Time course of cortically induced fos expression in auditory thalamus and midbrain after bilateral cochlear ablation

Neuroscience 160 (2009) 186 –197

TIME COURSE OF CORTICALLY INDUCED FOS EXPRESSION IN AUDITORY THALAMUS AND MIDBRAIN AFTER BILATERAL COCHLEAR ABLATION X. SUN,a,b,c,e1 Y. P. GUO,a1 D. K.-Y. SHUM,b,d Y.-S. CHANc,d* AND J. HEa**

Key words: denervation hypersensitivity, inferior colliculus, medial geniculate body, corticofugal activation, cochleostomy, bicuculline.

a

Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong b Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Sassoon Road, Hong Kong

The medial geniculate body (MGB) and the inferior colliculus (IC) relay auditory signals to the auditory cortex and also receive massive feedback projections from the auditory cortex (Andersen et al., 1980; Winer, 1984; Winer and Larue, 1987; Montero, 1991; Ojima, 1994; Saldana et al., 1996; Winer et al., 1999a,b, 2001). Corticofugal feedback is thought to exert a gain-control effect on the transmission of sensory information (Crick, 1984; Murphy and Sillito, 1987; Villa et al., 1991; He et al., 1997; Suga et al., 1997; He, 2003b). The goal of this study was to examine the differential effects of corticofugal influence on MGB and IC subnuclei, without the interference of ascending input from the cochlear nucleus. Animals received bilateral cochlear ablation. An advantage of this model was that the time course of the hypersensitivity of MGB and IC neurons to corticofugal inputs could, to a certain extent, reflect their dependence on ascending input from the cochlear nucleus. Previous studies have demonstrated that after central neurons lose a portion of their inputs, these central neurons show denervation hypersensitivity as they become more dependent on their remaining inputs (Creese et al., 1977; Darlington and Smith, 1996). Unilateral cochlear ablation resulted in weakening of inhibitory input and strengthening of the excitatory response in the contralateral IC (Mossop et al., 2000; Vale and Sanes, 2002; Vale et al., 2003, 2004), indicating hypersensitivity to the remaining cochlea. After bilateral cochlear ablation, neurons in the MGB, IC, and superior olivary complex showed denervation hypersensitivity to electrical stimulation of the respective nuclei (Gerken, 1979). The temporal features of MGB and IC neuronal responses to ascending denervation are not known. The MGB is subdivided into ventral (MGv), dorsal (MGd), and medial (MGm) divisions. The lemniscal (MGv) and non-lemniscal MGB (MGm and MGd) differ in their firing patterns and their corticofugal modulations and oscillatory properties (He, 1997, 2001, 2002, 2003a,b; Zhang and Suga, 1997; Jen and Zhang, 1999; He and Hu, 2002; He et al., 2002; Xiong et al., 2004; Yu et al., 2004a,b). Morphologically, MGv is further parceled into the pars ovoidea (OV) and pars lateralis (LV) nuclei (Romanski and Ledoux, 1993). These two subnuclei exhibit similar neuronal response properties and have been thought to be

c Department of Physiology, LKS Faculty of Medicine, The University of Hong Kong, Sassoon Road, Hong Kong d Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Sassoon Road, Hong Kong e Department of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, PR China

Abstract—Expression of c-fos in the medial geniculate body (MGB) and the inferior colliculus (IC) in response to bicuculline-induced corticofugal activation was examined in rats at different time points after bilateral cochlear ablation (4 hⴚ30 days). Corticofugal activation was crucial in eliciting Fos expression in the MGB after cochlear ablation. The pars ovoidea (OV) of the medial geniculate body ventral division (MGv) showed dense Fos expression 4 h after cochlear ablation; the expression declined to very low levels at 24 h and thereafter. In turn, strong Fos expression was found in the pars lateralis (LV) of the MGv 24 h after cochlear ablation and dropped dramatically at 14 days. The dorsal division of the MGB (MGd) showed high Fos expression 7 days after cochlear ablation, which persisted for a period of time. Using multi-electrode recordings, neuronal activity of different MGB subnuclei was found to correlate well with Fos expressions. The temporal changes in cortically activated Fos expression in different MGB subnuclei after bilateral cochlear ablation indicate differential denervation hypersensitivities of these MGB neurons and likely point to differential dependence of these nuclei on both auditory ascending and corticofugal descending inputs. After bilateral cochlear ablation, significant increases in Fos-positive neurons were detected unilaterally in all IC subnuclei, ipsilateral to the bicuculline injection. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. 1 Equal contribution. *Correspondence to: J. He and Y.-S. Chan. **Corresponding author. Tel: ⫹852-27666741; fax: ⫹852-23308656. E-mail addresses: [email protected] (J. He), yschan@hkucc. hku.hk (Y.-S. Chan). Abbreviations: ANOVA, analysis of variance; CIC, central nucleus of inferior colliculus; DAB, diaminobenzidine tetrahydrochloride; DCIC, dorsal cortex of inferior colliculus; IC, inferior colliculus; LCIC, lateral cortex of inferior colliculus; LV, pars lateralis of MGv; MGB, medial geniculate body; MGd, dorsal division of medial geniculate body; MGm, medial division of medial geniculate body; MGv, ventral division of medial geniculate body; NGS, normal goat serum; OV, pars ovoidea of MGv; PB, phosphate buffer; PBS, phosphate-buffered saline.

