Neuronal organization of the rat inferior colliculus participating in four major auditory pathways

Hearing Research

Hearing Research 218 (2006) 72–80

www.elsevier.com/locate/heares

Research paper

Neuronal organization of the rat inferior colliculus participating in four major auditory pathways Shigeo Okoyama a

a,*

, Masao Ohbayashi a, Makoto Ito b, Shinichi Harada

a

Laboratory of Neuroanatomy, Center for Biomedical Research and Education, Kanazawa University Graduate School of Medical Science, Kanazawa 920 8640, Japan b Division of Neuroscience, Clinical Neuroscience, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan Received 2 December 2005; received in revised form 19 April 2006; accepted 27 April 2006 Available online 11 July 2006

Abstract The central nucleus of the inferior colliculus (CNIC) contains different types of neurons and is a source of ascending projection to the medial geniculate body (MGB), commissural projection to the contralateral IC, direct descending projection to the cochlea nucleus (CN) and indirect projection to the CN via the superior olivary complex (SOC). Using a retrograde tracing technique, we examined what kind of neurons and what percentage of neurons of each type recognized in the CNIC participated in the above-mentioned four projection pathways. We also examined whether the individual CNIC neurons send the collateral to the MGB, the contralateral IC, the CN and the SOC. In the present study, we demonstrated that the neurons participating in the four projections could be morphologically classified into two types of neurons with soma size variation. The percentages of neurons of each type differed among the four projection pathways. Using a double-labeling technique, we found very few double-labeled neurons, indicating the collateral projections to the ipsilateral MGB and the contralateral IC. There were no double-labeled neurons in the collateral projections between the other combinations of targets. Therefore, we conclude that the ascending projection, the commissural projection and the descending projection to these targets arise from separate populations of neurons.  2006 Elsevier B.V. All rights reserved. Keywords: Neuronal classification; Collateral projections; Retrograde tracing; Ascending pathway; Descending pathway; Commissural pathway

1. Introduction Abbreviations: 7n, facial nerve; 8cn, cochlear root vestibulocochlear nerve; A-path, ascending pathway; CN, cochlea nucleus; CNIC, central nucleus of the inferior colliculus; C-path, commissural pathway; D1, direct descending projection to the CN; D2, indirect descending projection to the CN via the SOC; FG, Fluoro-Gold; FR, Fluoro-Ruby; HiF, hippocampal fissure; IC, inferior colliculus; INLL, intermediate nucleus of lateral lemniscus; MGB, medial geniculate body; MSO, medial superior olive; MVPO, medioventral zone of periolivary region; LSO, lateral superior olive; LVPO, lateroventral zone of periolivary region; py, pyramidal tract; SOC, superior olivary complex; sp5, spinal trigeminal tract; Tz, nucleus of trapezoid body; VCN, ventral cochlear nucleus; VNLL, ventral nucleus of the lateral lemniscus; VNTB, ventral nucleus of the trapezoid body * Corresponding author. Tel.: +81 762 265 2161. E-mail address: [email protected] (S. Okoyama). 0378-5955/$ - see front matter  2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.04.004

The inferior colliculus (IC) is an auditory midbrain relay station of the ascending projection to the medial geniculate body (MGB), the descending feedback to the CN, including the superior olivary complex (SOC) and the intercollicular commissural pathways (Coomes and Schofield, 2004; Faye-Lund, 1986; Faye-Lund, 1988; Gonzalez-Hernandez et al., 1996; Huffman and Henson, 1990; Malmierca et al., 2005; Oliver and Huerta, 1992; Schofield, 2001; Schofield and Cant, 1999). The ascending projection may transmit almost all auditory information destined to reach the cerebral cortex following the processing of information from the lower brainstem in the

