral
Caudal
40
20
0
20
10
0
Rostral
Caudal
4
2
10
Glycine puncta (/100 micra)
GABA puncta (/100 micra)
60
6
Caudal
GABA-IN
30
[*]
8
0
Rostral
80
Perimeter (micra)
10
[*]
Glycine puncta (/100 micra)
GABA puncta (/100 micra)
Perimeter (micra)
80
8
6
4
2
0
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
30
60
40
20
0
10
[*]
Glycine puncta (/100 micra)
[*]
GABA puncta (/100 micra)
Perimeter (micra)
80
[*]
20
10
0
CNIC
DCIC
LCIC
R283
8
R1119
[*]
6
4
2
0
CNIC
DCIC
LCIC
CNIC
DCIC
LCIC
GABA-IN
Perimeter (micra)
[*]
GABA puncta (/100 micra)
30
[*]
60
40
20
0
[*]
10
Glycine puncta (/100 micra)
80
[*]
20
10
0
CNIC
DCIC
LCIC
8
[*]
[*]
6
4
2
0
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
are similar in all respects (Fig. 6). The same holds true for
the GABA-IN neurons except that these neurons have
more perisomatic Gly puncta in the high frequency region
(Fig. 6).
Comparisons within laminae (Fig. 7). When seen en
face, each frequency-band lamina can be depicted as a
two-dimensional plane that adds a rostrocaudal dimension
to the lateromedial axis more commonly portrayed in coronal sections of the CNIC (cf., Fig. 17 in Malmierca et al.,
1993). Comparisons along these two axes allow us to
analyze the topographical distribution of GABA-IR and
GABA-IN neurons in all locations within a lamina. First we
compared GABA-IN and GABA-IR neurons from the ventrolateral region of the CNIC with their corresponding counterparts located in dorsomedial region of the CNIC (Fig. 7A).
These comparisons show that GABA-IR neurons are similar along the main ventrolateral-dorsomedial axis of the IC,
except for the fact that neurons in the ventral region receive more axosomatic glycinergic input (Fig. 7A). In addition, GABA-IN neurons in the ventral CNIC are larger,
receive more axosomatic glycinergic input and less axosomatic GABAergic input than those located in the dorsomedial portion of the CNIC (Fig. 7A).
Next, we compared the samples from the rostral and
caudal sections of the CNIC (Fig. 7B). The results show
that neurons located rostrally and caudally are very similar
in most respects with two exceptions: rostrally located
GABA-IR neurons receive more GABAergic axosomatic
input, and rostrally located GABA-IN neurons are larger
(Fig. 7B).
In summary, these data demonstrate that GABA-IR and
GABA-IN neurons tend to be alike regardless of the frequency region to which they belong. However, the data do
show some diversity of GABA-IR and GABA-IN neurons
within the CNIC laminae. For the same neural type, differences are more evident along the ventrolateral-to-dorsomedial
axis of the IC.
Densitometric analysis for the DCIC and LCIC
The R-283 sample from the collicular cortical regions includes 462 neurons (322 from the LCIC and 140 from the
DCIC; Tables 1 and 2; Figs. 4 and 8). In the analysis, we
excluded neurons from the rostral part of the external
cortex because this area is difficult to delineate and the
border with the CNIC and adjacent tegmentum is not clear
(Faye-Lund and Osen, 1985; Malmierca et al., 1993,
1995a,b).
On average, around 23% of the DCIC neurons are
GABA-IR. The OD distribution for the DCIC sample is
shown in Fig. 4. Similar to our findings for the CNIC, this
distribution is bimodal and reveals two distinct groups of
immunoreactive neurons. GABA-IR cells in the DCIC have
a mean perimeter of 49.58 ␮m with 13.02 perisomatic
GABA-IR puncta/100 ␮m, and 0.82 perisomatic Gly-IR
puncta/100 ␮m. The GABA-IN cells have a mean perimeter of 44.35 ␮m with 18.39 perisomatic GABA-IR puncta/
100 ␮m, and 0.91 perisomatic Gly-IR puncta/100 ␮m
918
M. Merchán et al. / Neuroscience 136 (2005) 907–925
%
&#
%
"%
%#
"$
%'
$#
#
%'
!
