Effects of Acetylcholine and Atropine on Plasticity of Central Auditory Neurons Caused by Conditioning in Bats

Weiqing Ji, Enquan Gao, and Nobuo Suga

Department of Biology, Washington University, St. Louis, Missouri 63130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ji, Weiqing, Enquan Gao, and Nobuo Suga. Effects of Acetylcholine and Atropine on Plasticity of Central Auditory Neurons Caused by Conditioning in Bats. J. Neurophysiol. 86: 211-225, 2001. In the big brown bat (Eptesicus fuscus), conditioning with acoustic stimuli followed by electric leg-stimulation causes shifts in frequency-tuning curves and best frequencies (hereafter BF shifts) of collicular and cortical neurons, i.e., reorganization of the cochleotopic (frequency) maps in the inferior colliculus (IC) and auditory cortex (AC). The collicular BF shift recovers 180 min after the conditioning, but the cortical BF shift lasts longer than 26 h. The collicular BF shift is not caused by conditioning, as the AC is inactivated during conditioning. Therefore it has been concluded that the collicular BF shift is caused by the corticofugal auditory system. The collicular and cortical BF shifts both are not caused by conditioning as the somatosensory cortex is inactivated during conditioning. Therefore it has been hypothesized that the cortical BF shift is mostly caused by both the subcortical (e.g., collicular) BF shift and the activity of nonauditory systems such as the somatosensory cortex excited by an unconditioned leg-stimulation and the cholinergic basal forebrain. The main aims of our present studies are to examine whether acetylcholine (ACh) applied to the AC augments the collicular and cortical BF shifts caused by the conditioning and whether atropine applied to the AC abolishes the cortical BF shift but not the collicular BF shift, as expected from the preceding hypothesis. In the awake bat, we made the following findings. ACh applied to the AC augments not only the cortical BF shift but also the collicular BF shift through the corticofugal system. Atropine applied to the AC reduces the collicular BF shift and abolishes the cortical BF shift which otherwise would be caused. ACh applied to the IC significantly augments the collicular BF shift but affects the cortical BF shift only slightly. ACh makes the cortical BF shift long-lasting beyond 4 h, but it cannot make the collicular BF shift long-lasting beyond 3 h. Atropine applied to the IC abolishes the collicular BF shift. It reduces the cortical BF shift but does not abolish it. Our findings favor the hypothesis that the BF shifts evoked by the corticofugal system, and an increased ACh level in the AC evoked by the basal forebrain are both necessary to evoke a long-lasting cortical BF shift.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the big brown bat (Eptesicus fuscus), repetitive focal electrical stimulation of the auditory cortex (AC) with 0.2 ms-long, 100-nA electric pulses for 30 min evokes changes in the best frequencies (BFs), frequency-tuning curves, and auditory responses of collicular and cortical neurons. These changes recover 180 min after the stimulation (Chowdhury and Suga 2000; Ma and Suga 2001; Yan and Suga 1998). Repetitive acoustic stimuli delivered to the bat without any electrical stimulation also evoke small short-lasting collicular (Gao and Suga 1998; Yan and Suga 1998) and cortical changes (Chowdhury and Suga 2000). Auditory conditioning with repetitive acoustic stimuli followed by electric leg-stimulation evokes the conditioned response of the bat and causes large collicular changes that recover 180 min after the conditioning (Gao and Suga 1998) and large cortical changes that last more than 26 h after the conditioning (Gao and Suga 2000). For these changes caused by the conditioning, the AC and the somatosensory cortex are both necessary (Gao and Suga 1998, 2000).

Gao and Suga (1998, 2000) incorporated their findings with the hypothesis proposed by Weinberger and his coworkers (1990) and proposed the following working hypothesis of the corticofugal function for cortical plasticity. When behaviorally irrelevant acoustic stimuli are delivered to an animal, the AC and the corticofugal system perform egocentric selection, which is a small and short-term modulation of subcortical signal processing. Accordingly, the small and short-term cortical change is evoked (Fig. 1, left column). When the acoustic stimuli are paired with electric leg stimulation, the auditory and somatosensory signals ascend from the periphery to the auditory and somatosensory cortices, respectively (Fig. 1, middle column), and then to the amygdala through association cortices. These signals are perhaps associated in the association cortices and the amygdala, which is essential for evoking a conditioned behavioral response. Therefore the acoustic stimuli become behaviorally relevant to the animal. The amygdala sends the "associated" signal to the cholinergic basal forebrain, which increases the cortical acetylcholine level. Then the change in the AC is augmented (Fig. 1, right column) (Kilgard and Merzenich 1998; Weinberger 1998). Accordingly, egocentric selection is augmented and the subcortical change becomes larger, so that the cortical change becomes larger and long term. This positive feedback loop is controlled by inhibition mediated by the thalamic reticular nucleus.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1. Block diagram to explain a working hypothesis for the adjustment and improvement of auditory signal processing according to associative learning. DCN, dorsal column nuclei in the spinal cord; FATS, frequency, amplitude, time, and space; Lat. lem. n., lateral lemniscal nuclei; SOC, superior olivary complex; TRN, thalamic reticular nucleus. The dashed lines with the solid arrowheads indicate the pathway essential for conditioned behavioral responses. The dashed lines with the open arrowheads indicate the pathway that may not be essential for cortical plasticity (Suga et al. 2000) (see the text).

The importance of the amygdala for conditioned response (reviews by LeDoux 2000; Weinberger 1998), of the cholinergic basal forebrain for plasticity of the AC (Bakin and Weinberger 1996; Bjordahl et al. 1998; Kilgard and Merzenich 1998; review by Rasmusson 2000), and of the corticofugal auditory system for collicular plasticity (Gao and Suga 1998, 2000) has been well demonstrated. However, it has not yet been studied whether ACh applied to the AC augments collicular and cortical plasticity caused by auditory conditioning as hypothesized by Gao and Suga (2000).

There has been a large number of papers on the auditory, somatosensory, and visual cortices indicating that the cholinergic basal forebrain and acetylcholine (ACh) in the cortex play an important role in cortical plasticity, as reviewed by Buonomano and Merzenich (1998), Sarter and Bruno (2000), and Rasmusson (2000). The papers on the somatosensory and visual systems are reviewed in DISCUSSION, but those on the auditory system are briefly reviewed in the following text.

In the AC of the guinea pig, plasticity in frequency tuning (i.e., BF shift) is caused by a tone burst paired with electrical stimulation of the cholinergic basal forebrain but not by the tone burst or the electrical stimulation alone, and it is similar to that caused by behavioral learning (Bakin and Weinberger 1996; Bjordahl et al. 1998). In the cat's AC, massive progressive reorganization of the cochleotopic map (i.e., BF shift) is caused by electrical stimulation of the basal forebrain paired with a tone burst (Kilgard and Merzenich 1998). In the big brown bat, electrical stimulation of the basal forebrain augments collicular and cortical BF shifts evoked by a train of acoustic stimuli or electrical stimulation of the AC (Ma and Suga 2000).

In the ACs of the squirrel monkey (Foote et al. 1975) and the guinea pig (Metherate et al. 1990), ACh iontophoretically applied to cortical neurons augments their responses to acoustic stimuli. In the ACs of cats (McKenna et al. 1989; Metherate and Weinberger 1989, 1990), an iontophoretic application of ACh to cortical neurons paired with tone bursts reduces their responses at the frequency of the tone bursts but increases their responses at other frequencies. Such effects of ACh are antagonized by atropine iontophoretically applied to these cortical neurons. In the inferior colliculus (IC) of the horseshoe bat (Rhinolophus ferrumequinum), ACh iontophoretically applied to collicular neurons does not change their frequency-tuning curves but increases the auditory responses of 37% of them and reduces the auditory responses of 16% of them. On the other hand, atropine decreases the auditory responses of 62% of the neurons studied (Habbicht and Vater 1996).