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.02.020

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complementary to each other (Winer et al., 1999a; Zhang et al., 2008). The corticofugal pathway has been widely investigated using either electrophysiological or histological approaches (Ryugo and Weinberger, 1976; Winer and Larue, 1987; Montero, 1991; Ojima, 1994; Saldaña et al., 1996; Zhang and Suga, 1997; He, 1997; He and Hashikawa, 1998; Jen and Zhang, 1999; Yu et al., 2004a,b), but little attempt has been directed to combine these techniques to understand the influence of the corticofugal pathway on functionally identified neuronal subpopulations. The expression of Fos protein has been used as an activity marker to locate functionally activated neurons in the auditory system (Sager et al., 1988; Keilmann and Herdegen, 1997; Saint Marie et al., 1999; Wu et al., 2003; Zhang et al., 2003; Sun et al., 2007). In the present study, we examined Fos expression, with a combination of extracellular recordings, to segregate subnuclei of the MGB and IC in terms of their corticofugal-induced neuronal activities and ascending denervation hypersensitivity. We demonstrate that different subnuclei of the MGB and IC show different time courses of responsiveness to this activation and denervation.

EXPERIMENTAL PROCEDURES Animals Thirty-eight adult male Sprague–Dawley rats with normal hearing (220 –380 g) were used in the immunohistochemical study. Animals were randomly assigned into an experimental group or a vehicle control group (see Table 1 for details). Another 10 rats were used in the electrophysiological study. After bilateral cochlea ablation, the animals were allowed to recover for 4 h (4 h, n⫽9), 24 h (24 h, n⫽11), 7 days (7 days, n⫽9), 14 days (14 days, n⫽8), or 30 days (30 days, n⫽8) before bicuculline (experimental group) or saline (vehicle control) was injected in the auditory cortex. The number of animals used in the present study was minimized. All experiments were conducted in accordance with the NIH Guidelines for Animals in Research (NIH publication no. 80-23, revised 1996). The experimental protocols were approved by the Animal Subjects Ethics Sub-committee of the Hong Kong Polytechnic University.

Animal preparation All animals were anesthetized with a combination of ketamine/ xylazine (100 and 10 mg/kg i.p.; Alfasan, Woerden, Holland). A heating pad was used to maintain body temperature during surgery and recovery from anesthesia. The skin behind the ear was Table 1. Number of animals used in study Time of bicuculline injection in AI after cochlear ablation

Experimental groups Fos expression

Electrophysiology

4h 24 h 7d 14 d 30 d

5 7 5 4 4

2 2 2 2 2

Vehicle control group (saline injection)

2 2 2 2 2

187

shaved and a postauricular incision was made under aseptic conditions. Subsequent surgical procedures were performed with the aid of an operation microscope (Olympus MTX, Tokyo, Japan). The tympanic membrane was removed. With the aid of a curved metal blade inserted into the round window, the cochlea was mechanically destroyed. The remaining cochlear contents were aspirated using a pipette connected to a suction pump. The bony cavity was packed with Gelfoam (absorbable gelatin sponge, Ferrosan, Denmark) and the overlying skin was sutured. In all experiments, the lesion was performed on both sides. The operated animals were then returned to single cages kept in a temperature-controlled room. For postoperative care, both lidocaine (Astra) and antibiotic ointment (Furacin, SmithKline Beecham Pharmaceuticals, Ltd., Johannesburg, South Africa) were applied four times daily to the skin wound for 3 consecutive days. If necessary, the analgesic buprenorphine (Sigma, St. Louis, MO, USA) was given s.c. The completeness of cochlear destruction was confirmed by postmortem examination of the temporal bone under a surgical microscope. The extent of cochlear destruction was also assessed by examining histological sections of the inner ear in randomly sampled operated animals. After removal of the brain, the temporal bones were removed from the skull and fixed in 4% paraformaldehyde in 0.1 M phosphate-buffer for 3–5 days. Bones were then decalcified in 0.1 M EDTA (Sigma) and 0.25% glutaraldehyde at least for 5 days. The decalcified tissues were transferred to distilled water for 1–2 h and stored in 70% ethanol (Merck, Darmstadt, Germany) for one night. Then, the bones were rinsed through a graded alcohol series, xylene, and embedded in paraffin for sectioning. Sections of 8 ␮m thickness were mounted on subbed slides, heated briefly, dried overnight, and placed in an oven at 56 °C to remove the paraffin. The sections were stained with hematoxylin and eosin to verify destruction of the basilar membrane (Lai et al., 2006).