$
&
$
#'
!"
'
#
"
'
!!
%
& '
Topographic distribution of GABA and Gly elements
in the IC
#
!
!
"
"
(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 variables under study (Table 2, Figs. 4 and 8).
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 distribution, we have produced gray-coded density (contour)
maps that illustrate the topographical distribution of immunoreactive 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 particularly 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 evident for perisomatic GABA puncta in both GABA-IR and
GABA-IN neurons (Fig. 10). Altogether, these findings suggest 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 evident. 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 different (Fig. 10). Although, this is reminiscent of a cluster-like
organization, it is obvious that most puncta are concentrated 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
Fig. 9. (A) Outline of the IC. Rectangles illustrate the location of the IC
fields considered in the study. The numbers by the rectangles correspond to the fields’ ID numbers (c.f. Fig. 3). (B) Dots illustrate the
approximate (x, y) coordinates assigned to each field in an arbitrary
calibrated Cartesian plane. The scales of the x and y axes are expressed 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
five neurones in the field. Linear interpolation between gray values for
adjacent fields was done automatically by the plotting application
DeltaGraph. (D) Resulting gray-coded map for the three regions of
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
B
Lamina on edge
Lamina en face
DL
DCIC
DM
LF
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 conclusions 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 representations 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 nonGABAergic; 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) differences 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 immunoreactive somata and GABAergic and glycinergic immunoreactive puncta in the IC, several technical problems
should be considered before discussing the functional implications 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 ultrastructural level, immunoreactive puncta can only be identified 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). Therefore, we have limited our study to the quantitative analysis
of perisomatic puncta following the example of other studies 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 identical concentrations of GABA or Gly could show different
degrees of immunostaining. Indeed, some GABA-IR neurons 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 immunostaining 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 phenomenon. Despite all these caveats, our methodology is powerful 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 consistent 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 neurons are GABAergic. Several reasons support this proposal: 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 material); 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 frequency 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 ascending projections to the MGB are both excitatory and inhibitory (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. Furthermore, 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 interneurons 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 somatosensory 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 complex, 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 colocalize GABA and Gly (Saint Marie et al., 1989; Saint
Marie and Baker, 1990; Riquelme et al., 2001). Our quantitative 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 synapses 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 results. It is known that some neurons in the VCLL colocalize 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 consecutive 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 studies.
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 synaptic 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 periodicity 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 peripheral). 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 distinguish 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 GABAergic 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).
REFERENCES
Adams JC (1979) Ascending projections to the inferior colliculus.
J Comp Neurol 183:519 –538.
Adams JC (1983) Multipolar cells in the ventral cochlear nucleus
project to the dorsal cochlear nucleus and the inferior colliculus.
Neurosci Lett 37:205–208.
Aitkin LM, Phillips SC (1984a) Is the inferior colliculus an obligatory
relay in the cat auditory system? Neurosci Lett 44:259 –264.
Aitkin LM, Phillips SC (1984b) The interconnections of the inferior
colliculi through their commissure. J Comp Neurol 228:210 –216.
Arcelli P, Frassoni C, Regondi MC, De Biasi S, Spreafico R (1997)
GABAergic neurons in mammalian thalamus: a marker of thalamic
complexity? Brain Res Bull 42:27–37.
Bajo VM, Merchán MA, López DE, Roullier EM (1993) Neuronal morphology and efferent projections of the dorsal nucleus of the lateral
lemniscus of the cat. J Comp Neurol 334:241–262.
Bartlett EL, Stark JM, Guillery RW, Smith PH (2000) Comparison of
the fine structure of cortical and collicular terminals in the rat medial
geniculate body. Neuroscience 100:811– 828.
Bauer CA, Brozoski TJ, Holder TM, Caspary DM (2000) Effects of
chronic salicylate on GABAergic activity in rat inferior colliculus.
Hear Res 147:175–182.
Beyerl BD (1978) Afferent projections to the central nucleus of the
inferior colliculus in the rat. Brain Res 145:209 –223.
Brodal P, Mihailoff G, Border B, Ottersen OP, Storm-Mathisen J (1988)
GABA-containing neurons in the pontine nuclei of rat cat and
monkey: an immunocytochemical study. Neuroscience 25:27– 45.
