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Auditory discrimination training rescues developmentally degraded directional selectivity and restores mature expression of GABAA and AMPA receptor subunits in rat auditory cortex
Behavioural Brain Research 229 (2012) 301–307
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Auditory discrimination training rescues developmentally degraded directional selectivity and restores mature expression of GABAA and AMPA receptor subunits in rat auditory cortex Fei Guo a , Jiping Zhang a , Xiaoqing Zhu a , Rui Cai a,b , Xiaoming Zhou a,∗ , Xinde Sun a,∗ a b
School of Life Science, Key Laboratory of Brain Functional Genomics, Ministry of Education, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA
a r t i c l e
i n f o
Article history: Received 20 November 2011 Received in revised form 27 December 2011 Accepted 28 December 2011 Available online 26 January 2012 Keywords: Directional selectivity Pulsed noise exposure Sensory discrimination training GABAA receptors AMPA GluR2 receptor Auditory cortex Rat
a b s t r a c t Auditory frequency discrimination training can remediate deteriorated frequency representations and temporal information processing in the adult primary auditory cortex induced by early post-natal pulsed noise exposure. In this study, we investigated the neural mechanisms underlying the restoration of directional selectivity by auditory spatial discrimination training. Rats exposed to pulsed noise during a post-natal critical period demonstrated reduced auditory directional selectivity but could be successfully trained to identify a target sound stimulus at a specific azimuth angle using a reward-contingent auditory discrimination task (EXP rats). In contrast, rats passively exposed to the training procedure but no reward for correct identification of the azimuth angle (PNR rats) showed no improvement and behavioral performance remained significantly below EXP rats and control (CON) rats reared under a normal sonic environment. The expression levels of GABAA receptor subunits ␣1, ␣3, 2, and 3, and the AMPA GluR2 subunit were significantly altered in the auditory cortex of untrained noise-raised (NR and PNR) rats compared to age-matched CON rats, while trained noise-raised (EXP) rats exhibited levels of expression not significantly different from CON rats. Thus, reward-contingent sound-azimuth discrimination training may remediate directional selectivity by restoring the proper expression profile of neurotransmitter receptor subunits in the auditory cortex, allowing for normal spatial selectivity by cortical neurons. The development of auditory directional selectivity depends on the regulated expression of these excitatory and inhibitory neurotransmitter receptor subunits; early pulsed noise may disrupt the normal development of directional selectivity by interfering with receptor expression. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Exposure to pulsed white noise during early post-natal development results is severely disrupted frequency representation in the adult auditory cortex and functional deficits in sound localization. While these deficits were once thought to be largely irreversible, recent studies indicate that intensive auditory training can rescue the deteriorated tonotopic organization of the cortex induced by early modulated noise exposure [1]. Furthermore, extensive sensory training can even reverse age-related functional and structural changes in older rats [2]. Sound localization is one of the most important tasks performed by the auditory system [3–8]. We recently demonstrated that, like
∗ Corresponding authors. Tel.: +86 21 62232775; fax: +86 21 62233754. E-mail addresses: [email protected] (X. Zhou), [email protected] (X. Sun). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.12.041
frequency discrimination, both behavioral performance for spatial discrimination and the directional selectivity of rat auditory cortical neurons were degraded by early pulsed noise exposure. Furthermore, intensive training in an auditory sound-azimuth discrimination task rescued both sound-azimuth discrimination performance and the directional selectivity of auditory cortical neurons [9]. The neurocellular and molecular changes that underlie degraded auditory discrimination and training-induced reversal remain unclear. The experience-dependent plasticity responsible for the development of sensory discrimination and cortical organization during the post-natal critical period both depends on and influences the expression of molecules that regulate GABAergic and glutamatergic neurotransmission [10–20]. Our previous study showed that early continuous noise exposure resulted in a significant decrease in the expression of the GABA synthesis enzyme GAD 65 and the GABAA receptor ␣1 subunit, as well as an increase in GABAA receptor ␣3 subunit expression [21,22]. Zhou et al. [23]
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F. Guo et al. / Behavioural Brain Research 229 (2012) 301–307 that also allows rats to escape the sound chamber. Nine speakers were installed in the interior wall at 10◦ intervals with a water spout under each. All speakers emitted tone bursts (white noise, 30-ms duration with 3-ms rise-decay time, 2/s) under the control of a computer. The sound pressure level (SPL) was about 70 dB SPL, and the water spouts were controlled by valves as described previously [3,9].
Fig. 1. Experimental time lines for auditory discrimination training. EXP—experimental noise-raised rats; NR—noise raised rats; PNR—passively noise-raised rats and CON—control rats.
recently reported that noise exposure significantly decreased the expression of GABAA receptor subunits ␣1 and 2/3 as well as NMDA receptor subunits NR2A and NR2B in rat auditory cortex. In light of these findings, we speculated that early noise exposure may disturb the normal development of GABA-mediated inhibition and glutamate-mediated excitation, thereby disrupting sensory processing in central auditory circuits. Conversely, intensive auditory training may restore impaired auditory spatial sensitivity by reestablishing the proper expression profile of molecules that regulate inhibitory and excitatory neurotransmission and excitability, including GABA and glutamate receptor subunits. To examine the molecular basis for degraded auditory spatial sensitivity and training-induced recovery, we trained developmentally impaired rats to identify a target auditory stimulus at a specific azimuthal angle (sound-azimuth discrimination training) and then compared the protein expression of GABA and AMPA receptor subunits in these trained rats (EXP group) with noise-raised but untrained rats (NR group), noise-raised but passively trained rats (PNR group), and control rats. Sound-azimuth discrimination training restored developmentally disrupted receptor subunit protein expression in auditory cortex, underscoring the importance of GABA and glutamate receptor expression profiles in the functional disruption and restoration of auditory discrimination. 2. Material and methods Sprague-Dawley (SD) rat pups and their mothers were exposed to pulsed white noise in a sound chamber from postnatal day 7 (P7) to postnatal day 35 (P35). After pulsed noise exposure, the rat pups were randomly divided onto three groups: (i) Noise-raised (NR) rats were moved to a normal environment; (ii) experimental noise-raised (EXP) rats were training to discriminate a target sound stimulus at a specific azimuth from P41 to P75; (iii) passively noise-raised (PNR) rats were exposed to the identical directional training equipment as EXP rats from P41 to P75 but were not required to identify the target sound azimuth for a water reward, and they were given free access to water across the training epoch (Fig. 1). A forth cohort of rats reared under a normal sonic environment served as the control (CON) group. The pulsed noise signals (50-ms duration, 5-ms rise-decay time, 65 dB sound pressure level, 1/s) were delivered from a speaker placed approximately 2 m above the rats. The sound energy was essentially flat across a broad frequency spectrum (0.2–30.0 kHz). A reversed 12-h light/dark cycle and constant humidity and temperature were maintained for all groups. No abnormalities in the behavior of either the pups or dames were detected during pulsed noise exposure. The weights of all pups and dames were continuously monitored, and there was no weight loss compared to the naive CON rats, and no noise-reared rats showed behavioral signs of stress.