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

neurons of the IC. This ascending projection is predominantly ipsilateral to the MGB. The commissural projection may be to modulate the response gain of the IC neurons to acoustic stimuli (Malmierca et al., 2005). The descending projections presumably serve to modify the processing of incoming information from the lower auditory center, including the CN (Huffman and Henson, 1990). Both sides of the CN received crossed and uncrossed direct descending projections from the IC. In addition to the direct projection from the IC to the CN, the IC has a second route of descending influence on CN activity via a synaptic relay in the SOC and/or the ventral nucleus of the trapezoid body (VNTB) (FayeLund, 1986; Huffman and Henson, 1990; Malmierca et al., 1996; Schofield and Cant, 1999). The IC also contributes to motor and vocalization functions through direct projections to the pons, superior colliculus, and the periaqueductal gray (Schuller et al., 1991). Understanding the functions of these various pathways, it is important to determine whether different populations of cells project to each target, or whether individual cells send axon collaterals to multiple targets. On the basis of the soma shape, size, dendritic morphology and orientation, the various types of neurons in the IC have been described in the rat (Faye-Lund and Osen, 1985; Malmierca et al., 1993), the mouse (Meininger et al., 1986), the cat (Oliver and Morest, 1984; Rockel and Jones, 1973a,b), the guinea pig (Schofield, 2001) and the squirrel monkey (FitzPatrick, 1975). The previous physiological studies showed that six or seven different types of neurons in the CNIC were classified according to their response to acoustic stimuli in the guinea pig (Le Beau et al., 1996, 2001; McAlpine et al., 1996; Rees et al., 1997; Syka et al., 2000), or by their intrinsic electrophysiological membrane properties in the rat (Bal et al., 2002; Peruzzi et al., 2000; Sivaramakrishnan and Oliver, 2001). As described above, it had been reported in the various species that a variety of neuronal types morphologically or physiologically were identified in the IC. However, it is still unclear about the relationship between the morphological classification of the neurons in the IC and their target nuclei. Since the central nucleus of the inferior colliculus (CNIC) is part of the lemniscal tonotopic pathway and is believed to be important in processing and coding auditory information for hearing (Irvine and Gago, 1990), it must be very important to investigate the morphological difference of the IC neurons and their target nuclei. Therefore, using a retrograde tracing technique, we examined what kind of neurons and what percentage of neurons of each type in the CNIC participated in the above-mentioned four projection pathways, the ascending projection to the ipsilateral MGB, the commissural projection to the contralateral IC, the descending projection to the ipsilateral CN and the CN via the SOC. We also examined whether the individual CNIC neurons send the collateral to the MGB, the contralateral IC, the CN and the SOC using a double-labeling technique.

73

2. Materials and methods 2.1. Animals and tracer injection A total of 38 adult male or female Spargue–Dawley rats were used in this study. The Animal Research Committee of Kanazawa University Graduate School of Medical Science approved the care and use of animals in the present study. The animals were anesthetized using an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). Prior to surgery, the animals were premedicated with atropine sulfate (0.08 mg/kg, i.m.) to decrease bronchial secretions. For tracer injection, the animals were fixed in a stereotaxic device. A hole was then drilled in the skull for each injection, guided by stereotaxic coordinates. The tracer was injected with a glass micropipette attached to a Picospritzer air-pressure injection system (General Valve). To cover a large region, a sufficient volume of each tracer (0.2–0.5 ll) was injected at multiple sites (usually four) within each target. Following all injections, the skin was sutured and the animal was returned to its cage. The following tracers were used: Fluoro-Gold (FG, 2% solution aqueous solution; FluoroChrome Inc. Englewood, CO) and Fluoro-Ruby (FR, 10% solution in saline; tetramethylrhodamine dextran, molecular weight = 10,000: Molecular Probes, Inc., Eugene OR). In the majority of retrograde tracers, the spread of the dye from the soma to the dendrites was too limited to reveal the detailed morphology. However, FG produces strongly intense cytoplasmic labeling with good visualization of dendrites in retrogradely labeled neurons, and it allows us to identify the morphological class of the labeled cell. To assess the distribution and morphology of labeled IC neurons, the FG was injected unilaterally into the MGB, IC, CN and SOC, respectively. In addition to the injections, these animals were received FR injections into the MGB, CN and IC (detailed tracers combination, injection and summary of analysis in Table 1). Then, we observed the right CNIC and examined five patterns of collateral projections from individual IC neurons: (1) to ipsilateral MGB and contralateral IC; (2) to ipsilateral MGB and ipsilateral CN; (3) to ipsilateral CN and contralateral IC; (4) ipsilateral CN and contralateral CN; (5) ipsilateral SOC and contralateral IC. 2.2. Fixation and tissue processing After an appropriate interval for the transport of the injected tracers (2–4 days), the animals were given an overdose of anesthetic and were perfused through the left ventricle with ice cold 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). The brains were removed and post-fixed with the same fixative overnight and stored in a 30% sucrose buffer for several days. Coronal sections were cut at 40 lm on a freezing microtome and collected in phosphate buffered-saline. The sections were counterstained with NeuroTrace 500/525 green-fluorescent Nissl

74

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

Table 1 Tracer injection and summary of observation in four major pathways FG injection MGB MGB IC IC CN CN CN SOC

FR injection (R) (R) (L) (L) (R) (R) (R) (R)

CN IC MGB CN MGB IC CN IC

(R) (L) (R) (R) (R) (L) (L) (L)

Analysis of neuron in each pathway

Combination collateral pathway

n (rat)

A-pathway A-pathway C-pathway C-pathway D1-pathway D1-pathway D1-pathway D2-pathway

A and D1 A and C C and A C and D1 D1 and A D1 and C D1 and D1 D2 and C

5 4 4 5 6 5 6 3

R, right; L, left; A, ascending; C, commissural; D1, direct descending projection to the CN; D2, indirect descending projection to the CN via the SOC; n, number of rat.