ACh apparently plays an important role in cortical plasticity and has excitatory and/or inhibitory effects on collicular and cortical neurons. However, there have been no direct studies about the role of ACh in BF shifts (i.e., reorganization of the cochleotopic map) caused by auditory conditioning. The aim of our present paper is to report the effects of ACh and/or atropine applied to the AC or IC on collicular and cortical BF shifts (plasticity) caused by the conditioning. We specifically examined the following four questions: whether ACh applied to the AC augments cortical plasticity as expected from the hypothesis that the cholinergic basal forebrain causes cortical plasticity (Weinberger 1998; Weinberger et al. 1990) and also collicular plasticity as expected from the hypothesis that the corticofugal system plays a role in collicular plasticity (Gao and Suga 1998, 2000); whether atropine applied to the AC abolishes or dramatically reduces cortical plasticity, as expected from the hypothesis proposed by Weinberger and his coworkers (1990), but not collicular plasticity as expected from both Gao and Suga's hypotheses (1998, 2000) and the fact that the corticofugal system as well as cortical signal processing does not depend on ACh (Tsumoto 1990); whether ACh applied to the IC does not augment cortical plasticity; and whether atropine applied to the IC does not abolish cortical BF shift.

Auditory conditioning causes short-term collicular plasticity and long-term cortical plasticity in the big brown bat (Gao and Suga 2000). It also causes short-term thalamic plasticity (Edeline and Weinberger 1991) and long-term cortical plasticity in the guinea pig (Weinberger et al. 1993). In the big brown bat, focal electric stimulation of the AC evokes brief-term collicular plasticity (Jen et al. 1998) or short-term collicular (Ma and Suga 2001; J Yan and Suga 1996; W Yan and Suga 1998; Zhang and Suga 2000) and cortical plasticity (Chowdhury and Suga 2000; Ma and Suga 2001). We have a hypothesis that ACh is necessary to change cortical plasticity from the short term to the long term. In the studies of plasticity, the time course as well as the magnitude and direction of plastic changes is the important measure, as demonstrated by Gao and Suga (2000). Therefore our present studies were particularly focused on the effects of ACh or atropine on the time course of collicular and cortical plasticity. We found that ACh and atropine have profound effects on collicular and cortical plasticity caused by auditory conditioning and that ACh changes short-term plasticity into long-term plasticity in the AC but not in the IC. (Here, the brief-, short-, and long-term plasticities are, respectively, defined as the changes that last not more than 10 s, not more than 4 h, and longer than 4 h after the cessation of cortical electric stimulation, auditory conditioning, or repetitive acoustic simulation.)


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation

Experiments were performed with 42 adult big brown bats, E. fuscus (body weight: 18-24g). Procedures for animal preparation, acoustic stimulation, electric leg-stimulation, recording of action potentials, and drug applications were the same as those previously described (Gao and Suga 1998, 2000). The protocol for this research was approved by the animal studies committee of Washington University.

Under neuroleptanalgesia (Innovar, 4.08 mg/kg body wt), a 15-mm-long metal post was glued to the dorsal surface of the bat's skull. Experiments for recording auditory responses from single neurons, conditioning animals, and drug application began 3-4 days after the surgery. The awake bat was placed in a polyethylene-foam body mold suspended by an elastic band at the center of a soundproof room maintained at a temperature of about 31°C. The temperature monitored with a thermister placed between the bat and body mold was 37°C. The head was immobilized by fixing the metal post glued on the skull onto a metal rod with set screws, and it was adjusted to face directly at a loudspeaker located 74 cm away. The bat was neither anesthetized nor tranquilized during experiments.

Electrodes for recording action potentials

For electrode penetrations into the AC or IC, holes of about 50 µm in diameter were made in the skull with the tip of a sharpened needle under a dissection microscope (40 magnification). [For a drug application, a hole of ~1.0 mm in diameter was made in the skull (see following text).] The awake animals showed no signs of distress with this procedure yet responded vigorously to accidental touching of the exposed surgical wounds or the face. This indicated their awareness and ability to express pain if so needed.

For recording of action potentials, a sharpened, vinyl-coated tungsten-wire microelectrode with a tip diameter of ~7 µm was inserted orthoganally into the AC or dorsoventrally into the IC through the holes. An indifferent tungsten-wire electrode was placed on the dura mater near the recording electrode.

Acoustic and/or electric stimuli

Acoustic stimuli (20-ms-long tone bursts with a 0.5-ms rise-decay time) were delivered to the bat at a rate of 5/s. Their frequency and amplitude were manually varied to measure the BF and minimum threshold of a given neuron. The tone bursts were also computer-controlled. The sharpness of the manually measured tuning curve was used to determine the step size (0.2-0.5 kHz) of a computer-controlled frequency scan: the sharper the tuning curve, the smaller the step. The frequency scan consisted of 34 150-ms-long time blocks. In each scan, a single tone burst was delivered at the beginning of each block, and the frequency of the tone burst was shifted in 33 steps across the BF of the neuron. In the last time block, no simulation was presented to count background discharges. The amplitude of the tone bursts in the scan was always set at 10 dB above the minimum threshold of the neuron so as to easily detect a BF shift. An identical frequency scan was delivered 50 times to obtain an array of peristimulus-time (PST) histograms as a function of frequency. BFs and frequency-response curves were obtained by counting the total numbers of impulses discharged to 50 identical tone bursts.

To evoke the BF shifts of cortical and collicular neurons, the bat was conditioned with a 1.0-s-long train of acoustic stimuli (ASt), followed by a 1.0-s gap and then by an electric leg-stimulus (ESl; hereafter, ASt + ESl). ASt and ESl were the conditioned and unconditioned stimuli, respectively. In ASt, the tone bursts were 50 dB SPL and 10-ms long and were delivered at a rate of 33/s. Their frequencies were set 5.0 kHz lower than the BF of a given cortical or collicular neuron to be studied because in the big brown bat, ASt evokes the largest BF shift for cortical (Chowdhury and Suga 2000) and collicular neurons (Gao and Suga 1998; Yan and Suga 1998) with a BF ~5.0 kHz higher than ASt. ESl was a 50-ms-long monophasic electric pulse. The intensity of ESl was 0.15-0.57 mA, just above the threshold for eliciting a leg flexion. A single (ASt or ESl) or a paired stimulus (ASt + ESl) was delivered every 30 s for 15 min (30 times in total) or 30 min (60 times in total). To minimize a cumulative effect of conditioning, only one neuron was studied in a 1-day experiment, and the same animal was used with a 1- to 3-day interval. In each 1-day experiment, tone bursts alone were delivered at a rate of 5/s over 2-3 h to record single-unit activity and to obtain data in the control condition. This period presumably caused extinction of BF shifts, if any, remaining after a previous conditioning experiment.

Drug applications

To investigate the role of ACh in BF shift caused by the conditioning, ACh and/or atropine were applied to the IC or AC before or after the conditioning. The AC was first electrophysiologically mapped to locate its approximate center (Dear et al. 1993). In the big brown bat, the dorsal surface of the IC was directly visible through the skull. A 1.0-mm-diam hole was made in the skull at the center of this visible portion for a drug application. The solutions of 1 M acetylcholine chloride (pH 4.5; dissolved in distilled water) and/or 0.4 M atropine sulfate (pH 5; dissolved in 0.9% saline) were applied to the exposed surface of the AC or IC with a 1.0 µl Hamilton syringe or syringes. The supplier of these drugs was Sigma Chemical in St. Louis, MO. A saline solution (0.9% sodium chloride) was also applied to the AC or IC for sham experiments.

Data acquisition and processing

The responses of single cortical and collicular neurons to tone bursts were respectively recorded at 200-700 µm depths (layers III-V) in the AC and 300-2,000 µm depths in the central nucleus of the IC with tungsten-wire microelectrodes (~7 µm tip diameter). A BAK time-amplitude window discriminator was used to select action potentials from a single neuron.

The waveform of an action potential of a single neuron was stored on a digital storage oscilloscope at the beginning of the data acquisition and was used as a template. Action potentials discharged by the neuron were continuously monitored together with the template on the screen of the digital storage oscilloscope during data acquisition: before, during, and after the conditioning and/or drug application. Data were acquired every 15 min over 225 min after the conditioning as far as action potentials visually matched the template. Data were stored on a hard drive of a personal computer and were used for off-line analysis.