2.1.2. Training All the rats used in the behavioral experiments had ad libitum access to food but restricted access to water. They were deprived of water before the experimental period and their body weights were maintained at about 90% of ad libitum body weight during the training period. The EXP, PNR, and CON rats were habituated for 40 min/day to the behavioral training apparatus from P36 to P40. During this period, the rats were allowed to move freely within the behavioral apparatus. They also learned to approach a water spout and lick it to obtaining water reward after presentation of a sound stimulus at a fixed sound azimuth. After this habituation and pretraining period, we started training each rat separately. The rats in the EXP group were trained to discriminate each sound azimuth (from left 40◦ to the right 40◦ in the chamber) by randomly delivering sounds from different angular directions. One of these sound azimuths was randomly chosen as the target at the beginning of each training session. At the beginning of a trial, the rat stayed in the starting box with head forward and was presented with a sound burst six times. Following presentation of this sound stimulus, the rat left the starting box and approached the source of the sound (a target speaker delivering the target sound azimuth), and had to lick the associated response spout to receive a water reward. The sound ceased whether or not the rat found the target speaker. The EXP and CON rats would obtain several drops of water if they approached the correct water spout, while incorrect responses were not rewarded. After an incorrect response, the sound was presented again from the same speaker as a corrective mechanism to guide the animal to the target location, but these results were not used for calculation of any of the training parameters and are not reported here. In addition to an untrained noise-raised group (NR), we included a passively noise-raised (PNR) group that was exposed to the same directional training equipment as EXP rats. These PNR rats were also trained to approach the corresponding water spout after presentation of the same sounds delivered to EXP rats, but they were given free access to water regardless of their initial orientation throughout the training period (P41–P75). Our reason for including this PNR group in the experimental design was to determine whether only passive exposure to the sound training regime could restore the developmentally degraded directional selectivity. For all groups, we measured the percent correct approaches, response latency, and azimuth deviation (directional error). Each rat was given 45 trials/day during the training period. 2.2. Quantitative immunoblots Following training, rats in all treatment groups were deeply anesthetized with an injection of sodium pentobarbital (75 mg/kg i.p.). Immediately after decapitation, the brain was obtained. The right and left AC regions (3–7 mm posterior of bregma and 2–6 mm ventral from the top of cortex, according to Polley et al. and our previous papers [4,20–22,36]) were dissected out and immediately homogenized in ice-cold homogenization buffer containing 0.5 mM dithiothreitol, 1 mM ethylene diamine tetraacetie acid, 2 mM ethylene glycol bis (-aminoethyether) N,N,N ,N -tetra acetic acid, 10 mM 2-hydroxyethylpiperazine-N-ethane -sulfonicacid, 10 mg/L leupeptin, 2 mg/L aprotinin, and 0.1 mM phenylmethanesulfonyl fluoride. Total protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). A portion of the whole homogenate was centrifuged at 14,000 × g for 10 min and retained for analysis of GABAA receptor subunit and GluR2 expression. These lysate samples were first resuspended in boiling 1% sodium-dodecyl-sulfate (SDS) and stored at −80 ◦ C. Quantitative immunoblotting assays were performed as described previously [4,20–22]. Primary antibodies used included rabbit anti-GABAAR ␣1 polyclonal antibody (AB5592, millipore), rabbit anti-GABAAR ␣3 polyclonal antibody (AB5594, millipore), rabbit anti-GABAAR 2 polyclonal antibody (AB5561, 50–56 kDa, millipore), rabbit anti-GABAAR 3 polyclonal antibody (AB5563, 50–56 kDa, millipore), rabbit anti-GluR2 polyclonal antibody (AB1768, 100 kDa, millipore) (all from upstate Biotechnology, Temecula, CA), and mouse anti--actin (BM0627, 42 kDa,millipore) (Sigma, St. Louis, MO). The relationship between optical density and protein concentration was linear within the ranges measured in this study. The density of each band on Western blots was measured using Image Processing and Analysis in Java (ImageJ) software, and the relative expression level of each target protein was calculated as the ratio of target protein band density to -actin density (the loading control). All immunoblots were performed blind to experimental conditions. 2.3. Data analysis
2.1. Behavioral training 2.1.1. Apparatus for sound-azimuth discrimination training As in earlier reports from our laboratory [3,9], sound-azimuth discrimination training was performed in a sound-attenuated chamber. The apparatus is made of wood, with a radius of 150 cm, and its interior is covered with black polyethylene sponge. At the front of the apparatus is a starting box (20L cm × 7W cm × 8H cm)
Results were analyzed by SPSS 16.0 software. One-way ANOVA used to analyze the differences in percent correct score, response latency, and azimuth deviation between the EXP and CON groups. An independent sample t-test was used to determine the differences between EXP and CON groups, and between PNR and CON groups. All data are expressed as mean ± SEM. A p < 0.05 was considered statistically significant.
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Fig. 2. Correct scores, response latencies, and azimuth deviations measured during sound-azimuth discrimination training for the EXP, PNR, and CON rats. (A) Average correct scores on the sound-azimuth discrimination task on each day for CON, EXP, and PNR rats. (B) Average response latencies on the sound-azimuth discrimination task on each training day. (C) Average azimuth deviations on the sound-azimuth discrimination task on each training day. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
3. Results 3.1. Auditory training restored the directional selectivity degraded by early pulsed noise exposure 3.1.1. Accuracy of sound-azimuth discrimination We assessed the percent correct on the sound-azimuth discrimination task in the three training groups (CON, EXP, PNR) to confirm that early pulsed noise exposure indeed impaired behavioral performance. The learning time courses during the training sessions were significantly different between CON, EXP, and PNR groups. The CON rats not exposed to pulsed noise from P7 to P35 took an average of 10 days to establish a 60% correct response rate in sound-azimuth discrimination, while the EXP rats exposed to pulsed noise required 20 days to attain the same 60% threshold, indicating that pulsed noise exposure disrupted spatial discrimination. In contrast, the PNR rats never reached the 60% performance criterion during this period (Fig. 2A). No significant differences were observed between training day 1 and training day 9 in the three groups. As expected, however, the CON group exhibited significantly higher correct response rates thereafter, in accord with our previous studies [3,9]. In the EXP group, the percent correct remained below 60% between training day 10 and training day 22 (except day 21) and was significant lower than CON rats (oneway ANOVA, F(1,42) = 133.234, p < 0.01). The percent correct for EXP rats increased sharply from training day 23 to training day 27, by which time the average correct response rate was not significantly different from CON rats (one-way ANOVA, F(1,75) = 2.423, p > 0.05), indicating that the EXP rats could match the performance level of the CON rats after a sufficient number of training trials. Conversely, PNR rats never attained the performance level of the
CON and EXP rats. Although the percent correct rose slightly from training day 1–10 in PNR rats, the correct response rate did not continue to increase as observed for EXP rats, and PNR rat performance remained below CON rat performance at all days after training day 10 (Fig. 2A). The improved accuracy of sound-azimuth discrimination for EXP rats compared to PNR rats after training day 21 indicates that active reward-contingent training is necessary for improved sound-azimuth discrimination.
3.1.2. Latency We also defined response latency as an index for sound-azimuth discrimination. As shown in Fig. 2B, there was no difference between training day 1 and training day 10 among the three training groups. After training day 5, the response latency of the CON rats decreased faster than EXP and PNR rats. The average response latency of the EXP group also decreased sharply, however, from 2.23 ± 1.10 s to 2.05 ± 0.06 s between training day 1 and training day 12, but there was still a significant difference between EXP and CON groups on training day 11 and training day 22 (one-way ANOVA, F(1,1973) = 51.617, p < 0.01). From training day 23 on, the average response latency of EXP rats was not significantly different from CON rats (one-way ANOVA, F(1,1874) = 0.516, p > 0.05), indicating training-dependent recovery of auditory spatial discrimination. The response latency of the PNR group did not decrease significantly after training day 10 and remained longer than the CON group over the remainder of the experimental period. The average response latency of EXP rats was significant shorter than PNR rats from day 23 to day 35, again indicating the necessity of active reward-contingent training for improved sound-azimuth discrimination (Fig. 2B).