Brunsø-Bechtold JK, Thompson GC, Masterton RB (1981) HRP study
of the organization of auditory afferents ascending to central nucleus of inferior colliculus in cat. J Comp Neurol 197:705–722.
Caspary DM, Holder TM, Hughes LF, Milbrandt JC, McKernan RM,
Naritoku DK (1999) Age-related changes in GABAA receptor subunit composition and function in rat auditory system. Neuroscience
93:307–312.
Casseday JH, Fremouw T, Covey E (2002) The inferior colliculus, a
hub in the midbrain. In: Integrative functions in the mammalian
auditory pathway (Oertel D, Fay RR, Popper AN, eds), pp 238 –
318. New York: Springer-Verlag.
Coleman JR, Clerici WJ (1987) Sources of projections to subdivisions
of the inferior colliculus in the rat. J Comp Neurol 262:215–226.
Coomes DL, Bickford ME, Schofield BR (2002) GABAergic circuitry in
the dorsal division of the cat medial geniculate nucleus. J Comp
Neurol 453:45–56.
Faingold CL (1999) Neuronal networks in the genetically epilepsyprone rat. Adv Neurol 79:311–321.
Faingold CL (2002) Role of GABA abnormalities in the inferior colliculus pathophysiology: audiogenic seizures. Hear Res 168:223–237.
Faingold CL, Boersma-Anderson CA, Caspary DM (1991) Involvement
of GABA in acoustically-evoked inhibition in inferior colliculus neurons. Hearing Res 52:201–216.
Faingold CL, Gehlbach G, Caspary DM (1989) On the role of GABA as
an inhibitory neurotransmitter in inferior colliculus neurons iontophoretic studies. Brain Res 500:302–312.
Faye-Lund H, Osen KK (1985) Anatomy of the inferior colliculus in rat.
Anat Embryol 171:1–20.
Fremouw T, Kleiser A, Heilman A, Casseday JH, Covey E (1999)
Intrinsic and commissural GABAergic connections in the inferior
colliculus of the big brown bat. Neurosci Abstr 25:1417.
923
Glendenning KK, Baker BN (1988) Neuroanatomical distribution of
receptors for three potential inhibitory neurotransmitters in the
brainstem auditory nuclei of the cat. J Comp Neurol 275:288 –308.
González-Hernández T, Mantolan B, González B, Pérez H (1996)
Sources of GABAergic input to the inferior colliculus of the rat.
J Comp Neurol 372:309 –326.
Hage SR, Ehret G (2003) Mapping responses to frequency sweeps
and tones in the inferior colliculus of house mice. Eur J Neurosci
18:2301–2312.
Irvine DRF (1992) Physiology of the auditory brainstem. In: Springer
handbook of auditory pathway neurophysiology (Popper AN, Fay
RR, eds), pp 153–231. New York: Springer-Verlag.
Kelly JB, Glenn SL, Beaver CJ (1991) Sound frequency and binaural
response properties of single neurons in rat inferior colliculus. Hear
Res 56:273–280.
Kelly JB, Liscum AL, van Abel B, Ito M (1998) Projections from the
superior olive and lateral lemniscus to tonotopic regions of the rat’s
inferior colliculus. Hear Res 116:43–54.
Kolston J, Osen KK, Hackney CM, Ottersen OP, Storm-Mathisen J
(1992) An atlas of glycine- and GABA-like immunoreactivity and
colocalization in the cochlear nuclear complex of the guinea pig.
Anat Embryol 186:443– 465.
Kulesza RJ Jr, Berrebi AS (2000) Superior paraolivary nucleus of the
rat is a GABAergic nucleus. J Assoc Res Otolaryngol 1:255–269.
Kuwada S, Batra R, Yin TCT, Oliver DL, Haberly LB, Stanford TR
(1997) Intracellular recordings in response to monaural and binaural stimulation of neurons in the inferior colliculus of the cat. J Neurosci 17:7565–7581.
Langner G, Albert M, Briede T (2002) Temporal and spatial coding of
periodicity information in the inferior colliculus of awake chinchilla,
Chinchilla laniger. Hear Res 168:110 –130.