Off-line data processing included plotting PST or peristimulus time-cumulative (PSTC) histograms displaying responses of a single collicular or cortical neuron to 50 identical acoustic stimuli and frequency-response curves based on 50 frequency scans obtained before, during, and after the conditioning and/or drug application. The magnitude of auditory responses was expressed by a number of impulses per 50 identical stimuli after subtracting background discharges counted in the last block of the frequency scan. The BF of the neuron was defined as the frequency at which the frequency-response curve was peaked (e.g., Fig. 2). To study the time course of BF shift, BFs determined from the frequency-response curves obtained every 15 min were plotted as a function of time. The time courses of BF shifts obtained from several neurons were averaged (e.g., Fig. 3). Therefore each data point in an averaged time course represents the mean and standard error based on 50 responses multiplied by a number of neurons (N) used for averaging. A t-test was used to test the difference between the auditory responses obtained before and after the conditioning and/or drug application and to test the difference between the responses of collicular and cortical neurons.

The following criteria were used for a shift in the frequency-response curve or BF of a collicular or cortical neuron evoked by the conditioning and/or drug application. If a shifted frequency-response curve or BF of a collicular neuron did not recover by more than 50%, the data were excluded from the analysis. In stable, long recording conditions, all curves shifted by the conditioning and/or drug application recovered by more than 50%. This recovery itself helped prove that the shift was significant and that the shift was not due to recording action potentials from different neurons. However, this criterion was not necessarily applied to cortical BF shift because it lasted several hours after a 30-min-long conditioning. Cortical BF shifts as well as collicular ones were highly specific to the frequency of a conditioned tone (Gao and Suga 1998, 2000; present study). This indicates that BF shifts were not due to random change in recording action potentials from different neurons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In 42 bats, 136 collicular and 149 cortical neurons were studied before and after auditory conditioning and/or drug application. Of the 285, different numbers of neurons were examined for the effect of ACh and/or atropine applied to the AC or IC as indicated in individual figures representing the time courses of BF shifts. BF shifts caused by the conditioning with or without an application of saline solution were obtained from 54 collicular and 51 cortical neurons. Effects of ACh applied to the AC or IC were studied on the BF shifts of 39 collicular and 44 cortical neurons; the effects of atropine applied to the AC or IC were studied on the BF shifts of 36 collicular and 49 cortical neurons; and the effects of ACh-atropine mixture applied to the AC or IC were studied on the BF shifts of 7 collicular and 5 cortical neurons. BF shifts at subthreshold (see DISCUSSION) were caused by a 15-min-long conditioning session to examine the effects of ACh on them, and BF shifts above threshold were caused by a 30-min-long conditioning session to examine the effects of atropine on them.

Collicular and cortical changes caused by conditioning with or without an application of saline solution (control data to drug-application experiments)

When a train of acoustic stimuli (ASt) followed by an electric leg-stimulation (ESl) was delivered to the animal for 30 min, collicular and cortical neurons with a BF 5.0 kHz higher than the frequency of ASt showed the largest BF shift toward the frequency of ASt, as previously reported by Gao and Suga (1998). Therefore all the data reported in the present paper were obtained from neurons tuned to a frequency 5.0 kHz higher than the frequency of ASt.

Figure 2A shows the responses to tone bursts and the frequency-response curves of a single collicular neuron which was tuned to 37.0 kHz (Fig. 2A, curve 1 in c). When the animal was conditioned with a 32.0-kHz ASt paired with ESl, the response (number of impulses per 50 stimuli) of the neuron decreased by 28% to a 37.0-kHz tone burst (Fig. 2A; compare a, 2 with 1), but increased by 103% to a 35.5-kHz tone burst (Fig. 2A; compare b, 2 with 1). As a result of these frequency-dependent changes, the frequency-response curve and BF of the neuron shifted from 37.0 to 35.5 kHz (Fig. 2A, curve 2 in c). Both the response and the frequency-response curve returned (hereafter recovered) to those in the control condition 170 min after the conditioning (Fig. 2A, a4, b4, and curve 4 in c). The response at the BF in the shifted condition was 10% less than that at the BF in the control condition as previously reported by Gao and Suga (1998). All 13 collicular neurons studied for the 30-min-long conditioning showed changes that were basically the same as those described in the preceding text. The mean BF shift and the mean decrease in response at the shifted BF were 1.35 ± 0.23 kHz and 11.6 ± 5.78% (n = 13), respectively.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2. Changes in the responses (a and b) and frequency-response curves (c) of a collicular (A) and a cortical neuron (B) caused by a 30-min-long conditioning session consisting of 60 stimulus pairs of a short train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). The data were obtained with tone bursts fixed at 10 dB above the minimum threshold of a given neuron. The frequency of ASt and the best frequency (BF) of the neuron were, respectively, 32.0 and 37.0 kHz in A and 30.0 and 35.0 kHz in B. 1-4, before (control condition, open circle ), 30 min after (), 90 or 180 min after (triangle ), and 170 or 360 min after (black-triangle), respectively, the conditioning. The poststimulus time histograms in the a and b, respectively, show changes in the responses at the best frequency in the control (BFc) and "shifted" conditions (BFs), which are indicated by the right-arrow in c. The BF shift of the collicular neuron recovered (i.e., BFs shifted back to BFc) 170 min after the conditioning but that of the cortical neuron did not recover even 6 h after the conditioning. AC, auditory cortex; IC, inferior colliculus.

Figure 2B shows the responses to tone bursts and the frequency-response curves of a single cortical neuron tuned to 35.0 kHz (Fig. 2B, curve 1 in c). When conditioned with a 30.0-kHz ASt followed by ESl, the response of the neuron immediately after the conditioning decreased by 14% to a 35.0-kHz tone burst (Fig. 2B; compare a, 2 with 1) and increased by 9.2% to a 34.5-kHz tone burst (Fig. 2B; compare b, 2 with 1). As a result, the BF of the neuron shifted down to 34.5 kHz from 35.0 kHz (Fig. 2B, curve 2 in c). About 180 min after the onset of the conditioning, the response decreased further by 27% at 35.0 kHz (Fig. 2B; compare a, 3 with 1) and increased by 22% at 33.5 kHz (Fig. 2B; compare b, 3 with 1). Because of these changes, the frequency-response curve shifted further down to 33.5 kHz (Fig. 2B, curve 3 in c). The response at the shifted BF was 10% less than that at the control BF. The changes in response and frequency-response curve of the neuron showed no sign of recovery even 360 min after the conditioning (Fig. 2B, a4, b4, and curve 4 in c). All 12 cortical neurons studied for the 30-min-long conditioning showed changes that were basically the same as those described in the preceding text. The mean BF shift and the mean decrease in response at the maximally shifted BF were 1.43 ± 0.19 kHz and 10.3 ± 2.1% (n = 12), respectively. These cortical changes were very similar to the collicular changes (P > 0.05). However, the time course of BF shift was dramatically different between collicular and cortical neurons.

The mean time courses of the BF shifts of 13 collicular (Fig. 3A, open circle ) and 12 cortical neurons studied (Fig. 3B, triangle ) showed the following differences between them: the collicular BF shift was largest at the end of the conditioning, and monotonically recovered ~180 min after the conditioning; the cortical BF shift developed slowly, reached a plateau ~60 min after the conditioning, and plateaued for more than 210 min; and immediately after the 30-min-long conditioning, the cortical BF shift was smaller than the collicular BF shift (0.83 ± 0.11 vs. 1.15 ± 0.09 kHz; P < 0.01).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. Time courses of the BF shifts of collicular (A and C) and cortical neurons (B and D) caused by a 15- or 30-min-long conditioning session under 3 different situations: conditioning only (open circle  and triangle ), conditioning after a saline application to the AC ( and black-triangle), and conditioning before a saline application to the AC (×). In each figure, , the time period for the conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). right-arrow, the time for an application of a saline solution (Sal.) to the AC, which was either pre- or postconditioning. Each data point represents the mean and SE of the values obtained from the number of neurons (N) listed on the right. The BF shifts caused by the 30-min-long conditioning session are not influenced at all by the saline application.