stain (Invitrogen) for the identification of the cytoarchitectural borders of CNIC. After the staining, the sections were mounted onto gelatin-coated slides and cover slipped with vector seal. 2.3. Data analysis The sections were viewed using an Olympus fluorescence microscope with equipped with epifluorescence and different filter systems for ultraviolet (for FG), rhodamine (for FR) and fluorescence (for Nissl counter stain) illumination. To identify the different neuronal types and/or doublelabeled neurons, sections were visualized through different filters at high magnifications (40· objective). Labeled neurons were photographed with a digital camera (Olympus DP70) and the soma of individual different neurons was then measured using computer software (DP Controller, Olympus). Although there were many methods to identify subdivisions of the IC, we used traditional Nissl staining technique to identify the CNIC in the present study (Faye-Lund and Osen, 1985; Oliver, 2005). FG-labeled neurons were observed in the sections throughout the entire right CNIC. We analyzed the FGlabeled neurons that project to the ipsilateral MGB, the contralateral IC, ipsilateral CN and SOC. The number of each type of FG labeled neurons and the total number of FG labeled neurons were counted to examine the percentages of different types of neurons participating in the four pathways. The difference in the percentage of individual neurons participating in the four pathways was compared statistically using Student’s t test, *p = 0.05, **p = 0.01. The number of double-labeled neurons (FR labeled neurons in the FG labeled neurons) was counted simultaneously with FG labeled neurons. 3. Results 3.1. Injection site and retrograde labeling neuron In order to label the maximum amount of neurons in the IC, we made large injections of FG into the MGB, the IC, the CN and the SOC (VNTB). Fig. 1 shows a representa-

tive injection site. In the case of MGB injection, the tracer spread throughout much of the MGB and encroached on some of the surrounding nuclei (Fig. 1A). Following the injections into the MGB, many labeled neurons were present in all three subdivisions in the ipsilateral side of the IC. Only a few labeled neurons were found in the contralateral IC. In the CNIC, the ratio of the number of labeled cells on the ipsilateral side versus the contralateral side was about 63:1 (average ratio of four rats). Therefore, we will only focus on labeled neurons within the ipsilateral CNIC that participate in the ipsilateral ascending pathway. Fig. 1B illustrates a typical injection site in which FG extended into the central nucleus, the external cortex and the dorsal cortex of the IC without encroaching on the dorsal nucleus of the lateral lemniscus, superior colliculus, or other nearby structures. FG labeled cells were observed in all subdivisions of the contralateral IC, as well as many brainstem nuclei, including the superior olivary complex, the cochlear nuclei, dorsal and intermediate nuclei of the lateral lemniscus. FG injections into the CN were located on the ventral and dorsal CN, and the granule cell areas. Our large injections spread throughout all subdivisions of the CN and extended medially and caudally to invade the spinal trigeminal tract (Fig. 1C). However, none of the injections spread into the superior olivary complex, nor the lateral lemniscus. In the CNIC, the labeled cells were more numerous on the ipsilateral side than on the contralateral side. The ratio of the number of labeled cells on the ipsilateral side versus that on the contralateral side was about 1.6:1 (average ratio of four rats). In the case of SOC and/or VNTB injection (Fig. 1D), the tracer infiltrated area involved in MSO, LSO, SPN, medioventral zone of periolivary region (MVPO) and lateroventral zone of periolivary region (LVPO). The ventral nucleus of the lateral lemniscus (VNLL) was free of tracer infiltration. All types of neurons distinguished in present study were labeled. The FG labeled cells were observed in the ipsilateral CNIC and the external cortex of the IC. The present result was in an agreement with the result of Faye-Lund (1986). We focused on the labeled neurons participating in the ipsilateral descending projection pathway in this study.

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

75

Fig. 1. Photomicrographs of representative Fluoro-Gold (FG) injection sites. (A) Injection of FG into the right medial geniculate body (MGB). (B) Injection of FG into the left inferior colliculus (IC). (C) Injection of FG into the right cochlear nucleus (CN). (D) Injection of FG into the right superior olivary complex (SOC) and/or ventral nucleus of the trapezoid body (VNTB). HiF, hippocampal fissure; MGB, medial geniculate body; SNR, substantia nigra reticular; IC, inferior colliculus; DNLL, dorsal nucleus of lateral lemniscus; INLL, intermediate nucleus of lateral lemniscus; 7n, facial nerve; sp5, spinal trigeminal tract; VCN, ventral cochlear nucleus; 8cn, cochlear root vestibulocochlear nerve; LSO, lateral superior olive; MSO, medial superior olive; MVPO, medioventral periolivary nucleus; Tz, nucleus trapezoid body; py, pyramidal tract.