LeBeau FEN, Malmierca MS, Rees A (1995) The role of inhibition in
determining neuronal responses properties in the inferior colliculus. In: Advances in hearing research (Manley GA, Klump G, Koppl
CH, Fastls H, Oekinghaus O, eds), pp 250 –257. Singapore: World
Scientific Press.
LeBeau FEN, Malmierca MS, Rees A (2001) Iontophoresis in vivo
demonstrates a key role for GABAA and glycinergic inhibition in
shaping frequency response areas in the inferior colliculus of
guinea pig. J Neurosci 21:7303–7312.
LeBeau FEN, Rees A, Malmierca MS (1996) The contribution of GABA
and Glycine mediated inhibition to the monaural temporal response
properties of neurons in the inferior colliculus. J Neurophysiol
75:902–919.
Lerma J, Paternain AV, Rodriguez-Moreno A, Lopez-Garcia JC (2001)
Molecular physiology of kainate receptors. Physiol Rev 81:
971–998.
Li L, Kelly JB (1992) Inhibitory influence of the dorsal nucleus of the
lateral lemniscus on binaural responses in the rat’s inferior colliculus. J Neurosci 12:4530 – 4539.
Ma CL, Kelly JB, Wu SH (2002) AMPA and NMDA receptors mediate
synaptic excitation in the rat’s inferior colliculus. Hear Res
168:25–34.
Malmierca MS (1991) Computer-assisted 3-D reconstructions of Golgiimpregnated cells in the rat inferior colliculus. Doctoral Thesis University of Oslo and Salamanca.
Malmierca MS (2003) The structure and physiology of the rat auditory
system: an overview. Int Rev Neurobiol 58:147–211.
Malmierca MS, Blackstad TW, Osen KK, Karagulle T, Molowny RL
(1993) The central nucleus of the inferior colliculus in rat: a Golgi
and computer reconstruction study of neuronal and laminar structure. J Comp Neurol 333:1–27.
Malmierca MS, Hernández O, Falconi A, López-Póveda EA, Merchán
MA, Rees A (2003) The commissure of the inferior colliculus
shapes frequency response areas in rat: an in vivo study using
reversible blockade with microinjection of kynurenic acid. Exp
Brain Res 153:522–529.
924
M. Merchán et al. / Neuroscience 136 (2005) 907–925
Malmierca MS, Leergaard TB, Bajo VM, Bjaalie JG (1998) Anatomic
evidence of a 3-D mosaic pattern of tonotopic organization in the
ventral complex of the lateral lemniscus in cat. J Neurosci
19:10603–10618.
Malmierca MS, Merchán M (2004) The auditory system. In: The rat
nervous system (Paxinos G, ed), pp 997–1082. San Diego: Academic Press.
Malmierca MS, Merchán M, Henkel CK, Oliver DL (2002) Direct projections from the dorsal cochlear nucleus to the auditory thalamus
in rat. J Neurosci 22:10891–10897.
Malmierca MS, Merchán MA, Oliver DL (1999a) Convergence of dorsal and ventral cochlear nuclei input onto frequency-band laminae
of the inferior colliculus a double tracer study in rat and cat. ARO
Abstr 22:221.
Malmierca MS, Oliver DL, Merchán MA (1999b) Convergence laminar
projections from dorsal DCN and ventral cochlear nucleus VCN to
inferior colliculus IC of rat and cat SFN. Abstr 25:1418.
Malmierca MS, Rees A, LeBeau FEN (1997) Ascending projections to
the medial geniculate body from physiologically identified loci in the
inferior colliculus. In: Acoustic signal processing in the central
auditory system (Syka J, ed), pp 295–302. New York: Plenum
Press.
Malmierca MS, Rees A, LeBeau FEN, Bjaalie JG (1995b) Laminar
organization of frequency-defined local axons within and between
the inferior colliculi of the guinea pig. J Comp Neurol 357:124 –144.
Malmierca MS, Seip KL, Osen KK (1995a) Morphological classification
and identification of neurons in the inferior colliculus a multivariate
analysis. Anat Embryol 191:343–350.
Marianowski R, Liao WH Van Den, Abbeele T, Fillit P, Herman P,
Frachet B, Huy PT (2000) Expression of NMDA AMPA and
GABA(A) receptor subunit mRNAs in the rat auditory brainstem: I.