As described later, ACh or atropine was applied to the AC or IC before or after the conditioning. Therefore sham experiments were performed with a saline solution. An application of saline solution to the AC 5 min prior to or 75 min after the onset of the 30-min-long conditioning session did not affect any of the auditory responses, frequency-response curves, and time courses of BF shifts of 15 collicular and 17 cortical neurons caused by the conditioning session. Figure 3, A and B, shows that the mean time courses of the collicular and cortical BF shifts caused by the 30-min-long conditioning were not affected by a saline application.

A 15-min-long conditioning session caused no changes in the responses, frequency-tuning curves, and BFs of 10 collicular and 8 cortical neurons studied (Fig. 3, C and D, open circle  and triangle ). A saline solution applied to the AC before or after the 15-min-long conditioning also evoked no changes in 16 collicular and 14 cortical neurons studied (Fig. 3, C and D, , black-triangle, and ×).

Effects of ACh or atropine applied to the AC on collicular and cortical responses

ACh applied to the AC augmented responses to tone bursts and background discharges of 26 collicular neurons studied. The mean maximal increases in response at the BF and background discharges of the collicular neurons were 49.8 ± 11.6 and 9.7 ± 6.5% (n = 26), respectively, which occurred 60 min after the ACh application. These augmentations disappeared ~150 min after the application (Fig. 4A, open circle ).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. Effects of acetylcholine (ACh) or atropine (Atr) applied to the AC (A) or IC (B) on the response magnitude of collicular ( and open circle ) and cortical neurons (black-triangle and triangle ). Response magnitude is expressed by the number of impulse per 50 tone bursts at the best frequency, and its change is expressed in percent. Each data point represents the mean and SE of the values obtained from the number of neurons (N) listed on the right. ACh evoked a prominent increase in response (open circle  and triangle ). Atropine evoked a small short-lasting decrease in response ( and black-triangle).

ACh applied to the AC also augmented auditory responses and background discharges of 36 cortical neurons studied. The mean maximal increases in response and background discharges were 61.7 ± 8.1 and 18.5 ± 7.8% (n = 36), respectively, which also occurred ~60 min after the ACh application. The augmentation disappeared ~150 min after the application (Fig. 4A, triangle ). The time course of the cortical augmentation was thus similar to that of the collicular augmentation.

ACh applied to the AC augmented the auditory responses of collicular and cortical neurons at all frequencies of tone bursts. Figure 5A shows the frequency-response curve of a collicular neuron tuned to 30.5 kHz (curve 1). The height of the curve gradually increased up to 51% over 60 min after the ACh application (curves 2 and 3) and then gradually decreased back to the original height over 120 min thereafter (curves 4 and 5). The overall shape of the frequency-response curve and the BF did not change regardless of a prominent change in response magnitude. Figure 5B shows the frequency-response curve of a cortical neuron tuned to 36.5 kHz (curve 1). Just like the collicular neuron described in the preceding text, the height of the curve of the cortical neuron increased up to 49% over 60 min after an ACh application (curves 2 and 3) and then decreased back to the original height over 120 min thereafter (curves 4 and 5). The overall shape of the frequency-response curve and BF did not change. Since a nonfocal application of ACh to the AC carried no frequency information, the preceding results were completely expected. As described later, however, when the ACh application was paired with a conditioned acoustic stimulus, ACh evoked BF shifts that were specifically related to the frequency of the conditioned acoustic stimulus.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5. Frequency-response curves of a collicular (A) and a cortical neuron (B). The curves were obtained before (control condition) and 30, 60, 90, and 180 min after an ACh application to the AC. ACh increased the height of the curve but did not change its overall shape and BF.

In the big brown bat, the IC protrudes between the cerebrum and the cerebellum so that its dorsal surface is located immediately posterior to the visual cortex. ACh applied to the visual cortex, which is located dorsoposteriorly to the AC, 5 min before the 15-min-long conditioning evoked neither BF shift nor increase in response and background discharges of any of six collicular neurons studied. Therefore the augmentation of auditory responses of collicular neurons by ACh applied to the AC was not due to ACh that might be diffused to the IC.

Atropine applied to the AC reduced collicular and cortical responses for 45 min (Fig. 4A,  and black-triangle). The amount of reduction was, however, statistically insignificant for 17 collicular neurons studied (4.7 ± 3.2%; P > 0.05), but was statistically significant for 32 cortical neurons studied (10.0 ± 6.8%; P < 0.05). The difference in reduction between the collicular and cortical neurons was insignificant (P > 0.05).

Effects of ACh or atropine applied to the IC on collicular and cortical responses

ACh applied to the IC augmented both collicular and cortical responses to tone bursts, as that applied to the AC did (Fig. 4B, open circle  and triangle ). Atropine applied to the IC reduced both collicular and cortical responses to tone bursts also as did that applied to the AC (Fig. 4B,  and black-triangle). The amount and time course of the augmentation or reduction were nearly the same for collicular and cortical responses (P > 0.05). Since ACh applied to the visual cortex had no effect on collicular responses, the augmentation and reduction of cortical auditory responses evoked by ACh or atropine applied to the IC were not due to the drugs, which might be diffused to the AC, but due to the change in the IC, i.e., the change in the ascending auditory system.

Effects of ACh applied to the AC on collicular and cortical plasticity

The 15-min-long conditioning session caused no BF shifts of collicular and cortical neurons (Fig. 3, C and D). However, it caused significant BF shifts of these neurons when ACh was applied to the AC 5 min prior to the conditioning. Figure 6A shows the responses (a and b) and frequency-tuning curves (c) of a single collicular neuron that was tuned to 28.0 kHz (curve 1 in c). When ACh was applied to the AC prior to the 15-min-long conditioning, frequency-dependent changes in responses were first evoked. That is, 30 min after the onset of the conditioning, the response was augmented at 27.5 kHz and frequencies lower than that, but was reduced at 28.0 kHz and frequencies higher than that (Fig. 6A, a2, b2, and curve 2 in c). Accordingly, the BF shifted toward the frequency of the conditioned tone burst, which was 23.0 kHz. This BF shift was, however, small and temporary. The response to the 27.5-kHz tone burst further increased with time because of ACh applied to the AC: 154% increase 60 min after and 95% increase 90 min after the onset of the conditioning (Fig. 6Ab, 3 and 4). The response to the 28.0-kHz tone burst also increased: 74% 60 min after and 43% 90 min after the onset of the conditioning (Fig. 6Aa, 3 and 4). Sixty minutes after the onset of the conditioning session, the frequency-response curve was raised and the small BF shift recovered (Fig. 6A, curve 3 in c). All 26 collicular neurons studied with ACh paired with the conditioning showed changes that were basically the same as those described in the preceding text. The mean BF shift and the mean maximal increase in the response at the BF were 0.82 ± 0.22 kHz and 52.7 ± 9.3% (n = 26), respectively.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 6. Changes in the responses (a and b) and frequency-response curves (c) of a collicular (A) and a cortical neuron (B) evoked by an ACh application to the auditory cortex (AC) before a 15-min-long conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). The data were obtained with tone bursts fixed at 10 dB above the minimum threshold of a given neuron. The frequency of ASt and the BF of the neuron were, respectively, 23.0 and 28.0 kHz in A and 21.0 and 26.0 kHz in B. 1-4, before (control condition, open circle ) and 30 min (), 60 min (triangle ), and 90 min (black-triangle), respectively, after the onset of the conditioning. The poststimulus time histograms in a and b, respectively, show changes in the responses at the best frequencies in the control (BFc) and shifted conditions (BFs), which are indicated by the right-arrow in c. The BF shift of the collicular neuron recovered (i.e., BFs shifted back to BFc) 60 min after the conditioning, but that of the cortical neuron did not recover 90 min after the conditioning. Both the collicular and cortical neurons showed auditory responses and BF shifts augmented by ACh.