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Fig. 3. Changes in GABAA ␣1 and ␣3 receptor subunit expression induced by early pulsed noise and subsequent sound-azimuth discrimination training. Representative Western blots and densitometric analysis of GABAA ␣1 and GABAA ␣3 expression (A) and the GABAA ␣1/GABAA ␣3 ratio (B) in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
3.1.3. Azimuth deviation The azimuth deviation was defined as the average difference between the rat approach angle and the target speaker sound angle (an azimuth deviation of 0◦ is the correct angle of approach). The initial average azimuth deviations were large (approximately 25–30◦ ) and there were no significant differences between CON, EXP, and PNR groups from training day 1 to day 7 (one-way ANOVA, F(1,1473) = 0.516, p > 0.05) (Fig. 2C). The azimuth deviation of CON rats rapidly decreased from 30.35 ± 1.48◦ on day 7 to 17.57 ± 1.85◦ on day 8, while the azimuth deviation of EXP rats decreased from 31.34 ± 1.12◦ to 19.05 ± 1.54◦ during this period. The average azimuth deviation of CON rats continued to decline
between training days 8 and 18 while that of EXP rats remained relatively stable such that there was a significant difference between EXP and CON groups from training day 11 to training day 21 (oneway ANOVA, F(1,1637) = 53.912, p < 0.01). After training day 21, however, the average azimuth deviation of EXP rats finally reached that of CON rats (one-way ANOVA, F(1,1589) = 0.654, p > 0.05), indicating that auditory training improved the sound localization of EXP rats to near control levels of accuracy. The azimuth deviation for PNR rats did not change significantly during the training period and was significantly higher than the CON rats after training day seven (one-way ANOVA, F(1,1894) = 53.219, p < 0.01) (Fig. 2C).
Fig. 4. Changes in GABAA receptor subunit 2 and 3 expression induced by early pulsed noise and sound-azimuth discrimination training. Representative Western blots and densitometric analysis of GABAA 2 and GABAA 3 expression (A) and the GABAA 2/GABAA 3 ratio (B) in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
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3.2. Auditory training restored GABAA and AMPA GluR2 receptor expression in auditory cortex To examine the molecular mechanisms responsible for disruption and restoration of sound-azimuth discrimination, we examined the expression of inhibitory and excitatory neurotransmitter receptor subunits. The receptor subunits chosen were either previously reported to exhibit experience-dependent regulation or specifically associated with spatial selectivity [4,11,13,21,22]. 3.2.1. Changes in GABAA receptor subunits Expression levels of GABAA ␣1, GABAA ␣3, GABAA 2, and GABAA 3 were quantified by immunoblotting of homogenates from the auditory cortices of all four treatment groups. Pulsed noise exposure resulted in a significant decrease in the expression of GABAA ␣1 subunit and a significantly increase in the expression of GABAA ␣3 in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test). The expression level of GABAA ␣1 was substantially higher in EXP rats than NR and PNR rats but not significantly different from age-matched control rats (Fig. 3A), while the expression of GABAA ␣3 in EXP auditory cortex was significantly lower than NR and PNR rats but again not significantly different from CON rats. The GABAA ␣1/GABAA ␣3 ratio represents the balance between different GABAA receptor subunits. Pulsed noise rearing caused a decrease in the GABAA ␣1/GABAA ␣3 ratio in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test) (Fig. 3B) that was reversed by reward contingent sound-azimuth discrimination training but not by passive exposure to the training task. We also examined the expression level of GABAA receptor subunits 2 and 3 (Fig. 4A). Both GABAA 2 and 3 subunits were significantly decreased by noise-rearing (NR rats) compared to controls (p < 0.05, n = 4 for each group, unpaired t-test), but were expressed normally after auditory training (EXP group) compared to age-matched CON rats. Passive auditory training did not reverse the decreased expression of GABAA 2 and 3 in PNR rats. There was no change in the GABAA 2 to 3 ratio between treatment groups (n = 4 for each group, p > 0.05, unpaired t-test, compared with CON rats) (Fig. 4B).
Fig. 5. The change in AMPA receptor GluR2 subunit expression induced by early pulsed noise and subsequent sound-azimuth discrimination training. Representative Western blots and densitometric analysis of expression in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
p < 0.05, unpaired t-test), while only GABAA 2/GluR2 differed between EXP and CON rats (all others p > 0.05, unpaired t-test). These results indicate that the developmental disruption in the inhibitory–excitatory receptor balance in the auditory cortex can be reversed by auditory training. 4. Discussion Infant rat pups reared under conditions of pulsed white noise demonstrated markedly attenuated behavioral performance for sound-azimuth discriminations, and neurons of the primary auditory cortex (A1) exhibited degraded directional selectivity. When these noise-raised animals were trained using a sound-azimuth discrimination task, both sound-azimuth discrimination performance and directional selectivity of A1 neurons were effectively normalized [9]. The present results confirm the restorative effect of sound-azimuth discrimination training, and further reveal correlations between loss and restoration of sound directional selectivity and the expression of GABAA receptor and AMPA receptor subunits. Rats with restored auditory discrimination had subunit expression profiles not significantly different from age-matched control rats, indicating that perceptual training in the adult can restore deteriorated auditory spatial localization caused by an early abnormal sonic environment at both the behavioral and synaptic/molecular level. It has been reported that the early acoustic environment and auditory experiences are critical for defining the basic functional organization and processing capabilities of the mammalian auditory cortex [19,24–28]. In particularly, the early sonic environment can result in tonotopic rearrangements in the central auditory system. Continuous white noise or pulsed noise exposure degraded the
3.2.2. Changes in the AMPA receptor GluR2 subunit We previously demonstrated that pulsed noise exposure led to a decrease in AMPA receptor GluR2 subunit expression in the auditory cortex of young and adult rats [38]. In this study, we examined if auditory training restored the changes in GluR2 expression induced by early pulsed noise exposure. As expected, although pulsed noise exposure decreased GluR2 subunit expression in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test), expression in EXP rats was not significantly different from CON rats (n = 4 for each group, p > 0.05, unpaired t-test) (Fig. 5). We also calculated the ratios of all GABAA receptor subunits (␣1, ␣3, 2, and 3) to GluR2 as indices of the inhibitory–excitatory balance (Table 1). Pulsed noise exposure not only caused significant changes in GABAA and AMPA GluR2 subunit expression levels, but also disturbed the balance of inhibitory and excitatory receptor expression. The GABAA ␣1/GluR2, GABAA ␣3/GluR2, GABAA 2/GluR2, and GABAA 3/GluR2 ratios were all significantly altered in noise-reared (NR) rats compared to age-matched controls (all
Table 1 The ratios of all GABAA receptor subunits examined to GluR2 subunit expression. All values are normalized against the mean of the control rats. *p < 0.05 and **p < 0.01. Ratio
CON (n = 4)
GABAA ␣ 1/GluR2 GABAA ␣ 3/GluR2 GABAA  2/GluR2 GABAA  3/GluR2
76.70 50.7 90.91 84.06
± ± ± ±
8.75 9.78 3.08 2.85
NR (n = 4) 56.49 96.43 59.78 47.96
± ± ± ±
13.40 2. 14* 0.94** 0.75**
EXP (n = 4) 76.28 47.48 102.41 84.59
± ± ± ±
9.79 6.44 2.17* 1.80
PNR (n = 4) 61.60 81.10 62.77 49.24
± ± ± ±
14.73 4.62* 3.01** 2.36**