3.2. Morphology of FG labeled neurons participating in four pathways In the majority of instances, the spread of the retrograde tracer FG from the soma into the dendrites was sufficient for the distinction of neuronal types as previously observed in Golgi material. On the basis of dendritic orientation and morphology, soma shape and size, two types of neurons with soma size variation were identified in the present retrograde FG labeling material. Most FG labeled neurons had fusiform to oval soma and dendritic fields oriented ventrolateral to dorsomedial in a transverse section of the

CNIC (Fig. 2A and B without arrows). The long axes of the dendritic fields were aligned in parallel arrays to produce a characteristic layered appearance in Fig. 2A. These neurons seemed to correspond to the disc-shaped and/or the flat type neurons (Fig. 2B without arrows) reported in the Golgi study (Malmierca et al., 1993; Morest and Oliver, 1984; Oliver and Morest, 1984). The other labeled neurons had rectangular or multipolar soma with dendrites that extended perpendicular to the laminae constituted by dendrites of disk-shaped neurons, as observed in Golgi sections, and seemed to correspond with the stellate and/or less flat type neurons (Fig. 2 arrows indicated in B).

Fig. 2. Photomicrograph of the right CNIC after FG injection into the contralateral IC (A) and (B) the appearance of the dendritic and somatic morphology in the high-power camera lucida drawing of the rectangular in the A. In the transverse section, the majority of dendrites and soma of discshaped neurons appeared to be arranged in parallel with each other and formed the ventrolateral to dorsomedial laminae. Note stellate neurons (arrows) with dendrites cutting across the laminae (B). Scale bar = 40 lm in A and 20 lm in B.

76

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

Fig. 3. Box and whisker plots summarizing the distribution of soma size of disc-shaped or stellate neurons participating in the four pathways. A box and whisker plot provides an excellent visual summary of a distribution. A box and whisker plot consists of rectangle showing the interquartile range (IQR) that is used to describe the dispersion of the data. Note that the IQR contains exactly 50% of the data within the distribution. Within the box the median is depicted as a separating line. To the left and right of the box are lines (called whiskers), which extend to the most extreme value with in 1.5 times IQR above or below the hinges of the box. Each whisker contains 25% of the data. Circles indicate outliers that are values farther removed from the median (outside values). n, Number of labeled neurons; Me, median; A, ascending pathway; C, commissural pathway; D1, direct projection to the CN; D2, indirect projection to the CN via the SOC.

The soma diameters of the two types of FG labeled neurons were measured using computer software (DP Controller, Olympus), and the result was presented in box and whisker plots summarizing the distribution of the soma sizes of the two types of neurons participating in the four pathways (Fig. 3). Considering the relative position of the median from the box and whisker plots, the disc-shaped labeled neurons could be subdivided according to the size of the cell body into small (range 7–12 lm), medium (13– 20) and large (21–31) neurons, and the stellate neurons into medium (12–20) and large (21–35) neurons. Comparison of the relative position of the median, the interquatile range (IQR) and the length of the whisker across the four pathways indicated that the populations of neuronal soma sizes differ among the four pathways. 3.3. Percentages of the different types of neurons that participate in the four pathways In order to assess the relative contribution of different types of neuron to the four projecting pathways, we counted each type of labeled neurons and expressed the results as the mean percentage of labeled cells in the CNIC (Table 2). Following FG injection into the target nuclei, all types of neurons recognized in this study were retrogradely labeled in the CNIC. In the ascending projection pathway to the ipsilateral MGB, the proportions of all disk-shaped and stellate neurons were 63.8% and 36.2%, respectively. In subdivision of the disc-shaped neurons, percentages were 30.0% (small), 26.7% (medium) and 7.1% (large) of all the constituent neurons, respectively. In the subdivision of the stellate neurons, the percentages of medium and large neurons were 30.8% and 5.3%. In the commissural projection pathway, the proportions of disk-shaped and stellate neurons were 67.1% and 32.9%. The proportions of the subdivision of disc-shaped neurons accounted for

38.4% in small, 23.3% in medium and 2.0% in large neurons, respectively. In the subdivision of the stellate neurons, the proportions of medium and large neurons were 26.8% and 6.0%, respectively, of all the constituent neurons. In the neurons participating in descending pathway from the IC to the direct ipsilateral CN (D1) or the CN via the SOC (D2), the proportions of disc-shaped and stellate neurons were 72.4% or 80.7%, and 27.6% or 19.3%, respectively. The proportions of the subdivision of discshaped neurons accounted for 47.9% or 60.5% in small, 22.0% or 18.6.0% in medium, and 2.6% or 1.6% in large neurons, respectively. In the subdivision of stellate neurons, the proportions of medium and large neurons were 26.0% or 1.6% and 17.1% or 2.2%, respectively. A comparison of the percentages of the different neurons participating in the four pathways and the statistical significance of the proportional difference in each neuron among the four pathways are shown in the Fig. 4. The proportion of small disc-shaped neurons was significantly higher in the descending pathways than in the other ascending and commissural pathways. Within the descending pathways, the proportions of small disc-shaped neurons were also significantly different between the direct (D1) and indirect (D2) pathways. On the other hand, the proportion of the medium stellate neuron was significantly high in the ascending pathway as compared with the indirect descending pathway. With regard to the proportions of the two different categories of neurons, the mean percentages of all disc-shaped neurons were 80.7%, 72.4%, 67.1% and 63.8% in neurons participating in D2, D1, C and A pathways, respectively (Table 2) and statistical differences were observed among the four different pathways (Fig. 4). As described above, a comparison of the percentages of the different types of neurons participating in the four pathways revealed that the contribution of each type of neuron was characteristic among the pathways (Fig. 4).