The cortical neuron in Fig. 6B was tuned to 26.0 kHz (curve 1 in c). When ACh was applied to the AC 5 min prior to the 15-min-long conditioning, its responses to tone bursts were augmented and its BF gradually shifted over 60 min. Sixty minutes after ACh application, augmentation was largest for the response to 24.5 kHz, a 143% increase (Fig. 6Bb3), and the frequency-response curve and BF shifted to 24.5 kHz from 26.0 kHz, that is, toward the frequency of the 21.0-kHz conditioned tone (Fig. 6B, curve 3 in c). Ninety minutes after the onset of the conditioning, the augmented responses to tone bursts slightly decreased, but the shifted BF stayed the same (Fig. 6B, a4, b4, and curve 4 in c). The augmented response returned to normal 150 min after the conditioning (Fig. 4A, triangle ), but the shifted BF stayed the same even 210 min after the conditioning (Fig. 8A, black-triangle). All 36 cortical neurons studied with ACh paired with the conditioning showed basically the same changes as described in the preceding text. The mean maximal cortical BF shift evoked by the conditioning paired with ACh was much larger than the mean maximal collicular BF shift: 1.52 ± 0.23 (n = 18) versus 0.82 ± 0.22 kHz (n = 8), P < 0.01.

The augmentation of auditory responses by ACh was mostly due to an increase not in initial discharges but in late discharges. In Fig. 6, an increase is the largest at the BF shifted by the conditioning paired with ACh: 2.5 times increase in A (b3 and curve 3 in c) and 2.4 times increase in B (b3 and curve 3 in c). In spite of such a large increase, the height of the PST histograms displaying the collicular and cortical responses stay nearly the same. That is, the initial discharges stayed almost unchanged. To further substantiate this observation, the PST histograms in Fig. 6, Ab and Bb, are replotted as PSTC histograms (Fig. 7). In Fig. 7, the PSTC histograms for the control condition and for 60 min after the conditioning with ACh are the same for the initial 2.0 ms (A) or 1.3 ms (B). Thereafter, the histogram for 60 min after the conditioning becomes 2.5 (A) or 2.4 times (B) higher than that for the control. When the response started to recover, the PSTC histogram becomes lower without a decrease in its initial portion (Fig. 7, curves 3 in A and B).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7. Peristimulus time-cumulative (PSTC) histograms displaying the responses of the collicular (A) and cortical neurons (B) shown in Fig. 6. The PST histograms in Fig. 6Ab, 1, 3, and 4, are shown as PSTC histograms in A and those in Fig. 6Bb, 1, 3, and 4, are shown as PSTC histograms in B. The arrows and vertical lines indicate the boundary between the initial discharges unaffected by ACh and the late discharges augmented by ACh. The frequency of a stimulus tone burst was 27.5 kHz for A and 24.5 kHz for B.

The mean time courses of BF shifts of 8 collicular and 18 cortical neurons caused by the 15-min-long conditioning preceded by an ACh application are shown in Fig. 8A. The collicular BF shift peaked 30 min after the onset of the conditioning and then recovered 60 min thereafter (). On the other hand, the cortical BF shift reached a plateau 60 min after the onset of the conditioning, that is, the time when the collicular BF shift had almost recovered. The cortical BF shift was much larger and lasted much longer than the collicular BF shift. The cortical BF shift showed little sign of recovery even 4 h after the conditioning. Therefore the time course and the amount of the cortical BF shift assisted by ACh are very similar to those caused by the 30-min-long conditioning alone (see Fig. 3B). It is also noticed that the time course of the cortical BF shift is quite different from that of the augmentation of auditory responses evoked by ACh applied to the AC (see Fig. 4A).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 8. Time courses of the BF shifts of collicular (IC; ) and cortical neurons (AC; black-triangle) caused by a 15- or 30-min-long conditioning session under 3 different situations: A, 15-min-long conditioning after an ACh or saline application to the auditory cortex (AC); B and C, 15- or 30-min-long conditioning before an ACh or saline application to the AC. In each figure, indicates the time period for the conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). right-arrow, the time when an ACh or saline solution (Sal.) was applied to the AC. Each solid curve with data points represents the effect or lack of effect of ACh on the time course of a BF shift. Each data point represents the mean and SE of the values obtained from the number of neurons shown in the parentheses. The saline application to the AC had no effect on the collicular ( · · · ) and cortical BF shifts (- - -; these curves are shown in Fig. 3 with data points). See the text.

ACh applied to the AC 5 min after the 15-min-long conditioning evoked neither collicular nor cortical BF shift (Fig. 8B), although it augmented collicular and cortical responses to tone bursts, and augmentation lasted ~150 min as shown in Fig. 4A.

The 30-min-long conditioning caused short-term collicular and long-term cortical BF shifts as reported by Gao and Suga (2000) and as shown in Fig. 3, A and B. ACh applied to the AC 40 min after the 30-min-long conditioning evoked no additional BF shifts of collicular and cortical neurons (Fig. 8C), although it augmented the responses of collicular and cortical neurons to tone bursts over ~150 min as shown in Fig. 4A.

An application of both atropine and ACh to the AC 5 min before the 15-min-long conditioning evoked neither BF shift nor increase in the auditory response of four cortical neurons studied but evoked a 0.5-kHz BF shift of one cortical neuron that lasted ~20 min. An application of both atropine and ACh to the IC 5 min before the 15-min-long conditioning also evoked neither BF shift nor increase in the auditory response of seven collicular neurons studied. Therefore the effects of ACh on collicular and cortical BF shifts and responses were antagonized by atropine.

Effects of atropine applied to the AC on collicular and cortical plasticity

Atropine applied to the AC 5 min prior to the 30-min-long conditioning session reduced the collicular BF shift caused by the conditioning slightly, whereas it completely abolished the cortical BF shift that otherwise would be caused by the conditioning. The collicular neuron in Fig. 9A was tuned to 23.0 kHz (curve 1 in c). When atropine was applied to the AC 5 min prior to the 30-min-long conditioning, the response of the neuron to a tone burst decreased by 47% at 23.0 kHz and increased by 77% at 21.5 kHz 30 min after the onset of the conditioning (Fig. 9A, a2 and b2). Such frequency-dependent changes shifted the BF of the neuron toward the frequency of the conditioned tone, 18.0 kHz. Figure 9Ac, curves 1-4, respectively, shows the frequency-response curves of the collicular neuron obtained before and 30, 60, and 90 min after the onset of the conditioning with an atropine application to the AC 5 min prior to the conditioning. These curves show that the BF of the neuron shifted from 23.0 to 21.5 kHz 30 min after (curve 2), recovered to 22.0 kHz 60 min after (curve 3), and recovered to 22.5 kHz 90 min after (curve 4) the onset of the conditioning. These changes were similar to, but not the same as, those caused by the conditioning without atropine (Fig. 2A) in amounts of changes in response and BF shift. The maximum decrease in response at the shifted BF from that at the control BF was 23.4 ± 6.85% (n = 8) for the conditioning with atropine, but it was 11.3 ± 4.72% (n = 7) for the conditioning with saline. The BF shift was 1.23 ± 0.23 kHz (n = 8) for the conditioning with atropine, but it was 1.62 ± 0.24 kHz (n = 7) for the conditioning with saline (Fig. 10A). These differences were statistically significant (P < 0.05).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 9. Changes in the responses (a and b in A; a in B) and frequency-response curves (c in A; b in B) of a collicular (A) and a cortical neuron (B) evoked by an atropine application to the AC before a 30-min-long conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). The data were obtained with tone bursts fixed at 10 dB above the minimum threshold of a given neuron. The frequency of ASt and the BF of the neuron were, respectively, 18.0 and 23.0 kHz in A and 21.0 and 26.0 kHz in B. 1-4: before (control condition, open circle ) and 30 min (), 60 min (triangle ), and 90 min (black-triangle), respectively, after the onset of the conditioning. The poststimulus time histograms in a and b, respectively, show changes in the responses at the best frequencies in the control (BFc) and shifted conditions (BFs), which are indicated by down-arrow  in c of A or b of B. The collicular neuron showed BF shift, but the cortical neuron did not.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 10. Time courses of the BF shifts of collicular (IC; ) and cortical neurons (AC; black-triangle) caused by a 30-min-long conditioning session under 3 different situations: A, conditioning after an atropine (Atr) or saline (Sal) application to the auditory cortex (AC); B and C, conditioning 45 or 180 min before an atropine or saline application to the AC. In each figure, , the time period for the conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). right-arrow, the time when an atropine or saline solution was applied to the AC. Each solid curve with data points represents the effect of atropine on the time course of BF shift. Each data point represents the mean and SE of the values obtained from the number of neurons shown in the parentheses. The saline application had no effect on the collicular ( · · · ) and cortical BF shifts (- - -; these lines are shown in Fig. 3 with data points). See the text.