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spectral and temporal response selectivity of A1 neurons, resulting in poorly developed cortical frequency receptive field structure and tonotopic organization, and decreased cortical responses to highfrequency bursts of repetitive sound stimuli [29–31]. Moreover, moderate acoustic noise exposure can disrupt previously established cortical organization in A1, thereby altering complex sound representations and FM sweep directional selectivity [23,32]. Consistent with these findings, our previous study suggested that early continuous noise rearing markedly attenuated the spatial selectivity of A1 neurons. The spike frequency of A1 neurons was much less sensitive to changes in stimulus location and the preferred location extended over a broader area (wider azimuth angle) [21]. In addition, the average angular ranges (ARs) of the neuronal azimuth selective curves were significantly less selective in A1 neurons from noise-raised rats [9]. It was once widely believed that unattended ambient auditory inputs can no longer alter the organization of tonotopic maps or the response properties of A1 neurons after the critical period [33–35,37,38,40]. Similarly, functional degradation caused by early sensory deprivation was thought to be irreversible despite a normal auditory environment during adulthood [1]. Recent studies, however, have clearly demonstrated that perceptual training in the juvenile–young adult period can significantly modify functional representations in auditory cortex, normalize the response properties of dysfunctional A1 neurons by increasing the spectral selectivity and response amplitude, and rescue the deteriorated frequency representation in adult A1 induced by early modulated noise exposure [1,34,35,39–43]. Other studies reported that perceptual training of adults rats resulted in a modest but enduring expansion of the cortical area representing the target auditory stimuli, and that age-related changes in A1 organization and neural response properties can be reversed by behavioral training [2]. In agreement with these findings, our most recent study also suggested that sound location discrimination training rescued the dysfunctional behavioral performance for sound-azimuth discrimination and the directional selectivity of adult A1 neurons degraded by early pulsed noise exposure [9]. All of these findings clearly indicate that perceptual training has the potential to induce therapeutic plasticity in the adult auditory cortex. What are the molecular factors underlying the restoration of degraded directional sensitivity? As the predominant fast inhibitory and excitatory neurotransmitter systems, changes in GABA-mediated inhibition and glutamate-mediated excitation are likely crucial for experience-dependent developmental plasticity. Indeed, loss of auditory spatial discrimination was correlated with decreased expression of glutamic acid decarboxylase 65 (GAD 65), GABAA ␣1, 2, and 3, and increased GABAA ␣3 expression in the auditory cortex [21–23]. Furthermore, an adult model of age-related hearing loss exhibited weakened inhibition associated with changes in GABA receptor function, subunit composition, and expression of presynaptic GAD and GABA [44,45]. A switch in relative dominance from GABAA ␣3 (the “immature” subunit) to ␣1 (the “mature” subunit) around the peak of the critical period has been observed in both the visual and auditory cortices [10]. Recently, it was reported that GABAA receptor subunit expression showed age-related changes in rat auditory cortex [46,47]. Early continuous white noise exposure decreased GAD 65 and GABAA receptor ␣1 subunit expression and increased GABAA receptor ␣3 subunit expression, which could be interpreted as a regression in the cortical GABAA receptor subunit expression profile to a less developed phenotype [21]. Our present study demonstrated precisely this pattern, with a significant decease in GABAA receptor ␣1 expression and an increase in GABAA ␣3 expression (and decrease in the GABAA ␣1/GABAA ␣3 ratio) in noise-raised (NR) rats. This suggests that pulsed noise hindered the maturation of the auditory cortex by delaying or inhibiting the switch in GABAA receptor isoforms.
Following intensive auditory training, however, this immature GABA receptor subunit expression profile was switched (normalized) to the typical adult state, as the expression levels of GABAA ␣1 and ␣3 (and the GABAA ␣1/GABAA ␣3 ratio) in EXP group were not significantly different from control rats. Furthermore, intensive auditory training also reversed the lower expression of GABAA 2, 3, and GluR2 subunits characteristic of both the immature cortex and rats exposed to early post-natal pulsed noise. We also previously reported that early enriched environment enhanced spatial sensitivity of A1 neurons and upregulated the expression of GABAA receptor subunits ␣1 and 3, underscoring the importance of GABAA receptors containing these subunits in fully developed auditory spatial discrimination [4]. On the basis of these findings, we suggest that early noise exposure may disturb the normal development of GABA-mediated inhibition and glutamate-mediated excitation. Specially, an aberrant post-natal sonic environment delays the development of GABAergic inhibition, thereby disrupting the balance between excitation and inhibition, and dampens the selective firing properties of cortical neurons. Conversely, intensive sensory training might restore normal synaptic transmission by normalizing GABA and glutamate receptor subunit expression, thereby reestablished the proper excitatory–inhibitory balance in auditory circuits and the response specificity of A1 cortical neurons. Magnusson et al. [48] reported that neurons in the lateral superior olive (LSO) adjusted their ILD sensitivity by regulating the balance between excitatory and inhibitory inputs, suggesting that adjustment of these inputs is also an adaptive response of auditory brainstem neurons to finetune sound localization. In summary, we have shown that auditory spatial discrimination training can restore the degradation of directional sensitivity induced by early pulsed noise exposure. Consistent with recovery of behavioral accuracy for sound-azimuth discrimination, expression levels of the inhibitory receptor GABAA subunits ␣1, ␣3, 1, 3 and the excitatory receptor GluR2 subunit in auditory cortex were restored to normal adult levels, indicating that normalization of GABAA and GluR2 receptor subunit expression likely contributes to the recovery of dysfunctional directional sensitivity. Acknowledgments This work was supported by Fundamental Research Funds for the Central University in China; Nature Science Foundation of China (NSFC no. 30970984; 31171058); New Century Excellent Talents in University of State Education Ministry of China, and the Shanghai Rising-Star Program (no. 09QH1400900). References [1] Zhou X, Merzenich MM. Intensive training in adults refines A1 representations degraded in an early postnatal critical period. Proc Natl Acad Sci USA 2007;104(40):15935–40. [2] de Villers-Sidani E, Alzghoul L, Zhou X, Simpson KL, Lin RCS. Recovery of functional and structural age-related changes in the rat primary auditory cortex with operant training. Proc Natl Acad Sci USA 2010;107(31):13900–5. [3] Cai R, Guo F, Zhang J, Xu J, Cui Y, Sun X. Environmental enrichment improves behavioral performance and auditory spatial representation of primary auditory cortical neurons in rat. Neurobiol Learn Mem 2009;91(4):366–76. [4] Cai R, Zhou X, Guo F, Xu J, Zhang J, Sun X. Maintenance of enriched environmentinduced changes of auditory spatial sensitivity and expression of GABAA, NMDA, and AMPA receptor subunits in rat auditory cortex. Neurobiol Learn Mem 2010;94:452–60. [5] Jenkins WM, Masterton RB. Sound localization: effects of unilateral lesions in central auditory system. J Neurophysiol 1982;47(6):987–1016. [6] Jenkins WM, Merzenich MM. Role of cat primary auditory cortex for soundlocalization behavior. J Neurophysiol 1984;52(5):819–47. [7] Kacelnik O, Nodal FR, Parsons CH, King AJ. Training-induced plasticity of auditory localization in adult mammals. PLoS Biol 2006;4(4):e71. [8] King AJ, Bajo VM, Bizley JK, Campbell RAA, Nodal FR, Schulz AL, et al. Physiological and behavioral studies of spatial coding in the auditory cortex. Hear Res 2007;229:106–15.