Injection of bicuculline into the auditory cortex To evaluate corticofugal influence on the thalamus and IC, we induced hyperactivity of the auditory cortex with bicuculline, a GABAA receptor antagonist that blocks intracortical inhibitory activity (Sillito, 1975; Grothe and Klump, 2000; Thiele et al., 2004). A craniotomy was performed over the right auditory cortex. The dura mater was left intact. Animals in the experimental groups were injected with bicuculline methobromide (0.3 ␮l, 15 mM, Sigma) diluted in 0.9% NaCl while animals in the vehicle control groups were injected with the same volume of saline (0.9% NaCl) through a Hamilton microinjection system. The injection needle was introduced perpendicular to the auditory cortex. The tip of the needle was placed at a depth of 1.0 mm below the pial surface. Animals were sacrificed 1 h after injection.

Electrophysiological recording Electrophysiological data were recorded in 10 rats. Each rat was mounted in a stereotaxic device (Narashige, Tokyo, Japan) following induction of anesthesia. Body temperature was maintained between 37.5 and 38.5 °C by a thermistor-controlled heating pad, with the thermistor attached to the animal’s abdomen. Two types of electrodes were used to record cortical and thalamic neuronal activities: (1) a single tungsten microelectrode (impedance: 1–3 M⍀, FHC Inc., Bowdoin, ME, USA) was placed in the cortex, and (2) an array of eight tungsten microelectrodes with constant inter-electrode distance of 0.5 mm (impedance: 1–3 M⍀, FHC Inc.) was placed in the thalamus. Spontaneous activity of multiunits was simultaneously recorded in the MGB and auditory cortex after the injection of bicuculline into the right auditory cortex. Electrodes were advanced using a stepping-motor micromanipulator (Narashige) that was controlled outside the soundproof room. Penetrations, according to the rat atlas (Paxinos et al.,

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1998; Paxinos and Watson, 1998), were made perpendicular to the pial surface of the auditory cortex and vertically from the top of the brain to the MGB. Signals were A/D-converted (Axon Digital, 1200, Molecular Devices, Sunnyvale, CA, USA) and filtered (300 Hz⫺5 kHz) before stored in a computer for offline analysis. At the end of the recording, a lesion was made by passing a constant electrical current of 1.5 ␮A for 20 s through one of the electrodes in the array for reconstruction of the recording positions.

Histology and Western blotting To confirm that the Fos antibody, PC38 (rabbit polyclonal antiserum against a synthetic N-terminal fragment at residues 4 –17 of human Fos; Calbiochem, La Jolla, CA, USA), was specifically reactive to Fos protein expressed in rats, Western blotting was performed on tissue extracts from the auditory thalamus as previously described (Sun et al., 2007). All animals were deeply anesthetized with an overdose of sodium pentobarbital (0.1 ml/100 g, 60 mg/ml i.p., Sagatal, RMB Animal Health, Ltd., Dagenham, UK) and perfused transcardially with 200 ml 0.9% NaCl, followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were quickly removed from the skull and post-fixed for 4 h in the same fixative. After post-fixation, the brains were cryoprotected in 30% sucrose in PB (0.1 M, pH 7.4) for 2 days at 4 °C. Frozen coronal sections were cut at a thickness of 40 ␮m with a freezing microtome and then collected in phosphate-buffered saline (PBS, 0.01 M). Sections collected consecutively from each animal were divided in series. Every third tissue section in the series was treated alike, and used for Fos immunostaining, Nissl staining and reserve, respectively (Chen et al., 2003). For immunohistochemical visualization of Fos expression, coronal brain sections were pretreated with a 0.3% hydrogen peroxide solution, followed by a 1% sodium borohydride solution, to remove peroxidase and aldehyde groups left in the tissue after paraformaldehyde fixation (Lai et al., 2004, 2006). Sections were then incubated in primary Fos antibody (1:4000; PC38; Calbiochem) in PBS with 2% normal goat serum (NGS) and 0.3% Triton X-100 (Sigma) overnight at room temperature. After incubation with the primary antibody, sections were placed in biotinylated goat antirabbit IgG secondary antibody (Vector, Burlingame, CA, USA; 1:200; in PBS with 2% NGS and 0.3% Triton X-100) for 2 h at room temperature, then in avidin– biotin peroxidase complex (ABC, Vector; 1:100 diluted by PBS) for 1 h at room temperature. Between each step, sections were rinsed three times with KPBS (5 min/rinse) and agitated on a rotator during each step. Finally, an intensified diaminobenzidine tetrahydrochloride (DAB) reaction (DAB, 0.02% w/v; H2O2, 0.002% v/v; in KPBS; Vector) was carried out for 10 min at room temperature (Lai et al., 2004, 2006). The reaction was stopped by washing with KPBS (three times). Control and experimental tissues from each group were processed in parallel (Zhang et al., 2006). No staining was observed in brain sections with omission of either the primary or secondary antibody. The sections were mounted on gelatincoated slides, allowed to air dry at room temperature, and dehydrated in a series of increasing concentrations of ethanol [50% (10 min), 70% (10 min), 90% (10 min), and absolute ethanol (2⫻10 min)] and then toluene (2⫻10 min). Finally, the sections were mounted in DPX (BDH Chemicals, Poole, UK) and covered with glass coverslips. The physiologically recorded thalamus was also cut in frontal sections (40 ␮m) and processed with Nissl staining (for details see He, 1997, 2001).