The cortical neuron in Fig. 9B was tuned to 26.0 kHz (curve 1 in b). Atropine applied to the AC 5 min prior to the 30-min-long conditioning slightly reduced the response of the neuron to 26.0 kHz (a) and abolished a BF shift of the neuron that otherwise would be caused by the conditioning (b). The data shown in Fig. 9 indicate that the collicular BF shift can be evoked without the cortical BF shift.

Compared with the collicular BF shift caused by the conditioning without atropine, the collicular BF shift caused by the conditioning with atropine developed slowly and peaked 45 min after the onset of the conditioning instead of 30 min after. The amount of the BF shift was 25% smaller (P < 0.05), and its recovery time was 60 min shorter (P < 0.05) than those caused by the conditioning without atropine (Fig. 10A, ).

Atropine applied to the AC 70 min after the onset of the 30-min-long conditioning (i.e., during the initial portion of the recovery phase of the collicular BF shift caused by the conditioning without atropine) had no effect on the collicular BF shift caused by the conditioning, so that the collicular BF shift monotonically recovered 180 min after the conditioning (Fig. 10B, ). On the other hand, the cortical BF shift that had nearly developed to the plateau after the conditioning was drastically affected by atropine. On the average from the data obtained from 10 cortical neurons, the BF shift recovered 73 ± 18.5% (n = 10) almost immediately after an atropine application and then developed back to the plateau 60 min thereafter. The plateau was maintained as was that which had been caused by the conditioning without atropine (Fig. 10B, black-triangle). When atropine was applied to the AC 210 min after the onset of the conditioning (i.e., after the recovery of the collicular BF shift), the cortical BF shift was unaffected by atropine (Fig. 10C, black-triangle). These data indicate that the cortical BF shift was not stabilized at ~70 min after the onset of the conditioning but was stabilized at ~210 min after.

Effects of ACh applied to the IC on collicular and cortical plasticity

ACh applied to the IC 5 min prior to the 15-min-long conditioning session augmented the responses to tone bursts and evoked the BF shifts of all 13 collicular neurons studied. The collicular neuron in Fig. 11A was tuned to 28.0 kHz (curve 1 in c). When ACh was applied to the IC 5 min prior to the 15-min-long conditioning, the neuron's response decreased at 28.0 kHz (a2) and increased at 27.0 kHz (b2), and its BF shifted from 28.0 to 27.0 kHz 30 min after the onset of the conditioning (curve 2 in c). Sixty minutes after the onset of the conditioning, the response of the neuron increased to all sounds from 23.5 to 31.5 kHz (a3, b3 and curve 3 in c). The shifted BF stayed at 27.0 kHz. These changes recovered ~150 min after the conditioning (Fig. 12A, ). On the average, the auditory response augmented by ACh was 56.0 ± 8.9% (n = 13) larger than that in the control condition, and the BF shift was 1.06 ± 0.32 kHz (n = 13), instead of no BF shift for the 15-min-long conditioning alone. The BF shift plateaued over ~45 min and then recovered 180 min after the end of the conditioning (Fig. 12A, ). The collicular and cortical BF shifts could, respectively, be evoked by the 15-min-long conditioning when ACh was applied to the IC or AC prior to the conditioning. However, there was a clear difference in duration of BF shift. That is, the collicular BF shift recovered in 3 h (Fig. 12A, ), but the cortical BF shift showed no sign of recovery even 3.5 h after the conditioning (Fig. 8A, black-triangle).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 11. Changes in the responses (a and b) and frequency-response curves (c) of a collicular (A) and a cortical neuron (B) evoked by an ACh application to the inferior colliculus before a 15-min-long conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). The data were obtained with tone bursts fixed at 10 dB above the minimum threshold of a given neuron. The frequency of ASt and the best frequency of the neuron are, respectively, 23.0 and 28.0 kHz in A and 28.0 and 33.0 kHz in B. 1-4, before (control condition, open circle ) and 30 min (), 60 min (triangle ), and 90 min (black-triangle), respectively, after the onset of the conditioning. The poststimulus time histograms in a and b, respectively, show changes in the responses at the best frequencies in the control (BFc) and shifted conditions (BFs), which are indicated by right-arrow in c. Sixty minutes after the conditioning, the collicular BF shift showed no sign of recovery but the cortical BF shift completely recovered. Both the collicular and cortical neurons showed increased responses evoked by ACh.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 12. Time courses of the BF shifts of collicular (IC; ) and cortical neurons (AC; black-triangle) caused by a 15- (A) or 30-min-long conditioning session (B and C) under 3 different situations: A and B, conditioning after an ACh (A) or atropine (Atr) application (B) to the inferior colliculus (IC); C, conditioning 40 min before an atropine application to the IC. In each figure, , the time period for the conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). right-arrow, the time for an application of an ACh, atropine, or saline solution (Sal.) to the IC, which was either pre- or postconditioning. Each solid curve with data points represents the effect of ACh or atropine on the time course of BF shift. Each data point represents the mean and SE of the values obtained from the number of neurons shown in the parentheses. The saline application had no effects on the collicular ( · · · ) and cortical BF shifts (- - -, these curves are shown in Fig. 3 with data points). See the text.

ACh applied to the IC 5 min prior to the 15-min-long conditioning augmented the auditory responses of all of eight cortical neurons studied, but it evoked the BF shifts of only three of the eight neurons. The cortical neuron in Fig. 11B was tuned to 33.0 kHz (curve 1 in c). When ACh was applied to the IC 5 min prior to the 15-min-long conditioning, the neuron shifted its BF from 33.0 to 32.5 kHz 30 min after the conditioning (curve 2 in c). The shifted BF quickly recovered to 33.0 kHz, but the response of the neuron increased to all sounds from 28.0 to 38.0 kHz 60 min after the conditioning (a, b, and curve 3 in c). On the average, the response augmented by ACh was 49.8 ± 11.7% (n = 8) larger than the response in the control condition. The BF shift observed in the three cortical neurons was largest 30 min after the onset of the conditioning, and the mean BF shift was 0.52 ± 0.29 kHz (n = 3). This BF shift was small but significant (P < 0.05). The data shown in Figs. 11B and 12A (black-triangle) indicate that the cortical BF did not shift according to the collicular BF shift.

Effects of atropine applied to the IC on collicular and cortical plasticity

Atropine applied to the IC 5 min prior to the 30-min-long conditioning session abolished the changes in response, frequency-response curve and BF that otherwise would be caused by the conditioning for all nine collicular neurons studied. In other words, they were not affected by the conditioning at all, and their function in encoding tone bursts was also not affected by atropine. The collicular neuron in Fig. 13A was tuned to 25.5 kHz (curve 1 in b). Its responses to a 25.5-kHz tone burst (a1) and frequency-response curve (curve 1 in b) were not changed by the 30-min-long conditioning following an atropine application to the IC (a2-a4 and curves 2-4 in b). The only change noticed was a small decrease in auditory response evoked by atropine.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 13. Changes in the responses (a in A; a and b in B) and frequency-response curves (b in A; c in B) of a collicular (A) and a cortical neuron (B) evoked by an atropine application to the inferior colliculus before a 30-min-long conditioning session consisting of pairs of a train of acoustic stimuli (ASt) and an electric leg-stimulation (ESl). The data were obtained with tone bursts fixed at 10 dB above the minimum threshold of a given neuron. The frequency of ASt and the BF of the neuron were, respectively, 20.5 and 25.5 kHz in A and 20.0 and 25.0 kHz in B. 1-4, before (control condition, open circle ) and 30 min (), 60 min (triangle ), and 90 min (black-triangle) after the onset of the conditioning. The poststimulus time histograms in a and b, respectively, show the changes in the responses at the best frequencies in the control (BFc) and shifted conditions (BFs), which are indicated by right-arrow in b of A or c of B. The collicular neuron showed no BF shift, but the cortical neuron did. The cortical BF shift recovered halfway 90 min after the conditioning.