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Research report
Auditory discrimination training rescues developmentally degraded directional selectivity and restores mature expression of GABAA and AMPA receptor subunits in rat auditory cortex Fei Guo a , Jiping Zhang a , Xiaoqing Zhu a , Rui Cai a,b , Xiaoming Zhou a,∗ , Xinde Sun a,∗ a b
School of Life Science, Key Laboratory of Brain Functional Genomics, Ministry of Education, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA
a r t i c l e
i n f o
Article history: Received 20 November 2011 Received in revised form 27 December 2011 Accepted 28 December 2011 Available online 26 January 2012 Keywords: Directional selectivity Pulsed noise exposure Sensory discrimination training GABAA receptors AMPA GluR2 receptor Auditory cortex Rat
a b s t r a c t Auditory frequency discrimination training can remediate deteriorated frequency representations and temporal information processing in the adult primary auditory cortex induced by early post-natal pulsed noise exposure. In this study, we investigated the neural mechanisms underlying the restoration of directional selectivity by auditory spatial discrimination training. Rats exposed to pulsed noise during a post-natal critical period demonstrated reduced auditory directional selectivity but could be successfully trained to identify a target sound stimulus at a specific azimuth angle using a reward-contingent auditory discrimination task (EXP rats). In contrast, rats passively exposed to the training procedure but no reward for correct identification of the azimuth angle (PNR rats) showed no improvement and behavioral performance remained significantly below EXP rats and control (CON) rats reared under a normal sonic environment. The expression levels of GABAA receptor subunits ␣1, ␣3, 2, and 3, and the AMPA GluR2 subunit were significantly altered in the auditory cortex of untrained noise-raised (NR and PNR) rats compared to age-matched CON rats, while trained noise-raised (EXP) rats exhibited levels of expression not significantly different from CON rats. Thus, reward-contingent sound-azimuth discrimination training may remediate directional selectivity by restoring the proper expression profile of neurotransmitter receptor subunits in the auditory cortex, allowing for normal spatial selectivity by cortical neurons. The development of auditory directional selectivity depends on the regulated expression of these excitatory and inhibitory neurotransmitter receptor subunits; early pulsed noise may disrupt the normal development of directional selectivity by interfering with receptor expression. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Exposure to pulsed white noise during early post-natal development results is severely disrupted frequency representation in the adult auditory cortex and functional deficits in sound localization. While these deficits were once thought to be largely irreversible, recent studies indicate that intensive auditory training can rescue the deteriorated tonotopic organization of the cortex induced by early modulated noise exposure [1]. Furthermore, extensive sensory training can even reverse age-related functional and structural changes in older rats [2]. Sound localization is one of the most important tasks performed by the auditory system [3–8]. We recently demonstrated that, like
∗ Corresponding authors. Tel.: +86 21 62232775; fax: +86 21 62233754. E-mail addresses: [email protected] (X. Zhou), [email protected] (X. Sun). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.12.041
frequency discrimination, both behavioral performance for spatial discrimination and the directional selectivity of rat auditory cortical neurons were degraded by early pulsed noise exposure. Furthermore, intensive training in an auditory sound-azimuth discrimination task rescued both sound-azimuth discrimination performance and the directional selectivity of auditory cortical neurons [9]. The neurocellular and molecular changes that underlie degraded auditory discrimination and training-induced reversal remain unclear. The experience-dependent plasticity responsible for the development of sensory discrimination and cortical organization during the post-natal critical period both depends on and influences the expression of molecules that regulate GABAergic and glutamatergic neurotransmission [10–20]. Our previous study showed that early continuous noise exposure resulted in a significant decrease in the expression of the GABA synthesis enzyme GAD 65 and the GABAA receptor ␣1 subunit, as well as an increase in GABAA receptor ␣3 subunit expression [21,22]. Zhou et al. [23]
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F. Guo et al. / Behavioural Brain Research 229 (2012) 301–307 that also allows rats to escape the sound chamber. Nine speakers were installed in the interior wall at 10◦ intervals with a water spout under each. All speakers emitted tone bursts (white noise, 30-ms duration with 3-ms rise-decay time, 2/s) under the control of a computer. The sound pressure level (SPL) was about 70 dB SPL, and the water spouts were controlled by valves as described previously [3,9].
Fig. 1. Experimental time lines for auditory discrimination training. EXP—experimental noise-raised rats; NR—noise raised rats; PNR—passively noise-raised rats and CON—control rats.
recently reported that noise exposure significantly decreased the expression of GABAA receptor subunits ␣1 and 2/3 as well as NMDA receptor subunits NR2A and NR2B in rat auditory cortex. In light of these findings, we speculated that early noise exposure may disturb the normal development of GABA-mediated inhibition and glutamate-mediated excitation, thereby disrupting sensory processing in central auditory circuits. Conversely, intensive auditory training may restore impaired auditory spatial sensitivity by reestablishing the proper expression profile of molecules that regulate inhibitory and excitatory neurotransmission and excitability, including GABA and glutamate receptor subunits. To examine the molecular basis for degraded auditory spatial sensitivity and training-induced recovery, we trained developmentally impaired rats to identify a target auditory stimulus at a specific azimuthal angle (sound-azimuth discrimination training) and then compared the protein expression of GABA and AMPA receptor subunits in these trained rats (EXP group) with noise-raised but untrained rats (NR group), noise-raised but passively trained rats (PNR group), and control rats. Sound-azimuth discrimination training restored developmentally disrupted receptor subunit protein expression in auditory cortex, underscoring the importance of GABA and glutamate receptor expression profiles in the functional disruption and restoration of auditory discrimination. 2. Material and methods Sprague-Dawley (SD) rat pups and their mothers were exposed to pulsed white noise in a sound chamber from postnatal day 7 (P7) to postnatal day 35 (P35). After pulsed noise exposure, the rat pups were randomly divided onto three groups: (i) Noise-raised (NR) rats were moved to a normal environment; (ii) experimental noise-raised (EXP) rats were training to discriminate a target sound stimulus at a specific azimuth from P41 to P75; (iii) passively noise-raised (PNR) rats were exposed to the identical directional training equipment as EXP rats from P41 to P75 but were not required to identify the target sound azimuth for a water reward, and they were given free access to water across the training epoch (Fig. 1). A forth cohort of rats reared under a normal sonic environment served as the control (CON) group. The pulsed noise signals (50-ms duration, 5-ms rise-decay time, 65 dB sound pressure level, 1/s) were delivered from a speaker placed approximately 2 m above the rats. The sound energy was essentially flat across a broad frequency spectrum (0.2–30.0 kHz). A reversed 12-h light/dark cycle and constant humidity and temperature were maintained for all groups. No abnormalities in the behavior of either the pups or dames were detected during pulsed noise exposure. The weights of all pups and dames were continuously monitored, and there was no weight loss compared to the naive CON rats, and no noise-reared rats showed behavioral signs of stress.