100.0

17.1 ± 1.3 2.2 ± 0.5 19.3 ± 1.4

100.0

3029

16.1 ± 0.3 22.5 ± 1.0 537 61 598 26.0 ± 0.6 1.6 ± 0.5 27.6 ± 1.8

6536 100.0

1725 112 1837

5950 100.0

1675 1537 375 3587

1736 312 2048

5635

Stellates Medium Large All Stellates

Total

16.7 ± 0.2 23.5 ± 0.8

30.8 ± 0.5 5.3 ± 0.9 36.2 ± 0.9

1586 351 1937

16.3 ± 0.2 24.6 ± 0.8

26.8 ± 1.5 6.0 ± 0.7 32.9 ± 2.1

15.7 ± 0.2 23.7 ± 1.5

60.5 ± 1.9 18.6 ± 1.3 1.6 ± 0.2 80.7 ± 1.4 9.9 ± 0.1 15.1 ± 0.3 23.6 ± 1.4 1814 571 46 2431 47.9 ± 2.0 22.0 ± 2.1 2.6 ± 0.4 72.4 ± 0.8 3075 1449 175 4699

To examine the distribution of single-labeled cells with different tracers simultaneously, or to determine whether individual neurons send collateral projections to innervate the four targets, two different tracers were injected into the targets shown in Table 1. All different types of neurons distinguished in the present study were labeled with both tracers, and the distributions of the single-labeled neurons with different tracers were similar to each other among the four pathways. Fig. 5 shows that the FG single-labeled cells ascending to the MGB (Fig. 5A) and the FR labeled cells commissural projection to the contralateral IC (Fig. 5B) were close to each other and intermingled within the CNIC (Fig. 5C). Only a few of the disc-shaped neurons were double-labeled with both FG and FR, indicating collateral projection to the ipsilateral MGB and the contralateral IC (arrow indicated in Fig. 5C). Quantitative analysis showed that the double-labeled neurons represented less than 1% of the single-labeled neurons of all types. Double-labeled neurons were not found in other combinations of collateral projection pathways: the ipsilateral MGB and CN; the ipsilateral CN and the contralateral IC; the ipsilateral SOC (VNTB) and the contralateral IC; the ipsilateral and the contralateral CN. Therefore, it is suggested in the present study that there may be several populations in each type of neurons that differ in their projections. 4. Discussion 4.1. Morphology and contribution of the different types neurons that participate in the four major pathways

Discs Small Medium Large All Discs

10.2 ± 0.1 15.9 ± 0.3 22.9 ± 0.2

30.0 ± 1.5 26.7 ± 2.1 7.1 ± 1.5 63.8 ± 0.9

2475 1413 125 4013

10.2 ± 0.1 14.9 ± 0.2 23.4 ± 0.9

38.4 ± 2.0 23.3 ± 2.1 2.0 ± 0.6 67.1 ± 2.1

10.0 ± 0.1 15.5 ± 0.2 23.0 ± 0.8

Diameter (mean ± SE) Cell examined Diameter (mean ± SE) Cell examined Diameter (mean ± SE)

77

3.4. Distribution and collateral projection of neurons

% (mean ± SE) Diameter (mean ± SE) Cell examined Cell examined

% (mean ± SE)

C-pathway A-pathway Cell type

Table 2 Mean percentage and diameter of different types of neurons that participating in the four pathways

D1-pathway (CN)

% (mean ± SE)

D2-pathway (SOC)

% (mean ± SE)