On the other hand, atropine applied to the IC decreased, but did not abolish, a cortical BF shift caused by the conditioning. In other words, the cortical BF shift could be evoked without the collicular BF shift. The cortical neuron in Fig. 13B was tuned to 25.0 kHz (curve 1 in c). For the conditioning following an atropine application, the neuron's response decreased at 25.0 kHz (a, 2 and 3) but increased at 23.0 kHz (b, 2 and 3). Its frequency-response curve and BF shifted from 25.0 to 23.0 kHz, i.e., toward the frequency of the conditioned tone, 20.0 kHz (Fig. 13B, curve 3 in c). Compared with the BF shifts of the 10 cortical neurons caused by the conditioning with a saline application (Fig. 3B), the BF shifts of 10 cortical neurons caused by the conditioning with atropine (Fig. 12B, black-triangle) were small and short-lasting. The mean maximal BF shift was 1.10 ± 0.21 kHz (n = 10) instead of 1.75 ± 0.15 kHz (n = 10). This difference is significant (P < 0.05). The mean recovery time of the BF shift was ~70 min (Fig. 12B, black-triangle) instead of longer than 3 h, i.e., showing no sign of recovery (Fig. 3B). The amount and time course of the cortical BF shift caused by the 30-min-long conditioning with atropine (Fig. 12B, black-triangle) were also small and short compared with those caused by the 15-min-long conditioning following a cortical ACh application (Fig. 8A, black-triangle). Therefore the collicular BF shift, as well as an increased ACh level in the AC, is necessary to evoke the large and long-lasting cortical BF shift.

When atropine was applied to the IC 40 min after the end of the 30-min-long conditioning session, i.e., at the middle of the recovery period of the collicular BF shift, the collicular BF shift rapidly recovered (Fig. 12C, ). On the other hand, the cortical BF shift was unaffected and showed no sign of recovery even 210 min after the conditioning (Fig. 12C, black-triangle). In other words, the cortical BF shift that has developed to the plateau does not require the collicular BF shift for its maintenance.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the following, we discuss the significance of our individual data in relation to the model proposed by Gao and Suga (1998, 2000) and Weinberger and his coworkers (1990). We then briefly review the data obtained from the auditory system of non-bat species and from nonauditory systems in relation to our present studies.

Effects of ACh and/or atropine applied to the AC or IC on collicular and cortical responses

ACh applied to the AC augmented not only cortical, but also collicular, responses to tone bursts (Fig. 4A). ACh applied to the visual cortex, which is located immediately anterior to the colliculus, did not affect collicular responses to tone bursts at all. Therefore collicular augmentation by ACh applied to the AC was not due to diffusion of ACh from the AC to the IC but was due to the augmentation of the corticofugal activity because there was no other neural pathway from the AC to the IC. This collicular augmentation agrees with the data that cortical neurons augment subcortical auditory responses through positive feedback mediated by the corticofugal system (Gao and Suga 1998; Yan and Suga 1996, 1999; Zhang and Suga 1997; Zhang et al. 1997).

ACh applied to the IC augmented not only collicular but also cortical auditory responses (Fig. 4B). The augmentation developed at the same time and to the same amount for collicular and cortical neurons. The augmented cortical response was apparently evoked by the augmented collicular response transmitted from the IC to the AC through the medial geniculate body. The augmentation of collicular and cortical responses by ACh was antagonized by atropine applied together with ACh to the IC or AC so that ACh affects collicular or cortical neurons via muscarinic receptors.

Atropine applied to the AC (Fig. 4A) or IC (Fig. 4B) reduced the auditory responses of collicular and cortical neurons only slightly and for a short time. Therefore the auditory responses of collicular and cortical neurons only slightly depend on ACh. However, as discussed later, BF shifts (plasticity) of the neurons caused by the conditioning greatly depend on ACh.

Nonfocal ACh application to the IC or AC, without auditory conditioning, always augmented both collicular and cortical auditory responses but did not evoke BF shifts (Fig. 5). This is quite reasonable because without auditory conditioning, there is no frequency information necessary for BF shifts. Augmentation of auditory responses by iontophoretically applied ACh was also found in the ACs of the squirrel monkey (Foote et al. 1975) and the guinea pig (Metherate et al. 1990). In addition to augmentation, however, suppression of auditory responses by iontophoretically applied ACh was found in the IC of the horseshoe bat (Habbicht and Vater 1996).

It was also found that whether ACh evokes augmentation or suppression of auditory responses of cortical neurons in the cat and guinea pig depends on the frequency of acoustic stimuli: suppression at the frequency of a tone burst paired with an iontophoretic ACh application but augmentation at other frequencies (McKenna et al. 1989; Metherate and Weinberger 1989, 1990). In our present experiments, ACh applied to the IC or AC immediately prior to the conditioning augmented the changes in the frequency-response curves of collicular and cortical neurons (Fig. 6). Such changes resulted from frequency dependent suppression and facilitation of their auditory responses caused by the conditioning. Therefore our findings are not contradictory to those by others.

Effects of ACh or atropine applied to the AC on collicular and cortical plasticity

A 30-min-long conditioning session without an ACh application to the AC evokes noticeable collicular and cortical BF shifts (Gao and Suga 1998, 2000) (Figs. 2 and 3, A and B). A nonfocal ACh application to the AC or IC alone did not evoke collicular and cortical BF shifts at all (Fig. 5). A 15-min-long conditioning alone also did not evoke the BF shifts (Fig. 3, C and D). However, the 15-min-long conditioning following the ACh application evoked BF shifts (Figs. 6 and 8A). The frequency information necessary for the BF shifts was not in the ACh applied, but in the conditioning, i.e., in the conditioned tone bursts that excited auditory neurons from the periphery through the AC. ACh would not evoke the BF shifts if nothing was evoked in the IC and AC by the 15-min-long conditioning. It was most likely that small BF shifts evoked by the 15-min-long conditioning would be detected if the step-size of the frequency scan was smaller than 0.2 kHz. Therefore it may be defined that the 15-min-long conditioning evoked "subthreshold" BF shifts in the IC and AC.

ACh applied to the AC immediately prior to the 15-min-long conditioning augmented the subthreshold collicular and cortical BF shifts caused by the conditioning: the collicular BF shift developed up to half of that caused by the 30-min-long conditioning alone, and the cortical BF shift became almost the same in amount and duration as that caused by the 30-min-long conditioning alone (Figs. 6 and 8A). Atropine applied to the AC prior to the 30-min-long conditioning reduced the collicular BF shift by 43% and completely abolished the cortical BF shift that otherwise would be caused by the conditioning (Figs. 9 and 10A). These data indicate that ACh is necessary to evoke the long-lasting cortical BF shift, that ACh augments the collicular BF shift through augmented activity of the corticofugal system, and that the collicular BF shift only partially depends on the cortical BF shift. The importance of the corticofugal system in the collicular plasticity can be justified because it has been known that the corticofugal system forms highly frequency-specific feedback loops and evokes collicular BF shifts (Yan and Suga 1998; Zhang and Suga 2000; Zhang et al. 1997), that no pathways other than the corticofugal system have been known to evoke collicular changes due to cortical changes, and that the collicular plasticity caused by auditory conditioning is abolished as the AC is inactivated (Gao and Suga 2000).

An ACh application to the AC after the 15-min-long conditioning augmented neither the subthreshold collicular BF shift nor the subthreshold cortical BF shift (Fig. 8B). ACh applied to the AC 45 min after the 30-min-long conditioning did not affect the collicular and cortical BF shifts (Fig. 8C). Therefore an increase in ACh level during the conditioning is essential. It has been hypothesized that an increase in ACh level in the AC caused by the excitation of the cholinergic basal forebrain is essential for auditory cortical plasticity (Weinberger 1998; Weinberger et al. 1990). Electrical stimulation of the basal forebrain indeed evokes large plastic changes in the AC (Bakin and Weinberger 1996; Kilgard and Merzenich 1998). Our present data favor the hypotheses that an increase in ACh level in the AC is necessary for the development of cortical plasticity caused by conditioning (Weinberger 1998; Weinberger et al. 1990), that collicular plasticity is caused by the corticofugal system (Gao and Suga 1998, 2000), and that collicular and cortical plasticity evoked by a behaviorally irrelevant sound is augmented by an increase in the cortical ACh level as the sound becomes behaviorally relevant.