Influence of early auditory deprivation. Hear Res 150:1–11.
Merchán MA, Berbel P (1996) Anatomy of the ventral nucleus of the
lateral lemniscus in rats: a nucleus with a concentric laminar organization. J Comp Neurol 372:245–263.
Merchán MA, Saldaña E, Plaza I (1994) Dorsal nucleus of the lateral
lemniscus in the rat concentric organization and tonotopic projection to the inferior colliculus. J Comp Neurol 342:259 –278.
Moore JK, Osen KK, Størm-Mathisen J, Ottersen OP (1996) gammaAminobutyric acid and glycine in the baboon cochlear nuclei an
immunocytochemical colocalization study with reference to interspecies differences in inhibitory systems. J Comp Neurol 369:
497–519.
Morest DK (1964) The laminar structure of the inferior colliculus of the
cat. Anat Rec 148:314.
Mulders WHAM, Robertson D (2000) Effects on cochlear responses of
activation of descending pathways from the inferior colliculus. Hear
Res 149:11–23.
Nelson PG, Erulkar SD (1963) Synaptic mechanisms of excitation and
inhibition in the central auditory pathway. J Neurophysiol 26:
908 –923.
Oliver DL (1984a) Dorsal cochlear nucleus projections to the inferior
colliculus in the cat a light and electron microscopic study. J Comp
Neurol 224:155–172.
Oliver DL (1984b) Neurons types in the central nucleus of the inferior
colliculus that project to the medial geniculate body. Neuroscience
11:409 – 424.
Oliver DL (1987) Projections to the inferior colliculus from the anteroventral cochlear nucleus in the cat possible substrates for binaural
interaction. J Comp Neurol 264:24 – 46.
Oliver DL (2000) Ascending efferent projections of the superior olivary
complex. Microsc Res Tech 51:355–363.
Oliver DL, Beckius GE, Bishop DC, Kuwada S (1997) Simultaneous
anterograde labeling of axonal layers from lateral superior olive
and dorsal cochlear nucleus in the inferior colliculus of cat. J Comp
Neurol 382:215–229.
Oliver DL, Beckius GE, Shneiderman A (1995) Axonal projections from
the lateral and medial superior olive to the inferior colliculus of the
cat: a study using electron microscopic autoradiography. J Comp
Neurol 360:17–32.
Oliver DL, Huerta M (1992) Inferior and superior colliculi. In: The
mammalian auditory pathway neuroanatomy (Webster DB, Popper
AN, Fay RR, eds), pp 168 –221. Berlin: Springer-Verlag.
Oliver DL, Kuwada S, Yin TCT, Haberly LB, Henkel CK (1991) Dendritic and axonal morphology of HRP-injected neurons in the inferior colliculus of the cat. J Comp Neurol 303:75–100.
Oliver DL, Morest DK (1984) The central nucleus of the inferior colliculus in the cat. J Comp Neurol 222:237–264.
Oliver DL, Ostapoff EM, Beckius GE (1999) Direct innervation of
identified tectothalamic neurons in the inferior colliculus by axons
from the cochlear nucleus. Neuroscience 93:643– 658.
Oliver DL, Shneiderman A (1991) The anatomy of the inferior
colliculus: a cellular basis for integration of monaural and binaural
information. In: Neurobiology of hearing (Altschuler RA, Bobbin
RP, Clopton BM, Hoffmann DW, eds), pp 195–222. New York:
Raven Press.
Oliver DL, Winer JA, Beckius GE, Saint Marie RL (1994) Morphology
of GABAergic neurons in the inferior colliculus of the cat. J Comp
Neurol 340:27– 42.
Ornung G, Shupliakov O, Linda H, Ottersen OP, Storm-Mathisen J,
Ulfhake B, Cullheim S (1996) Qualitative and quantitative analysis
of glycine and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: a postembedding
electron microscopic study. J Comp Neurol 365:413– 426.
Osen KK, Ottersen OP, Størm-Mathisen J (1990) Colocalization of
glycine-like and GABA-like immunoreactivities a semiquantitative
study of individual neurons in the dorsal cochlear nucleus of cat. In:
Glycine neurotransmission (Ottersen OP, Størm-Mathissen J,
eds), pp 417– 451. Chichester: Wiley.