Cell counting and statistical analysis The goal of this study was to identify trends of increasing or decreasing Fos-positive neurons of each subcortical auditory nucleus following administration of bicuculline at different periods

after cochleostomy. This study was not designed to estimate the absolute cell density of a given nucleus. Thus, we counted the number of Fos-positive neurons of each nucleus per tissue section without using stereological techniques (Coggeshall and Lekan, 1996; Saper, 1996; Sun et al., 2007). The presence and locations of Fos-positive neurons in subcortical auditory nuclei, including the MGB and IC, were examined under a light microscope (Axioplan II Imaging, Carl Zeiss, Germany) equipped with a CCD camera (Spot; Diagnostic Instrument, Sterling Heights, MI, USA). The photomicrographs presented in this manuscript (e.g. Fig. 3) were captured using a 10⫻ objective. For image analysis of Fos-labeled neurons within the confines of each anatomically demarcated nucleus, all images were captured using a 40⫻ objective of the microscope (e.g. Fig. 3 insets). Digital image-analysis software (Image-J 1.61; W. Rasband, NIH, Bethesda, MD, USA) (Lai et al., 2004; Sun et al., 2007) was adopted for objective identification of Fos-positive neurons. Tissue background signals were averaged over defined areas of cell aggregate in Fos poor areas. Average tissue background was set as the minimum on a gray scale of 255 shades. Threshold was then set at the gray level of 130 U. Similar thresholding techniques have been used by other researchers (Moratalla et al., 1996; Janusonis and Fite, 2001). Image montages of captured files were assembled using Adobe Photoshop 6.0 (Lai et al., 2008; Tse et al., 2008). Subnuclei of the MGB and IC were parceled based on the atlases of Paxinos and colleagues (1998); Clerici and Coleman (1990, 1998); Faye-Lund and Osen (1985), and Malmierca and colleagues (Malmierca et al., 1993, 1995; Loftus et al., 2008). Counts for each sub-nucleus were obtained by summation of Fos-positive neurons in all sections of that nucleus. Data are presented as means⫾standard error of the mean (SEM). The mean number of Fos-positive neurons in each group was compared with others using one-way analysis of variance (ANOVA), followed by Tukey–Kramer multiple-comparisons tests (SigmaStat 2.0, SYSTAT Software Inc., Richmond, CA, USA). In all analyses, a probability value of P⬍0.05 was considered statistically significant.

RESULTS Previous Western blot experiments indicated that immunopositivity of Fos was selectively neutralized by preincubation of MGB extracts with the immunizing Fos peptide (Sun et al., 2007). The spread of bicuculline-induced Fos labeling was confined within the entire auditory cortex. Fig. 1 shows an example from the 4-h group, in which an injection of 0.3 ␮l bicuculline was made 4 h after destruction of the bilateral cochlea. The effective activation of the auditory cortical neurons, as indicated by Fos expression, was only detected in the ipsilateral hemisphere to the bicuculline injection, but not in the contralateral hemisphere (Fig. 1A). A pilot experiment showed that the contralateral auditory cortex could not be activated even with an injection of 1.0 ␮l of bicuculline into the unilateral auditory cortex (Sun et al., 2007). Fos expression in the MGB varies with survival periods following cochlear ablation In the auditory thalamus, Fos-positive neurons were detected only in the MGB ipsilateral to the bicuculline-injected side (right panel of Figs. 1 and 3), especially in the MGv and MGd. In vehicle control animals, no Fos-positive neu-

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number of Fos-positive neurons decreased drastically to 764.4⫾155.8 in the 24-h group (n⫽7, P⬍0.001; ⬍20%, compared with the 4-h group), and further decreased to 109.6⫾25.69 in the 7-day group (n⫽5, P⬍0.001) and to 98.75⫾6.125 in the 14-day group (n⫽4, P⬍0.001). In the 30-day group, the number of Fos-positive neurons was 119.3⫾17.90 (n⫽4, P⬍0.001). In the LV, there were 68.2⫾20.71 (n⫽5) Fos-positive neurons in the 4-h group. The number of neurons increased by 50-fold to 3531⫾147.8 in the 24-h group (n⫽7, P⬍0.001, compared with the 4-h group) and to 2991⫾ 387.9 in the 7-day group (n⫽5, P⬎0.05, compared with the 24-h group; P⬍0.001, compared with the 4-h group). The number of Fos-positive neurons then decreased drastically to 574.3⫾30.68 (n⫽4) and 574.3⫾78.61 (n⫽4) in the 14and 30-day groups, respectively (both P⬍0.001, compared with the 24-h and 7-day groups). In summary, Fos expression in the OV was evident during an early time window of 4 h to 24 h, with maximal expression approximately 4 h after bilateral cochlear ablation and bicuculline injection in the auditory cortex (Fig. 6). In contrast, Fos expression in the LV was most prominent from 24 h to 1 week after cochlear ablation. Fos expression in the MGm

Fig. 1. Fos expression in the cortex and the MGB was detected only on the side ipsilateral to bicuculline injection. The bicuculline injection was made on the right side. (A) Fos-positive neurons were seen only in the ipsilateral auditory cortex. The boxed areas in both panels are displayed at a higher magnification in the insets. (B–D) Fos expression in MGB at different rostrocaudal levels. The right and left panels are from the same section. The number at the upper left in each panel indicates the distance from Bregma (mm). Abbreviations: Au, auditory cortex; d, MGd; m, MGm; vLGN, ventral subnucleus of lateral geniculate nucleus. The above abbreviations also apply to Figs. 2– 4. Scale bars⫽400 ␮m.