2.1.2. Training All the rats used in the behavioral experiments had ad libitum access to food but restricted access to water. They were deprived of water before the experimental period and their body weights were maintained at about 90% of ad libitum body weight during the training period. The EXP, PNR, and CON rats were habituated for 40 min/day to the behavioral training apparatus from P36 to P40. During this period, the rats were allowed to move freely within the behavioral apparatus. They also learned to approach a water spout and lick it to obtaining water reward after presentation of a sound stimulus at a fixed sound azimuth. After this habituation and pretraining period, we started training each rat separately. The rats in the EXP group were trained to discriminate each sound azimuth (from left 40◦ to the right 40◦ in the chamber) by randomly delivering sounds from different angular directions. One of these sound azimuths was randomly chosen as the target at the beginning of each training session. At the beginning of a trial, the rat stayed in the starting box with head forward and was presented with a sound burst six times. Following presentation of this sound stimulus, the rat left the starting box and approached the source of the sound (a target speaker delivering the target sound azimuth), and had to lick the associated response spout to receive a water reward. The sound ceased whether or not the rat found the target speaker. The EXP and CON rats would obtain several drops of water if they approached the correct water spout, while incorrect responses were not rewarded. After an incorrect response, the sound was presented again from the same speaker as a corrective mechanism to guide the animal to the target location, but these results were not used for calculation of any of the training parameters and are not reported here. In addition to an untrained noise-raised group (NR), we included a passively noise-raised (PNR) group that was exposed to the same directional training equipment as EXP rats. These PNR rats were also trained to approach the corresponding water spout after presentation of the same sounds delivered to EXP rats, but they were given free access to water regardless of their initial orientation throughout the training period (P41–P75). Our reason for including this PNR group in the experimental design was to determine whether only passive exposure to the sound training regime could restore the developmentally degraded directional selectivity. For all groups, we measured the percent correct approaches, response latency, and azimuth deviation (directional error). Each rat was given 45 trials/day during the training period. 2.2. Quantitative immunoblots Following training, rats in all treatment groups were deeply anesthetized with an injection of sodium pentobarbital (75 mg/kg i.p.). Immediately after decapitation, the brain was obtained. The right and left AC regions (3–7 mm posterior of bregma and 2–6 mm ventral from the top of cortex, according to Polley et al. and our previous papers [4,20–22,36]) were dissected out and immediately homogenized in ice-cold homogenization buffer containing 0.5 mM dithiothreitol, 1 mM ethylene diamine tetraacetie acid, 2 mM ethylene glycol bis (-aminoethyether) N,N,N ,N -tetra acetic acid, 10 mM 2-hydroxyethylpiperazine-N-ethane -sulfonicacid, 10 mg/L leupeptin, 2 mg/L aprotinin, and 0.1 mM phenylmethanesulfonyl fluoride. Total protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). A portion of the whole homogenate was centrifuged at 14,000 × g for 10 min and retained for analysis of GABAA receptor subunit and GluR2 expression. These lysate samples were first resuspended in boiling 1% sodium-dodecyl-sulfate (SDS) and stored at −80 ◦ C. Quantitative immunoblotting assays were performed as described previously [4,20–22]. Primary antibodies used included rabbit anti-GABAAR ␣1 polyclonal antibody (AB5592, millipore), rabbit anti-GABAAR ␣3 polyclonal antibody (AB5594, millipore), rabbit anti-GABAAR 2 polyclonal antibody (AB5561, 50–56 kDa, millipore), rabbit anti-GABAAR 3 polyclonal antibody (AB5563, 50–56 kDa, millipore), rabbit anti-GluR2 polyclonal antibody (AB1768, 100 kDa, millipore) (all from upstate Biotechnology, Temecula, CA), and mouse anti--actin (BM0627, 42 kDa,millipore) (Sigma, St. Louis, MO). The relationship between optical density and protein concentration was linear within the ranges measured in this study. The density of each band on Western blots was measured using Image Processing and Analysis in Java (ImageJ) software, and the relative expression level of each target protein was calculated as the ratio of target protein band density to -actin density (the loading control). All immunoblots were performed blind to experimental conditions. 2.3. Data analysis
2.1. Behavioral training 2.1.1. Apparatus for sound-azimuth discrimination training As in earlier reports from our laboratory [3,9], sound-azimuth discrimination training was performed in a sound-attenuated chamber. The apparatus is made of wood, with a radius of 150 cm, and its interior is covered with black polyethylene sponge. At the front of the apparatus is a starting box (20L cm × 7W cm × 8H cm)
Results were analyzed by SPSS 16.0 software. One-way ANOVA used to analyze the differences in percent correct score, response latency, and azimuth deviation between the EXP and CON groups. An independent sample t-test was used to determine the differences between EXP and CON groups, and between PNR and CON groups. All data are expressed as mean ± SEM. A p < 0.05 was considered statistically significant.
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Fig. 2. Correct scores, response latencies, and azimuth deviations measured during sound-azimuth discrimination training for the EXP, PNR, and CON rats. (A) Average correct scores on the sound-azimuth discrimination task on each day for CON, EXP, and PNR rats. (B) Average response latencies on the sound-azimuth discrimination task on each training day. (C) Average azimuth deviations on the sound-azimuth discrimination task on each training day. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
3. Results 3.1. Auditory training restored the directional selectivity degraded by early pulsed noise exposure 3.1.1. Accuracy of sound-azimuth discrimination We assessed the percent correct on the sound-azimuth discrimination task in the three training groups (CON, EXP, PNR) to confirm that early pulsed noise exposure indeed impaired behavioral performance. The learning time courses during the training sessions were significantly different between CON, EXP, and PNR groups. The CON rats not exposed to pulsed noise from P7 to P35 took an average of 10 days to establish a 60% correct response rate in sound-azimuth discrimination, while the EXP rats exposed to pulsed noise required 20 days to attain the same 60% threshold, indicating that pulsed noise exposure disrupted spatial discrimination. In contrast, the PNR rats never reached the 60% performance criterion during this period (Fig. 2A). No significant differences were observed between training day 1 and training day 9 in the three groups. As expected, however, the CON group exhibited significantly higher correct response rates thereafter, in accord with our previous studies [3,9]. In the EXP group, the percent correct remained below 60% between training day 10 and training day 22 (except day 21) and was significant lower than CON rats (oneway ANOVA, F(1,42) = 133.234, p < 0.01). The percent correct for EXP rats increased sharply from training day 23 to training day 27, by which time the average correct response rate was not significantly different from CON rats (one-way ANOVA, F(1,75) = 2.423, p > 0.05), indicating that the EXP rats could match the performance level of the CON rats after a sufficient number of training trials. Conversely, PNR rats never attained the performance level of the
CON and EXP rats. Although the percent correct rose slightly from training day 1–10 in PNR rats, the correct response rate did not continue to increase as observed for EXP rats, and PNR rat performance remained below CON rat performance at all days after training day 10 (Fig. 2A). The improved accuracy of sound-azimuth discrimination for EXP rats compared to PNR rats after training day 21 indicates that active reward-contingent training is necessary for improved sound-azimuth discrimination.
3.1.2. Latency We also defined response latency as an index for sound-azimuth discrimination. As shown in Fig. 2B, there was no difference between training day 1 and training day 10 among the three training groups. After training day 5, the response latency of the CON rats decreased faster than EXP and PNR rats. The average response latency of the EXP group also decreased sharply, however, from 2.23 ± 1.10 s to 2.05 ± 0.06 s between training day 1 and training day 12, but there was still a significant difference between EXP and CON groups on training day 11 and training day 22 (one-way ANOVA, F(1,1973) = 51.617, p < 0.01). From training day 23 on, the average response latency of EXP rats was not significantly different from CON rats (one-way ANOVA, F(1,1874) = 0.516, p > 0.05), indicating training-dependent recovery of auditory spatial discrimination. The response latency of the PNR group did not decrease significantly after training day 10 and remained longer than the CON group over the remainder of the experimental period. The average response latency of EXP rats was significant shorter than PNR rats from day 23 to day 35, again indicating the necessity of active reward-contingent training for improved sound-azimuth discrimination (Fig. 2B).