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

In many previous studies about IC neurons, multiple cell types have been distinguished either by morphological or physiological criteria (Faye-Lund, 1985; Gonzalez Hernandez et al., 1986, 1991; Le Beau et al., 1996; Malmierca et al., 1993; Oliver, 1984; Oliver and Morest, 1984; Peruzzi et al., 2000; Reetz and Ehret, 1999; Ribak and Roberts, 1986; Schofield, 2001; Sivaramakrishnan and Oliver, 2001). In the present retrograde tracing experiment, the spread of retrograde tracer FG from the soma into the dendrites was sufficient for the distinction of neuronal types, as previously observed in the Golgi study. According to the visual criteria of the Golgi study (Malmierca et al., 1993; Morest and Oliver, 1984; Oliver and Morest, 1984), the present FG labeled neurons could be divided into two groups: disc-shaped cells that contribute to form the dendritic lamina, oriented ventrolateral to dosomedial, and stellate cells defined as having dendrites cutting across the laminae in the camera lucida drawing (Fig. 2B indicated arrows). It also appeared that several varieties were defined according to cell size in the box and whisker plots, summarizing the distribution of soma size of the two types of neurons (Fig. 3). These present results showed that five different types of neurons were classified in the CNIC after

78

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

Fig. 4. Proportion of distinct types of neurons and statistical significance of proportional difference in each neuron among the four pathways. Bar graph shows the percentages of different types of neurons within each projection pathway. Note the difference in the percentages of each neuron and the significant difference among the four projection pathways. Projection pathways are shown in the key. The values (%) presented are the means. *p < 0.05, **p < 0.01 Student’s t test.

Fig. 5. Fluorescent photomicrographs of labeled neurons in the CNIC. (A) FG labeled neurons project to the ipsilateral MGB. (B) FR labeled neurons project to the contralateral IC. Paired photomicrographs were taken using different filter systems. (C) Fluorescent overlay of single labeled neurons. Note FG and FR labeled neurons are close to each other and intermingled in the CNIC. The arrow indicated the double-labeled neuron, suggesting collateral projection to the MGB and the IC. Scale bar = 20 lm.

FG injection, and were largely corresponded with the other morphological studies cited above. It is reasonable to speculate that morphologically different type of neurons may have different functions. As a matter of fact, neurons in the IC have different responses to acoustic stimulation (Covey et al., 1996; Kuwada et al., 1997; Peruzzi et al., 2000; Popelar and Syka, 1982). Many of the differences in response may be due to complex interactions of multiple neuronal parameters i.e. the location in a particular synaptic domain, the axonal target, the neuronal morphology, the intrinsic membrane properties, and the types of synaptic input (Kelly and Zhang, 2002; Oliver, 2000, 2005; Sivaramakrishnan and Oliver, 2001; Wu et al., 2002). Since there is evidence suggesting that differences in CNIC neuronal cell size may correlate differences in neurotransmitters or in patterns of synaptic organiza-

tion (Oliver et al., 1994; Ribak and Roberts, 1986; Roberts and Ribak, 1987; Shiraishi et al., 2001), diversity in organization of the IC neurons projecting to the major four targets as well as the diversity in response of the IC neurons may play important role for auditory information processing. However, it is unclear organization of the IC projection neurons i.e. what kind of neurons or how much percentage of neurons of each type participates in four major pathways. Therefore, we examined the morphology of neuron type, its distribution, and percentages of neuron of each type participating in four pathways after FG injection into the target nuclei. All types of neurons recognized in the present study contributed to four pathways. The distribution of the neurons overlapped and intermingled with each other, but the percentages of specific neuronal types differed among the four projection pathways. In

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

the present study, it appeared that the percentage of all stellate cells was about 36.2% in the ascending, 32.9% in the commissural, and low in the descending pathways (27.6% in D1, 19.3% in D2), respectively. As noted earlier, the disc-shaped cells have narrow dendritic field parallel the incoming fibers from lower brain stem auditory nuclei and create fibrodendritic laminae, and dendrites of the stellate cells typically extend beyond the single fibrodendritic lamina into adjacent laminae. With respect to the dendritic patterns, it may be expected that disc-shaped cells and stellate cells would transmit a relatively restricted and wide range of acoustic information, respectively, through the four projection pathways to the targets. Therefore, it appeared that within the ascending pathway, wide range of acoustic information was preferentially transmitted, and within the descending pathway, a restricted range of acoustic information (72.4–80.7%) was dominantly transmitted to their targets. 4.2. Collateral projection to four major targets With respect to understanding the functions of the four major auditory pathways, it is important to determine whether different populations of cells project to each target or whether individual cells send axon collaterals to multiple targets. For collateral projections, it would be expected that the same information would be sent simultaneously to each target. In contrast, separate origins of projections may receive different combinations of inputs, and send different information to their targets. As demonstrated in the present study, the collateral projections were found in between the ascending and the commissural projections (less than 1% of FG single labeled cells), but not in between other projections. These findings were largely congruent with the results of previous doublelabeling studies (Coomes and Schofield, 2004; GonzalezHernandez et al., 1991; Schofield, 2001). The present results showed the existence of the neuronal circuit, that the same auditory information ascending to the ipsilateral higher auditory center simultaneously traverses to the contralateral side at the IC. This circuit may be important, considering that there were few neurons projecting contralateral MGB, as revealed in the present results, or it may be that the commissural projection of the IC modulates the response gain of IC neurons to acoustic stimulation (Malmierca et al., 2005, 2003). However, the small number of double-labeled cells indicated in the present study makes it difficult to determine whether this small population serves a significant function of collateral projections. The present study also indicated that there were no double-labeled cells in the other collateral projections between the ascending and descending or, between the descending and commissural, or between both cochlea nuclei. What is the functional significance of the three projections arising from separate populations of cells? With regard to the collateral projection between the ascending and descending pathways, the following possibility is considered here.