The proportion of Fos-labeled neurons in the MGm was approximately 10% of the total number of Fos-positive neurons in the MGB for all groups. The number of Fospositive neurons in the MGm ipsilateral to the bicuculline injection was significantly higher than that of the contralateral side. The number of Fos-positive neurons in the MGm contralateral to the bicuculline injection was low in all

rons were detected on either side of the MGv and MGd, except a small number in the MGm. These results were similar in all vehicle control animals with different survival periods after bilateral cochlear ablation. Fig. 2 shows an example from a vehicle control animal of 7-day group. After different survival periods, however, the Fos labeling pattern within different MGB subnuclei was significantly different. Fig. 3A–E shows the distribution of Fos expression in the ipsilateral MGB of five experimental groups. Nissl staining result corresponding to sections in Fig. 3 is shown in Fig. 4. A greater number of Fos-positive neurons were labeled in animals with shorter survival periods after bilateral cochlear ablation (Fig. 5; 4-h group: 4866⫾407.6, 24-h group: 5021⫾320.0, 7-day group: 3899⫾397.9, 14day group: 1007⫾36.77, and 30-day group: 1076⫾120.0). Fos expression in the MGv In the OV of the MGv, there were 4186⫾360.7 (n⫽5) Fos-positive neurons in the 4-h experimental group. The

Fig. 2. Fos expression in the MGB in vehicle-injected animals after bilateral cochlear ablation. No differences in Fos expression were found between bilateral MGBs. Image shown is from an animal 7 days after bilateral cochlear ablation. Scale bar⫽400 ␮m.

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Fig. 3. Different Fos expression patterns in the MGB from different experimental groups. Survival periods after cochlear ablations were 4 h (A1–A3), 24 h (B1–B3), 7 days (C1–C3), 14 days (D1–D3), and 30 days (E1–E3). Areas bound by the squares are shown at higher magnification in insets. Bicuculline was injected into the right auditory cortex 1 h before the animal was sacrificed. Scale bar⫽400 ␮m (100 ␮m for inset).

groups, ranging between 87.5⫾52.5 and 111.2⫾12.5. This is similar to the results from the vehicle groups. The number of Fos-positive neurons in the MGm ipsilateral to bicuculline injection at 4 h 24 h, and 7 days after bilateral cochlear ablation was 499.2⫾84.91, 497.1⫾75.88, and 536.2⫾62.70, respectively. These counts were significantly higher than those of the corresponding vehicle control groups. The number of Fos-positive neurons decreased significantly in the 14-day (99.75⫾20.18) and 30-day groups (178.3⫾26.24) (both P⬍0.05) compared with the 4-, 24-h, and 7-day groups. It is of interest to note that Fos-positive neurons in the MGm were darker than those in other regions of the thalamus (Fig. 3). In vehicle control groups, only a small number of Fospositive neurons were expressed in the MGm (Fig. 2), and no differences were found between the two hemispheres. Fos expression in the MGd MGd showed the lowest amount of Fos labeling neurons, compared with other thalamic subnuclei. Fos-positive neurons were found between the middle portion (bregma ⫺5.50 mm) and rostral end of the MGB. The number of Fos-positive neurons in the 4-h groups after bilateral cochlear ablation (112.0⫾19.75, n⫽5) accounted for 2.39% of all Fos-positive neurons within the auditory thalamus. The number of Fos-positive neurons increased significantly in the 24-h and 7-day groups (227.9⫾24.24, n⫽7 and 262.6⫾27.86, n⫽5, both P⬍0.05 when compared with the 4-h group, Fig. 7B), and remained at a relatively high level in the 14-day (233.8⫾10.91, n⫽4) and the 30-day groups (204.5⫾35.53, n⫽4).