Ottersen OP (1987) Postembedding light- and electron microscopic
immunocytochemistry of amino acids: description of a new model
system allowing identical conditions for specificity testing and
tissue processing. Exp Brain Res 69:167–174.
Ottersen OP, Hjelle OP, Osen KK, Laake JH (1995) Amino acid
transmitters. In: The rat nervous system, 2nd ed (Paxinos G, ed),
pp 1017–1037. San Diego: Academic Press.
Ottersen OP, Storm-Mathisen J (1984) Glutamate- and GABAcontaining neurons in the mouse and rat brain as demonstrated
with a new immunocytochemical technique. J Comp Neurol
229:374 –392.
Peruzzi D, Bartlett E, Smith PH, Oliver DL (1997) A monosynaptic
GABAergic input from the inferior colliculus to the medial geniculate body in rat. J Neurosci 17:3766 –3777.
Rees A, Sarbaz A, Malmierca MS, LeBeau FEN (1997) Regularity of
firing of neurons in the inferior colliculus of the guinea-pig. J Neurophysiol 77:2945–2965.
Reichenberger I, Straka H, Ottersen OP, Streit P, Gerrits NM,
Dieringer N (1997) Distribution of GABA glycine and glutamate
immunoreactivities in the vestibular nuclear complex of the frog.
J Comp Neurol 377:149 –164.
Riquelme R, Saldaña E, Osen KK, Ottersen OP, Merchán MA (2001)
Colocalization of GABA and glycine in the ventral nucleus of the
lateral lemniscus in rat: an in situ hybridization and semiquantitative immunocytochemical study. J Comp Neurol 432:409 – 424.
Roberts RC, Ribak CE (1987) An electron microscopic study of
GABAergic neurons and terminals in the central nucleus of the
inferior colliculus of the rat. J Neurocytol 16:333–345.
Roberts RC, Ribak CE, Kitzes LM, Oertel WH (1985) Regional distribution of GABAergic neurons and axon terminals in the brainstem
auditory nuclei of the gerbil. Anat Rec 211:161A.
Rose JE, Greenwood DO, Golderberg JM, Hind JE (1963) Some
discharge characteristic of single neurons in the inferior colliculus
of the cat: I. Tonopic organization relation of spike-counts to tone
intensity and firing patterns of single elements. J Neurophysiol
26:294 –320.
M. Merchán et al. / Neuroscience 136 (2005) 907–925
Ryan AF, Furlow Z, Wool NK, Keithley EM (1988) The spatial representation of frequency in the rat dorsal cochlear nucleus and
inferior colliculus. Hear Res 36:181–189.
Ryugo DK, Willard FH, Fekete DM (1981) Differential afferent projections to the inferior colliculus from the cochlear nucleus in the
albino mouse. Brain Res 210:342–349.
Saint Marie RL, Baker RA (1990) Neurotransmitter-specific uptake and
retrograde transport of [3H] glycine from the inferior colliculus by
ipsilateral projections of the superior olivary complex and nuclei of
the lateral lemniscus. Brain Res 524:244 –253.
Saint Marie RL, Ostapoff EM, Morest DK, Wenthold RJ (1989)
Glycine-immunoreactive projection of the cat lateral superior olive:
possible role in midbrain ear dominance. J Comp Neurol 279:
382–396.
Saint Marie RL, Stanforth DA, Jubelier EM (1997) Substrate for rapid
feedforward inhibition of the auditory forebrain. Brain Res
765:173–176.
Sanes DH, Geary WA, Wooten GF, Rubel EW (1987) Quantitative
distribution of the glycine receptor in the auditory brain stem of the
gerbil. J Neurosci 711:3793–3802.
Schreiner CE, Langner G (1988) Coding of temporal patterns in the
central auditory nervous system. In: Auditory function (Edelman
GM, Gall WE, Cowan WM, eds), pp 337–340. New York: Wiley.
Shiraishi S, Shiraishi Y, Oliver DL, Altschuler RA (2001) Expression of
GABA(A) receptor subunits in the rat central nucleus of the inferior
colliculus. Brain Res Mol Brain Res 96:122–132.