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Fig. 3. Changes in GABAA ␣1 and ␣3 receptor subunit expression induced by early pulsed noise and subsequent sound-azimuth discrimination training. Representative Western blots and densitometric analysis of GABAA ␣1 and GABAA ␣3 expression (A) and the GABAA ␣1/GABAA ␣3 ratio (B) in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
3.1.3. Azimuth deviation The azimuth deviation was defined as the average difference between the rat approach angle and the target speaker sound angle (an azimuth deviation of 0◦ is the correct angle of approach). The initial average azimuth deviations were large (approximately 25–30◦ ) and there were no significant differences between CON, EXP, and PNR groups from training day 1 to day 7 (one-way ANOVA, F(1,1473) = 0.516, p > 0.05) (Fig. 2C). The azimuth deviation of CON rats rapidly decreased from 30.35 ± 1.48◦ on day 7 to 17.57 ± 1.85◦ on day 8, while the azimuth deviation of EXP rats decreased from 31.34 ± 1.12◦ to 19.05 ± 1.54◦ during this period. The average azimuth deviation of CON rats continued to decline
between training days 8 and 18 while that of EXP rats remained relatively stable such that there was a significant difference between EXP and CON groups from training day 11 to training day 21 (oneway ANOVA, F(1,1637) = 53.912, p < 0.01). After training day 21, however, the average azimuth deviation of EXP rats finally reached that of CON rats (one-way ANOVA, F(1,1589) = 0.654, p > 0.05), indicating that auditory training improved the sound localization of EXP rats to near control levels of accuracy. The azimuth deviation for PNR rats did not change significantly during the training period and was significantly higher than the CON rats after training day seven (one-way ANOVA, F(1,1894) = 53.219, p < 0.01) (Fig. 2C).
Fig. 4. Changes in GABAA receptor subunit 2 and 3 expression induced by early pulsed noise and sound-azimuth discrimination training. Representative Western blots and densitometric analysis of GABAA 2 and GABAA 3 expression (A) and the GABAA 2/GABAA 3 ratio (B) in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
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3.2. Auditory training restored GABAA and AMPA GluR2 receptor expression in auditory cortex To examine the molecular mechanisms responsible for disruption and restoration of sound-azimuth discrimination, we examined the expression of inhibitory and excitatory neurotransmitter receptor subunits. The receptor subunits chosen were either previously reported to exhibit experience-dependent regulation or specifically associated with spatial selectivity [4,11,13,21,22]. 3.2.1. Changes in GABAA receptor subunits Expression levels of GABAA ␣1, GABAA ␣3, GABAA 2, and GABAA 3 were quantified by immunoblotting of homogenates from the auditory cortices of all four treatment groups. Pulsed noise exposure resulted in a significant decrease in the expression of GABAA ␣1 subunit and a significantly increase in the expression of GABAA ␣3 in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test). The expression level of GABAA ␣1 was substantially higher in EXP rats than NR and PNR rats but not significantly different from age-matched control rats (Fig. 3A), while the expression of GABAA ␣3 in EXP auditory cortex was significantly lower than NR and PNR rats but again not significantly different from CON rats. The GABAA ␣1/GABAA ␣3 ratio represents the balance between different GABAA receptor subunits. Pulsed noise rearing caused a decrease in the GABAA ␣1/GABAA ␣3 ratio in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test) (Fig. 3B) that was reversed by reward contingent sound-azimuth discrimination training but not by passive exposure to the training task. We also examined the expression level of GABAA receptor subunits 2 and 3 (Fig. 4A). Both GABAA 2 and 3 subunits were significantly decreased by noise-rearing (NR rats) compared to controls (p < 0.05, n = 4 for each group, unpaired t-test), but were expressed normally after auditory training (EXP group) compared to age-matched CON rats. Passive auditory training did not reverse the decreased expression of GABAA 2 and 3 in PNR rats. There was no change in the GABAA 2 to 3 ratio between treatment groups (n = 4 for each group, p > 0.05, unpaired t-test, compared with CON rats) (Fig. 4B).
Fig. 5. The change in AMPA receptor GluR2 subunit expression induced by early pulsed noise and subsequent sound-azimuth discrimination training. Representative Western blots and densitometric analysis of expression in the auditory cortex of CON, NR, EXP, and PNR rats. Values shown are mean ± SEM. *p < 0.05 and **p < 0.01 compared with the CON rats.
p < 0.05, unpaired t-test), while only GABAA 2/GluR2 differed between EXP and CON rats (all others p > 0.05, unpaired t-test). These results indicate that the developmental disruption in the inhibitory–excitatory receptor balance in the auditory cortex can be reversed by auditory training. 4. Discussion Infant rat pups reared under conditions of pulsed white noise demonstrated markedly attenuated behavioral performance for sound-azimuth discriminations, and neurons of the primary auditory cortex (A1) exhibited degraded directional selectivity. When these noise-raised animals were trained using a sound-azimuth discrimination task, both sound-azimuth discrimination performance and directional selectivity of A1 neurons were effectively normalized [9]. The present results confirm the restorative effect of sound-azimuth discrimination training, and further reveal correlations between loss and restoration of sound directional selectivity and the expression of GABAA receptor and AMPA receptor subunits. Rats with restored auditory discrimination had subunit expression profiles not significantly different from age-matched control rats, indicating that perceptual training in the adult can restore deteriorated auditory spatial localization caused by an early abnormal sonic environment at both the behavioral and synaptic/molecular level. It has been reported that the early acoustic environment and auditory experiences are critical for defining the basic functional organization and processing capabilities of the mammalian auditory cortex [19,24–28]. In particularly, the early sonic environment can result in tonotopic rearrangements in the central auditory system. Continuous white noise or pulsed noise exposure degraded the
3.2.2. Changes in the AMPA receptor GluR2 subunit We previously demonstrated that pulsed noise exposure led to a decrease in AMPA receptor GluR2 subunit expression in the auditory cortex of young and adult rats [38]. In this study, we examined if auditory training restored the changes in GluR2 expression induced by early pulsed noise exposure. As expected, although pulsed noise exposure decreased GluR2 subunit expression in NR rats compared to CON rats (n = 4 for each group, p < 0.05, unpaired t-test), expression in EXP rats was not significantly different from CON rats (n = 4 for each group, p > 0.05, unpaired t-test) (Fig. 5). We also calculated the ratios of all GABAA receptor subunits (␣1, ␣3, 2, and 3) to GluR2 as indices of the inhibitory–excitatory balance (Table 1). Pulsed noise exposure not only caused significant changes in GABAA and AMPA GluR2 subunit expression levels, but also disturbed the balance of inhibitory and excitatory receptor expression. The GABAA ␣1/GluR2, GABAA ␣3/GluR2, GABAA 2/GluR2, and GABAA 3/GluR2 ratios were all significantly altered in noise-reared (NR) rats compared to age-matched controls (all
Table 1 The ratios of all GABAA receptor subunits examined to GluR2 subunit expression. All values are normalized against the mean of the control rats. *p < 0.05 and **p < 0.01. Ratio
CON (n = 4)
GABAA ␣ 1/GluR2 GABAA ␣ 3/GluR2 GABAA  2/GluR2 GABAA  3/GluR2
76.70 50.7 90.91 84.06
± ± ± ±
8.75 9.78 3.08 2.85
NR (n = 4) 56.49 96.43 59.78 47.96
± ± ± ±
13.40 2. 14* 0.94** 0.75**
EXP (n = 4) 76.28 47.48 102.41 84.59
± ± ± ±
9.79 6.44 2.17* 1.80
PNR (n = 4) 61.60 81.10 62.77 49.24
± ± ± ±
14.73 4.62* 3.01** 2.36**