Neuronal activity in the MGB In the present study, we recorded neuronal activity in bicuculline-injected auditory cortex and different subnuclei of the ipsilateral MGB at different survival periods. A periodic burst firing was observed in the auditory cortex after bicuculline injection (Guo et al., 2007). The locations of recording electrodes were reconstructed based on the lesion and traces of the electrode tracks. The recorded neuronal activity correlated well with Fos expression when comparing animals with different survival times. A representative sample is shown in Fig. 8. In a rat from the 4-h group, neurons in the OV showed strong activity (Th4 and Th5 in Fig. 8A), while MGm and LV neurons showed relatively weak activity (Th3 and Th6, respectively, in Fig. 8A). Neuronal activity of the MGB was synchronous with activity in the auditory cortex, while neurons outside the auditory thalamus showed no synchronous activity. In the 24-h group, however, only very low amplitude neuronal activity could be recorded in the OV (Th4 in Fig. 8B). In contrast, LV neurons showed the strongest synchronized activity (Th5 in Fig. 8B), followed by MGm neurons (Th3 in Fig. 8B). Additionally, activity in MGm was similar in both animals (Th3 in both cases). This finding is consistent with the presence of Fos-positive neurons in the MGm at 4 h and 24 h. Fos-positive neurons in the IC Fos-positive neurons were found in all IC subnuclei, even in the vehicle control groups (data not shown). There were no differences in the number of Fos-positive neurons in the IC between the two hemispheres in the vehicle control groups.

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In the experimental groups, the number of Fos-positive neurons in the IC contralateral to the bicuculline-injected cortex (left panel of Fig. 9 and Fig. 10A) was similar to the bilateral IC of the control groups. A significantly higher number of Fos-positive neurons were found in the ipsilateral IC, compared to the contralateral IC, at all time points after cochlear ablation. This suggests a stronger corticocollicular effect ipsilaterally than contralaterally (Figs. 9 and 10). The difference between the two hemispheres was greater in the dorsal cortex (DCIC) and lateral cortex (LCIC) of inferior colliculus than that in the central nucleus of the inferior colliculus (CIC).

DISCUSSION In the present study, we demonstrated that MGB subnuclei showed different patterns of activation in response to corticofugal input. Bicuculline was used to block intracortical inhibitory activity. We examined the variation in the number of Fos-positive neurons in different MGB and IC subnuclei at different time points after bilateral cochlear ablation. Thus, we were able to assess this yet uncharacterized hypersensitivity of MGB and IC to cortical inputs. Corticofu-

Fig. 4. Nissl-stained sections corresponding to the rostocaudal levels as shown in each subgroup of Fig. 3. Scale bar⫽400 ␮m. d, MGd; m, MGm.

Fig. 5. Histogram showing the total numbers of Fos-positive neurons in the MGB of different experimental groups. Data are shown as mean⫾standard error of the mean (SEM). Labeled neurons were counted from serial sections. The MGB of one animal spanned 12 serial sections. The number of animals in each group is indicated above the bars. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 (ANOVA), # : compared with the 4-h group; &: compared with the 24-h group; ^: compared with the 7-day group.

gal activation was crucial in eliciting differential Fos expression in MGB subnuclei at different time points after cochlear ablation. The OV showed dense Fos expression 4 h after cochlear ablation. Expression dropped to a very low level at 24 h and remained at a low level thereafter. In the LV, Fos expression was strong 24 h after cochlear ablation and dramatically dropped at 14 days after cochlear ablation. In the MGd, Fos expression reached a high plateau at 7 days after cochlear ablation and maintained a high level. Multi-electrode recordings in different experimental groups further suggested that neuronal responses in dif-

Fig. 6. Histograms showing the numbers of Fos-positive neurons in the OV (A) and the LV (B) subnuclei of different experimental groups. Fos expression peaked at 4 h in the OV and 24 h in the LV * P⬍0.05, ** P⬍0.01, *** P⬍0.001 (ANOVA), #: compared with the 4-h group; &: compared with the 24-h group; ^: compared with the 7-day group.

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well as angiogenesis and the interaction between neural circuits and blood supply. Further investigations are necessary to delineate the precise mechanisms underlying the dramatic change in Fos expression during longer survival times after bilateral cochlear ablation. Time course of Fos expression in different subnuclei of the MGB

Fig. 7. Histograms showing the numbers of Fos-positive neurons in the medial and dorsal divisions of the MGB (A, MGm, B, MGd) of different experimental groups. Fos expression peaked at 7 days in the MGd. Fos expression dramatically decreased in the MGm between 7 and 14 days. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 (ANOVA), #: compared with the 4-h group; &: compared with the 24-h group; ^: compared with the 7-day group.

ferent MGB nuclei might correlate with Fos expression in these subnuclei. After bilateral cochlear ablation, a significant increase in Fos-positive neurons was detected in all IC subnuclei ipsilateral to the bicuculline injection. Methodological limitation In the present study, the number of Fos-positive neurons (or profiles) of each nucleus per tissue section was counted without using stereological techniques (Coggeshall and Lekan, 1996; Saper, 1996). Although the cell numbers reported here do not represent the total number of Fos-positive neurons contained in each tissue section, these results did reveal relative differences in Fos-positive neurons between hemispheres in the auditory system structures investigated here across different survival periods after cochleostomy. It can be assumed that after bilateral cochlear ablation, ascending inputs to the IC and MGB were immediately abolished. However, the innervation of these nuclei becomes more complicated as the survival period increases. During the course of losing one of the major inputs, i.e. the ascending input, neurons in the IC and MGB would become hypersensitive to other inputs, particularly corticofugal ones. However, factors other than denervation hypersensitivity and corticofugal activation might be confounded in the induction of Fos expression in the MGB, especially in preparations with long survival time. These factors may include regeneration and remodeling of neural circuits, as