79

Since all ascending auditory information converges in the IC, the ascending projection from the IC may participate in the majority of auditory functions. On the other hand, descending projections are known to have a feedback function (Huffman and Henson, 1990), whereby a higher auditory center may affect the lower center processing in the ascending auditory pathways. Therefore, the descending signals must be different from those in the ascending pathways. It may be reasonable to speculate from the present study that separate origins for ascending, descending and commissural projections could be a general characteristic of auditory pathways. Acknowledgment We thank Prof. Shoichi Iseki, of the Department of Histology and Embryology, Kanazawa University Graduate School of Medical Science, for his critical reading of the manuscript. References Bal, R., Green, G.G., Rees, A., Sanders, D.J., 2002. Firing patterns of inferior colliculus neurons-histology and mechanism to change firing patterns in rat brain slices. Neurosci. Lett. 317, 42–46. Coomes, D.L., Schofield, B.R., 2004. Separate projections from the inferior colliculus to the cochlear nucleus and thalamus in guinea pigs. Hear. Res. 191, 67–78. Covey, E., Kauer, J.A., Casseday, J.H., 1996. Whole-cell patch-clamp recording reveals subthreshold sound-evoked postsynaptic currents in the inferior colliculus of awake bats. J. Neurosci. 16, 3009–3018. Faye-Lund, H., 1985. The neocortical projection to the inferior colliculus in the albino rat. Anat. Embryol. (Berl) 173, 53–70. Faye-Lund, H., 1986. Projection from the inferior colliculus to the superior olivary complex in the albino rat. Anat. Embryol. (Berl) 175, 35–52. Faye-Lund, H., 1988. Inferior colliculus and related descending pathways in rat. Ups J. Med. Sci. 93, 1–17. Faye-Lund, H., Osen, K.K., 1985. Anatomy of the inferior colliculus in rat. Anat. Embryol. (Berl) 171, 1–20. FitzPatrick, K.A., 1975. Cellular architecture and topographic organization of the inferior colliculus of the squirrel monkey. J. Comp. Neurol. 164, 185–207. Gonzalez Hernandez, T.H., Meyer, G., Ferres-Torres, R., 1986. The commissural interconnections of the inferior colliculus in the albino mouse. Brain Res. 368, 268–276. Gonzalez-Hernandez, T.H., Galindo-Mireles, D., Castaneyra-Perdomo, A., Ferres-Torres, R., 1991. Divergent projections of projecting neurons of the inferior colliculus to the medial geniculate body and the contralateral inferior colliculus in the rat. Hear. Res. 52, 17–21. Gonzalez-Hernandez, T., Mantolan-Sarmiento, B., Gonzalez-Gonzalez, B., Perez-Gonzalez, H., 1996. Sources of GABAergic input to the inferior colliculus of the rat. J. Comp. Neurol. 372, 309–326. Huffman, R.F., Henson Jr., O.W., 1990. The descending auditory pathway and acousticomotor systems: connections with the inferior colliculus. Brain Res. Brain Res. Rev. 15, 295–323. Irvine, D.R., Gago, G., 1990. Binaural interaction in high-frequency neurons in inferior colliculus of the cat: effects of variations in sound pressure level on sensitivity to interaural intensity differences. J. Neurophysiol. 63, 570–591. Kelly, J.B., Zhang, H., 2002. Contribution of AMPA and NMDA receptors to excitatory responses in the inferior colliculus. Hear. Res. 168, 35–42.