Shneiderman A, Henkel CK (1987) Banding of lateral superior olivary
nucleus afferents in the inferior colliculus: a possible substrate for
sensory integration. J Comp Neurol 266:519 –534.
Shneiderman A, Oliver DL, Henkel CK (1988) Connections of the
dorsal nucleus of the lateral lemniscus an inhibitory parallel pathway in the ascending auditory system? J Comp Neurol 276:
188 –208.
Shneiderman A, Chase MB, Rockwood JM, Benson CG, Potashner SJ
(1998) Evidence for a GABAergic projection from the dorsal nucleus of the lateral lemniscus to the inferior colliculus. J Neurochem
60:72– 82.
Somogyi P, Hodgson AJ, Smith AD, Nunzi MG, Gorio A, Wu JY (1984)
Different populations of GABAergic neurons in the visual cortex
and hippocampus of cat contain somatostatin- or cholecystokininimmunoreactive material. J Neurosci 4:2590 –2603.
Somogyi P, Halasy K, Somogyi J, Storm-Mathisen J, Ottersen OP
(1986) Quantification of immunogold labeling reveals enrichment
of glutamate in mossy and parallel fibre terminals in cat cerebellum. Neuroscience 19:1045–1050.
925
Spirou GA, Berrebi AS (1997) Glycine immunoreactivity in the lateral
nucleus of the trapezoid body of the cat. J Comp Neurol 383:
473– 488.
Suneja SK, Benson CG, Potashner SJ (1998) Glycine receptors in
adult guinea pig brain stem auditory nuclei: regulation after unilateral cochlear ablation. Exp Neurol 154:473– 488.
Thompson GC, Cortez AM, Man-Kit, Lam D (1985) Localization of
GABA immunoreactivity in the auditory brainstem of guinea pigs.
Brain Res 339:119 –122.
Vater M, Kossl M, Horn AK (1992) GAD- and GABA-immunoreactivity
in the ascending auditory pathway of horseshoe and mustached
bats. J Comp Neurol 325:183–206.
Vetter DE, Mugnaini E (1984) Immunocytochemical localization of
GABAergic elements in the rat inferior colliculus. Society for Neuroscience Abstr 10:1148.
Vetter DE, Mugnaini E (1985) Discrete bilateral GABAergic neuron
pools at the commissures of superior and inferior colliculi in the rat.
Society for Neuroscience Abstr 11:246.
von Bèkèsy G (1960) Experiments in hearing. New York: McGraw-Hill.
Walberg F, Ottersen OP (1989) Demonstration of GABA immunoreactive cells in the inferior olive of baboons (Papio papio and Papio
anubis). Neurosci Lett 101:149 –155.
Walberg F, Ottersen OP (1992) Neuroactive amino acids in the area
postrema: an immunocytochemical investigation in rat with some
observations in cat and monkey (Macaca fascicularis). Anat Embryol (Berl) 185:529 –545.
Wenthold RJ, Huie D, Altschuler RA, Reeks KA (1987) Glycine immunoreactivity localized in the cochlear nucleus and superior olivary
complex. Neuroscience 22:897–912.
Winer JA, Larue DT (1988) Anatomy of glutamic acid decarboxylase
immunoreactive neurons and axons in the rat medial geniculate
body. J Comp Neurol 278:47– 68.
Winer JA, Larue DT, Pollak GD (1995) GABA and glycine in the central
auditory system of the mustache bat: structural substrates for
inhibitory neural organization. J Comp Neurol 352:1–37.
Winer JA, Saint Marie RL, Laure DT, Oliver DL (1996) Gabaergic
feedforward projections from the inferior colliculus to the medial
geniculate body. Proc Natl Acad Sci USA 93:80005– 80010.
Yang Y, Surette AM, Bishop D, Oliver DL (2000) Properties of local
circuits in the central nucleus of the IC (CIC). SFN Abstr 26:674.
Zhang H, Kelly JB (2001) AMPA and NMDA receptors regulate responses of neurons in the rat’s inferior colliculus. J Neurophysiol
86:871– 880.
Zhang H, Kelly JB (2003) Glutamatergic and GABAergic regulation of
neural responses in inferior colliculus to amplitude-modulated
sounds. J Neurophysiol 90:477– 490.
(Accepted 30 December 2004)