Fos expression in the MGv and MGd could not be induced only with ascending auditory input (Zhang et al., 2003; Sun et al., 2007), but could be induced when there was corticofugal activation (Guo et al., 2007). However, Fos expression in the MGB could be modulated by ascending auditory inputs in the presence of descending corticofugal input (Sun et al., 2007). With cochlear ablation, different MGB subnuclei exhibited different time courses of Fos expression in response to the activation of auditory cortex, suggesting the differential dependence of the MGB subnuclei on both the ascending inputs from the periphery and the descending projections arising from the auditory cortex. Since the MGv relays primary auditory inputs from the brainstem to the auditory cortex (Romanski and LeDoux, 1993; Winer et al., 1999a,b), bilateral cochlear ablation would cause a major loss of input to this core nucleus of the MGB. After losing one major input arising from the brainstem, neurons in the MGv should increase their sensitivity to remaining inputs from the cortex and the thalamic reticular nucleus, and rebuild excitatory–inhibitory balance within a few hours (Mossop et al., 2000; Vale et al., 2004). This would result in earlier peaks of Fos expression in the OV and LV of MGv (at 4 h and 24 h, respectively) (Fig. 6A). Late reaching of the high Fos expression plateau (at approximately 7 days) and fewer Fos-positive neurons were found in the MGd, suggesting their secondary dependence on ascending and descending inputs, as well as their complementary role in information transmission and processing (Winer et al., 1999a). Since elevated Fos expression was observed in the MGm after the activation of auditory cortex, auditory information is likely to be one of its major inputs. However, Fos expression was also seen in the MGm of cochlear-ablated animals even without cortical activation (Sun et al., 2007), indicating that MGm neurons are less dependent on audiospecific stimulation. This is consistent with previous studies demonstrating that MGm was involved in multisensory integration (Gerren and Weinberger, 1983). The dramatic decrease of Fos expression in the MGm between 7 and 14 days is probably due to the strengthening of other inputs from non-auditory modalities and/or from the amygdala and basal ganglia (Russchen, 1982). The occurrence of early peaks of Fos expression in the OV and LV after bilateral cochlear ablation might be explained by the strong corticofugal facilitation in the MGv. Morphologically, the corticofugal projection to the MGv is five times heavier than the projection to the MGd and 10 times heavier than that to the MGm (Winer and Larue, 1987). In MGB subnuclei that were packed with Fos-positive neurons, neurons showed a synchronized oscillation with those in auditory cortex (Fig. 8). This corticothalamic

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Fig. 8. Corticothalamic synchronizations were observed in regions containing large numbers of Fos-positive neurons. Simultaneous recordings of the auditory cortex and thalamus from a 4-h rat (A) and a 24-h rat (B). Nissl-stained sections in the upper panels show the locations of the electrode array in the thalamus. An electrolytic lesion was made at electrode Th1 in each animal. Scale bar⫽400 ␮m. Middle panels show neuronal activities recorded from the auditory cortex and multi-channel electrodes in the thalamus. AC represents a recording from the auditory cortex and Th1⫺7 represents recordings from the thalamus and hippocampus. Bottom panels show the autocorrelation (AC–AC) or cross-correlation analysis between AC site and other thalamic sites. The numbers over the bars indicate the generalized correlation coefficient. Scale bar⫽1.0 s.

oscillation was proven to be derived from direct excitatory input of the corticofugal projection (Guo et al., 2007). These results echoed our previous findings that strong corticofugal facilitation prevails over the inhibition effects in the MGv (He et al., 2002; He, 2003c; Guo et al., 2007). Functional segregation of the MGv It is not fully understood whether subnuclei OV and LV of the MGv are functionally segregated. Previous studies have shown that neurons in the OV and LV had similar physiological properties: sharp tuning curves, short response latency and tonotopic organization (Aitkin and Webster, 1972; Imig and Morel, 1984, 1985; Morel et al., 1987; Clarey and Irvine, 1990). However, the morphological differences between OV and LV are obvious: (1) neurons in the OV are spherical and uniform in size while those in the LV are oval and generally smaller in size, (2) neuronal density in the OV is higher than in the LV, (3) dendrites of OV neurons show a tendency of lining up in the laminar structure while the somata of LV neurons tend to form columns, and (4) OV projects to AI while LV projects to AI and surrounding areas (Calford, 1983; LeDoux et al., 1985; Morest and Winer, 1986; Winer et al., 1988; Clerici and Coleman, 1990, 1998; Romanski and LeDoux, 1993; Bartlett et al., 2000; Olucha-Bordonau et al., 2004). Our results demonstrate that OV and LV had different time windows of Fos-expression in response to activation