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spectral and temporal response selectivity of A1 neurons, resulting in poorly developed cortical frequency receptive field structure and tonotopic organization, and decreased cortical responses to highfrequency bursts of repetitive sound stimuli [29–31]. Moreover, moderate acoustic noise exposure can disrupt previously established cortical organization in A1, thereby altering complex sound representations and FM sweep directional selectivity [23,32]. Consistent with these findings, our previous study suggested that early continuous noise rearing markedly attenuated the spatial selectivity of A1 neurons. The spike frequency of A1 neurons was much less sensitive to changes in stimulus location and the preferred location extended over a broader area (wider azimuth angle) [21]. In addition, the average angular ranges (ARs) of the neuronal azimuth selective curves were significantly less selective in A1 neurons from noise-raised rats [9]. It was once widely believed that unattended ambient auditory inputs can no longer alter the organization of tonotopic maps or the response properties of A1 neurons after the critical period [33–35,37,38,40]. Similarly, functional degradation caused by early sensory deprivation was thought to be irreversible despite a normal auditory environment during adulthood [1]. Recent studies, however, have clearly demonstrated that perceptual training in the juvenile–young adult period can significantly modify functional representations in auditory cortex, normalize the response properties of dysfunctional A1 neurons by increasing the spectral selectivity and response amplitude, and rescue the deteriorated frequency representation in adult A1 induced by early modulated noise exposure [1,34,35,39–43]. Other studies reported that perceptual training of adults rats resulted in a modest but enduring expansion of the cortical area representing the target auditory stimuli, and that age-related changes in A1 organization and neural response properties can be reversed by behavioral training [2]. In agreement with these findings, our most recent study also suggested that sound location discrimination training rescued the dysfunctional behavioral performance for sound-azimuth discrimination and the directional selectivity of adult A1 neurons degraded by early pulsed noise exposure [9]. All of these findings clearly indicate that perceptual training has the potential to induce therapeutic plasticity in the adult auditory cortex. What are the molecular factors underlying the restoration of degraded directional sensitivity? As the predominant fast inhibitory and excitatory neurotransmitter systems, changes in GABA-mediated inhibition and glutamate-mediated excitation are likely crucial for experience-dependent developmental plasticity. Indeed, loss of auditory spatial discrimination was correlated with decreased expression of glutamic acid decarboxylase 65 (GAD 65), GABAA ␣1, 2, and 3, and increased GABAA ␣3 expression in the auditory cortex [21–23]. Furthermore, an adult model of age-related hearing loss exhibited weakened inhibition associated with changes in GABA receptor function, subunit composition, and expression of presynaptic GAD and GABA [44,45]. A switch in relative dominance from GABAA ␣3 (the “immature” subunit) to ␣1 (the “mature” subunit) around the peak of the critical period has been observed in both the visual and auditory cortices [10]. Recently, it was reported that GABAA receptor subunit expression showed age-related changes in rat auditory cortex [46,47]. Early continuous white noise exposure decreased GAD 65 and GABAA receptor ␣1 subunit expression and increased GABAA receptor ␣3 subunit expression, which could be interpreted as a regression in the cortical GABAA receptor subunit expression profile to a less developed phenotype [21]. Our present study demonstrated precisely this pattern, with a significant decease in GABAA receptor ␣1 expression and an increase in GABAA ␣3 expression (and decrease in the GABAA ␣1/GABAA ␣3 ratio) in noise-raised (NR) rats. This suggests that pulsed noise hindered the maturation of the auditory cortex by delaying or inhibiting the switch in GABAA receptor isoforms.
Following intensive auditory training, however, this immature GABA receptor subunit expression profile was switched (normalized) to the typical adult state, as the expression levels of GABAA ␣1 and ␣3 (and the GABAA ␣1/GABAA ␣3 ratio) in EXP group were not significantly different from control rats. Furthermore, intensive auditory training also reversed the lower expression of GABAA 2, 3, and GluR2 subunits characteristic of both the immature cortex and rats exposed to early post-natal pulsed noise. We also previously reported that early enriched environment enhanced spatial sensitivity of A1 neurons and upregulated the expression of GABAA receptor subunits ␣1 and 3, underscoring the importance of GABAA receptors containing these subunits in fully developed auditory spatial discrimination [4]. On the basis of these findings, we suggest that early noise exposure may disturb the normal development of GABA-mediated inhibition and glutamate-mediated excitation. Specially, an aberrant post-natal sonic environment delays the development of GABAergic inhibition, thereby disrupting the balance between excitation and inhibition, and dampens the selective firing properties of cortical neurons. Conversely, intensive sensory training might restore normal synaptic transmission by normalizing GABA and glutamate receptor subunit expression, thereby reestablished the proper excitatory–inhibitory balance in auditory circuits and the response specificity of A1 cortical neurons. Magnusson et al. [48] reported that neurons in the lateral superior olive (LSO) adjusted their ILD sensitivity by regulating the balance between excitatory and inhibitory inputs, suggesting that adjustment of these inputs is also an adaptive response of auditory brainstem neurons to finetune sound localization. In summary, we have shown that auditory spatial discrimination training can restore the degradation of directional sensitivity induced by early pulsed noise exposure. Consistent with recovery of behavioral accuracy for sound-azimuth discrimination, expression levels of the inhibitory receptor GABAA subunits ␣1, ␣3, 1, 3 and the excitatory receptor GluR2 subunit in auditory cortex were restored to normal adult levels, indicating that normalization of GABAA and GluR2 receptor subunit expression likely contributes to the recovery of dysfunctional directional sensitivity. Acknowledgments This work was supported by Fundamental Research Funds for the Central University in China; Nature Science Foundation of China (NSFC no. 30970984; 31171058); New Century Excellent Talents in University of State Education Ministry of China, and the Shanghai Rising-Star Program (no. 09QH1400900). References [1] Zhou X, Merzenich MM. Intensive training in adults refines A1 representations degraded in an early postnatal critical period. Proc Natl Acad Sci USA 2007;104(40):15935–40. [2] de Villers-Sidani E, Alzghoul L, Zhou X, Simpson KL, Lin RCS. Recovery of functional and structural age-related changes in the rat primary auditory cortex with operant training. Proc Natl Acad Sci USA 2010;107(31):13900–5. [3] Cai R, Guo F, Zhang J, Xu J, Cui Y, Sun X. Environmental enrichment improves behavioral performance and auditory spatial representation of primary auditory cortical neurons in rat. Neurobiol Learn Mem 2009;91(4):366–76. [4] Cai R, Zhou X, Guo F, Xu J, Zhang J, Sun X. Maintenance of enriched environmentinduced changes of auditory spatial sensitivity and expression of GABAA, NMDA, and AMPA receptor subunits in rat auditory cortex. Neurobiol Learn Mem 2010;94:452–60. [5] Jenkins WM, Masterton RB. Sound localization: effects of unilateral lesions in central auditory system. J Neurophysiol 1982;47(6):987–1016. [6] Jenkins WM, Merzenich MM. Role of cat primary auditory cortex for soundlocalization behavior. J Neurophysiol 1984;52(5):819–47. [7] Kacelnik O, Nodal FR, Parsons CH, King AJ. Training-induced plasticity of auditory localization in adult mammals. PLoS Biol 2006;4(4):e71. [8] King AJ, Bajo VM, Bizley JK, Campbell RAA, Nodal FR, Schulz AL, et al. Physiological and behavioral studies of spatial coding in the auditory cortex. Hear Res 2007;229:106–15.
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