80

S. Okoyama et al. / Hearing Research 218 (2006) 72–80

Kuwada, S., Batra, R., Yin, T.C., Oliver, D.L., Haberly, L.B., Stanford, T.R., 1997. Intracellular recordings in response to monaural and binaural stimulation of neurons in the inferior colliculus of the cat. J. Neurosci. 17, 7565–7581. Le Beau, F.E., Rees, A., Malmierca, M.S., 1996. Contribution of GABAand glycine-mediated inhibition to the monaural temporal response properties of neurons in the inferior colliculus. J. Neurophysiol. 75, 902–919. LeBeau, F.E., Malmierca, M.S., Rees, A., 2001. Iontophoresis in vivo demonstrates a key role for GABA(A) and glycinergic inhibition in shaping frequency response areas in the inferior colliculus of guinea pig. J. Neurosci. 21, 7303–7312. Malmierca, M.S., Blackstad, T.W., Osen, K.K., Karagulle, T., Molowny, R.L., 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, M.S., Le Beau, F.E., Rees, A., 1996. The topographical organization of descending projections from the central nucleus of the inferior colliculus in guinea pig. Hear. Res. 93, 167–180. Malmierca, M.S., Hernandez, O., Falconi, A., Lopez-Poveda, E.A., Merchan, M., 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. Malmierca, M.S., Hernandez, O., Rees, A., 2005. Intercollicular commissural projections modulate neuronal responses in the inferior colliculus. Eur. J. Neurosci. 21, 2701–2710. McAlpine, D., Jiang, D., Palmer, A.R., 1996. Interaural delay sensitivity and the classification of low best-frequency binaural responses in the inferior colliculus of the guinea pig. Hear. Res. 97, 136–152. Meininger, V., Pol, D., Derer, P., 1986. The inferior colliculus of the mouse. A Nissl and Golgi study. Neuroscience 17, 1159–1179. Morest, D.K., Oliver, D.L., 1984. The neuronal architecture of the inferior colliculus in the cat: defining the functional anatomy of the auditory midbrain. J. Comp. Neurol. 222, 209–236. Oliver, D.L., 1984. Neuron types in the central nucleus of the inferior colliculus that project to the medial geniculate body. Neuroscience 11, 409–424. Oliver, D.L., 2000. Ascending efferent projections of the superior olivary complex. Microsc. Res. Technol. 51, 355–363. Oliver, D.L., 2005. Neural Organization in the Inferior Colliculus. Springer-Verlag, New York. Oliver, D.L., Huerta, M.F., 1992. Inferior and Superior Colliculi. Springer, New York. Oliver, D.L., Morest, D.K., 1984. The central nucleus of the inferior colliculus in the cat. J. Comp. Neurol. 222, 237–264.

Oliver, D.L., Winer, J.A., Beckius, G.E., Saint Marie, R.L., 1994. Morphology of GABAergic neurons in the inferior colliculus of the cat. J. Comp. Neurol. 340, 27–42. Peruzzi, D., Sivaramakrishnan, S., Oliver, D.L., 2000. Identification of cell types in brain slices of the inferior colliculus. Neuroscience 101, 403– 416. Popelar, J., Syka, J., 1982. Response properties of neurons in the inferior colliculus of the guinea-pig. Acta Neurobiol. Exp. (Wars) 42, 299–310. Rees, A., Sarbaz, A., Malmierca, M.S., Le Beau, F.E., 1997. Regularity of firing of neurons in the inferior colliculus. J. Neurophysiol. 77, 2945– 2965. Reetz, G., Ehret, G., 1999. Inputs from three brainstem sources to identified neurons of the mouse inferior colliculus slice. Brain Res. 816, 527–543. Ribak, C.E., Roberts, R.C., 1986. The ultrastructure of the central nucleus of the inferior colliculus of the Sprague–Dawley rat. J. Neurocytol. 15, 421–438. Roberts, R.C., Ribak, C.E., 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. Rockel, A.J., Jones, E.G., 1973a. Observations on the fine structure of the central nucleus of the inferior colliculus of the cat. J. Comp. Neurol. 147, 61–92. Rockel, A.J., Jones, E.G., 1973b. The neuronal organization of the inferior colliculus of the adult cat I. The central nucleus. J. Comp. Neurol. 147, 11–60. Schofield, B.R., 2001. Origins of projections from the inferior colliculus to the cochlear nucleus in guinea pigs. J. Comp. Neurol. 429, 206–220. Schofield, B.R., Cant, N.B., 1999. Descending auditory pathways: projections from the inferior colliculus contact superior olivary cells that project bilaterally to the cochlear nuclei. J. Comp. Neurol. 409, 210–223. Schuller, G., Covey, E., Casseday, J.H., 1991. Auditory pontine grey: connections and response properties in the horseshoe bat. Eur. J. Neurosci. 3, 648–662. Shiraishi, S., Shiraishi, Y., Oliver, D.L., Altschuler, R.A., 2001. Expression of GABA(A) receptor subunits in the rat central nucleus of the inferior colliculus. Brain Res. Mol. Brain Res. 96, 122–132. Sivaramakrishnan, S., Oliver, D.L., 2001. Distinct K currents result in physiologically distinct cell types in the inferior colliculus of the rat. J. Neurosci. 21, 2861–2877. Syka, J., Popelar, J., Kvasnak, E., Astl, J., 2000. Response properties of neurons in the central nucleus and external and dorsal cortices of the inferior colliculus in guinea pig. Exp. Brain Res. 133, 254–266. Wu, S.H., Ma, C.L., Sivaramakrishnan, S., Oliver, D.L., 2002. Synaptic modification in neurons of the central nucleus of the inferior colliculus. Hear. Res. 168, 43–54.