of the auditory cortex after cochlear ablation. The time window of hypersensitivity of LV neurons to cortical activation after cochlear ablation started later and lasted longer (24 h to 7 days) than that of OV neurons (4 h, Fig. 6). The delayed onset of hypersensitivity of LV neurons might imply that these neurons are less dependent on direct ascending inputs. This indicates that OV and LV are functionally segregated in terms of their differential dependence on the ascending inputs from the brainstem and on the descending projections arising from the auditory cortex. Furthermore, we recently demonstrated that OV neurons showed uniform, transient phasic firing patterns and sharper frequency selectivity, while LV neurons exhibited various firing patterns and more robust spontaneous discharges (Zhang et al., 2008). Taken together, our findings indicate that OV and LV are segregated both morphologically and functionally. These two subnuclei are, however, complementary to each other. The OV tends to act as the primary buffer to simplify the processing of auditory information from periphery to the cortex. On the other hand, LV neurons most likely provide more encoding power to enrich other embedded components of sound. Plastic changes in the auditory pathway after cochlear ablation After unilateral ablation of the cochlea, neurons in the auditory pathway, especially those in the ipsilateral cochlear nucleus and contralateral IC, are partly denervated.

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for ⬎35 days (Robertson and Irvine, 1989) and in the thalamus for ⬎40 days (Kamke et al., 2003). Bilateral cochlear ablation reduced inhibitory synaptic strength and increased excitatory synaptic strength in the IC (Vale and Sanes, 2002). These changes are related to homeostatic regulation of the excitation–inhibition balance that maintains stable neuronal activities in different regions of the brain (Luo et al., 1999; Maffei et al., 2004; Holt et al., 2005). The ipsilateral CIC showed a significant increase in Fos-positive neurons over the contralateral CIC 4 h 24 h, and 7 days after cochlear ablation. In animals without cochlear ablation, no difference was found between the ipsi- and contralateral CICs after unilateral bicuculline injection in the auditory cortex (Sun et al., 2007). The present results might be explained by the strong dependence of the CIC on ascending auditory input. The loss of the ascending input, assumingly to be mainly excitatory in nature, probably triggers homeostatic regulation in maintaining the excitation–inhibition balance through the suppression of inhibitory inputs or the attenuation of other excitatory input. The finding that the ipsilateral DCIC and LCIC showed greater increases in Fos-positive neurons than the CIC is consistent with findings in animals with cortical injection of bicuculline but without cochlear ablation (Sun et al., 2007). This finding echoes the presence of heavier corticofugal projections to the DCIC and LCIC than to the CIC (Andersen et al., 1980; Herbert et al., 1991; Winer et al., 1998). The large difference in the number of

Fig. 9. Comparison of Fos expression in ipsilateral and contralateral IC from different experimental groups. The ipsilateral IC (right panel) showed more Fos-positive neurons than the contralateral (left panel) side on the same section in each group. Scale bar⫽400 ␮m.

Plastic changes at different time intervals after cochlear ablation include the following: (1) downregulation of inhibition in the contralateral IC of neonatal and adult animals (24 h or 7 days, Mossop et al., 2000; 1–7 days; Vale et al., 2004; 2–3 days; Vale and Sanes, 2002; ⬎6 months, Kitzes, 1984; Kitzes and Semple, 1985), and (2) an increased projection from the contralateral ventral cochlear nucleus to the ipsi- and contralateral medial superior olive, lateral superior olive, and medial nucleus of the trapezoid body in neonatal animals (from 24 h to 11 days, Kitzes et al., 1995). Partial ablation of the unilateral cochlea also resulted in frequency reorganization in the auditory cortex

Fig. 10. Histogram showing the number of Fos-positive neurons in the IC (A) and its subnuclei (B) in different experimental groups. Abbreviations: Contra- and ipsi are in reference to the side of bicuculline injection. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 (t-test, compared with the same nucleus in the contralateral IC).

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Fos-labeled neurons between the MGB and IC probably reflects the differential degrees of corticofugal control (Winer et al., 2001). It is interesting to note that the IC showed a relatively long temporal window of Fos expression after cochlear ablation and cortical injection of bicuculline when compared with the MGB. This result again points to the functional differentiation of corticofugal projections to the MGB (thalamus) and IC (midbrain). The corticofugal pathway traditionally has been thought to be important in shifting tuning frequency and delay-tuning curves or sharpening tuning curves. Recent studies indicate that the corticofugal pathway may also be involved in physiological functions such as novelty detecting and startle behavior (Winer et al., 2002; Perez-Gonzalez et al., 2005).

CONCLUSION In the present study, the time course of Fos expression in individual central auditory relays may reflect the degree and order of dependence of different subnuclei of MGB and IC on both auditory ascending and corticofugal descending inputs. While earlier electrophysiological studies reported that neurons in the OV and LV had similar properties, the present results, together with our recent intracellular findings (Zhang et al., 2008), suggest that OV and LV are functionally segregated. The present study also demonstrates for the first time that denervation hypersensitivity could be associated with Fos expression in the MGB and IC. Acknowledgments—We thank Simon S. M. Chan and Kimmy F. L. Tsang of the University of Hong Kong for their excellent technical assistance in the experiments. This work was supported by the Hong Kong Research Grants Council (CERG PolyU 5467/05 M).

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(Accepted 10 February 2009) (Available online 13 February 2009)