Atropine applied to the AC 45 min after the 30-min-long conditioning did not affect the collicular BF shift at all but temporarily reduced the cortical BF shift (Fig. 10B). Atropine applied to the AC 180 min after a 30-min-long conditioning affects neither the collicular BF shift nor the cortical BF shift (Fig. 10C). This suggests that the cortical BF shift was not yet consolidated at ~1.5 h after the conditioning, i.e., at the time when the BF shift just reached a plateau, and that the recovery phase of the collicular BF shift is not influenced by the cortical BF shift but totally depends on a collicular change itself.

In the cat's AC, activation of muscarinic receptors by acetyl-beta-methylcholine (MCh) enhances N-methyl-D-aspartate (NMDA)-mediated neurotransmission and reduces GABA-mediated inhibition (Aramakis et al. 1997a,b) so that ACh facilitates the NMDA dependent plasticity. We have not yet studied the effects of NMDA and 2-amino-5-phosphonovaleric acid on the collicular and cortical BF shifts caused by conditioning.

Effects of ACh or atropine applied to the IC on cortical and collicular plasticity

ACh applied to the IC 5 min prior to the 15-min-long conditioning augmented the subthreshold collicular BF shift to be somewhat similar to that caused by the 30-min-long conditioning, but the augmentation of the cortical BF shift was very small and short-lasting (Figs. 11 and 12A). This means that the collicular BF shift alone is not enough to evoke a large and long-lasting cortical BF shift and that an increase of ACh level in the AC is essential for the long-lasting cortical BF shift. Therefore our data support Gao and Suga's (1998) hypothesis that the collicular BF shift augments the cortical BF shift, which is further augmented by an increased ACh level (Fig. 1). It also supports Weinberger's hypothesis (Weinberger 1998; Weinberger et al. 1990) that an increase in an ACh level in the AC is essential for cortical plasticity caused by fear conditioning (Fig. 1).

Atropine applied to the IC prior to the 30-min-long conditioning did not change the auditory responses and frequency-response curves of collicular neurons but abolished the collicular BF shift that otherwise would be caused by the conditioning (Figs. 12B and 13A). It reduced the cortical BF shift caused by the conditioning (Figs. 12B and 13B). These data indicate that endogenous ACh is important for the development of the collicular BF shift, that the collicular BF shift is not a result of BF shifts that might be caused in the subcollicular nuclei and the cochlea by the conditioning, and that a small and short-lasting cortical BF shift can be caused by conditioning without collicular BF shift. The corticofugal system forms feedback loops not only through the IC but also through the medial geniculate body. It is likely that the cortical BF shift is augmented by both the cortico-thalamic and -collicular feedback loops. One of the critical experiments for further testing of Gao and Suga's hypothesis (1998), in which the BF shifts in the IC and medial geniculate body both are abolished during the conditioning with atropine and the cortical BF shift is examined, remains to be performed.

Several studies have demonstrated that the excitatory neurotransmitters in the cerebral cortex (Tsumoto 1990) and the neurotransmitters of the corticofugal neurons are glutamate and/or aspartate but not acetylcholine (Dori et al. 1992; Fonnum et al. 1981; Karlsen and Fonnum 1978; Nieoullon and Dusticier 1983; Tsumoto 1990). Therefore it is explainable that atropine applied to the AC (Fig. 9B) and the IC (Fig. 13A), respectively, did not affect cortical and collicular auditory responses and frequency-response curves, and it is expected that atropine applied to the AC or IC did not abolish the activity of the corticofugal fibers descending to and beyond the IC. Atropine applied to the AC prior to the 30-min-long conditioning completely abolishes the cortical BF shift but not the collicular BF shift (Figs. 9 and 10A). The collicular BF shift that would be caused by the 30-min-long conditioning is completely abolished by inactivation of the AC with muscimol (Gao and Suga 1998). These data clearly indicate that the collicular BF shift is not the result of the cortical BF shift and that it is evoked by the corticofugal system. As already discussed, the collicular BF shift is not a result of a BF shift that might be evoked in the subcollicular auditory nuclei and the cochlea by the conditioning. The data presented in our paper support the hypothesis proposed by Gao and Suga (1998, 2000).

Subcortical plasticity based on corticofugal modulation in the visual, somatosensory, and auditory systems

It has been well known that the auditory, visual, and somatosensory systems, respectively, have cochleotopic (frequency), retinotopic, and somatotopic maps in their central neural pathways and that these topographic maps are modified by deprivation, injury, and experience even in adult animals (reviews by Buonomano and Merzenich 1998; Irvine and Rajan 1996; Kaas et al. 1990; Weinberger 1998). Such plasticity has been explained by changes in divergent and convergent projections of the ascending sensory system. The contribution of the massive corticofugal system to the plasticity of sensory systems had been given little consideration until very recently although there had been a great deal of data demonstrating the corticofugal modulation of the responses of subcortical neurons to sensory stimuli.

In the visual system, the contribution of the corticofugal system to visual signal processing has been extensively studied (e.g., Murphy et al. 1999; Sillito et al. 1993, 1994; Tsumoto et al. 1978). However, reorganization of the retinotopic map (i.e., shift in receptive field) in the thalamus by the corticofugal system has not yet been shown.

In the somatosensory system, reorganization of the somatotopic map due to somatosensory experience is evident in the thalamus as well as in the cortex (Garraghty and Kaas 1991a,b; Pollin and Albe-Fessard 1979). A treatment of the somatosensory cortex for several months with an NMDA receptor agonist induces a large change in the somatotopic map in the thalamus (Ergenzinger et al. 1998). Inactivation of the somatosensory cortex by muscimol (GABA agonist) infused through a cannula immediately increases or decreases the size of the receptive fields of thalamic neurons (Krupa et al. 1999). These findings indicate that the corticofugal system modulates the thalamic somatotopic map.

In the auditory system, it has been known for a long time that electric stimulation of the AC evokes excitation or inhibition of collicular and thalamic auditory neurons (e.g., Jen et al. 1998; Sun et al. 1989, 1996; Watanabe et al. 1966). However, the finding that the corticofugal system shifts the tuning curves of subcortical neurons (i.e., reorganizes the physiological maps in the subcortical nuclei) was rather recent. Yan and Suga (1996) found that focal inactivation of the FM-FM area of the AC of the mustached bat with lidocaine immediately shifts the delay-response curves of collicular FM-FM neurons. Their finding indicates that the normal functional organization of the IC is maintained by the corticofugal system. They also found that focal activation of the FM-FM area with electric pulses shifts the delay-response curves of collicular neurons and that the shifts last a few hours after the cessation of the electric stimulation. Therefore it is clear that the corticofugal system is involved in the plasticity of the IC. The plasticity of the central auditory system has been further studied by Suga and his coworkers in relation to corticofugal modulation. It has been demonstrated that reorganization of the cochleotopic map in the IC is evoked by focal electric stimulation of the AC (Ma and Suga 2001; Yan and Suga 1998; Zhang and Suga 2000), repetitive acoustic stimulation (Gao and Suga 1998; Yan and Suga 1998), or auditory conditioning (Gao and Suga 1998, 2000). Our present data also indicate that the corticofugal system plays an important role in collicular plasticity.

Comparative studies with the mustached bat (Sakai and Suga 2001; Zhang and Suga 2000), big brown bat (Chowdhury and Suga 2000; Gao and Suga 1998, 2000; Ma and Suga 2001; Yan and Suga 1998), and Mongolian gerbil (Sakai and Suga 2001) indicate that reorganization of the cochleotopic maps in the IC and AC is different between species and between the specialized and nonspecialized areas of the same species (Sakai and Suga 2001; review by Suga et al. 2000).

Effects of ACh on neurons in the sensory cortices of non-bat species

ACh facilitates excitatory responses in the somatosensory cortex (Donoghue and Carroll 1987; Krujevic and Phillis 1963; Lamour et al. 1988; Metharate et al. 1987) and those in the visual cortex (Murphy and Sillito 1991; Sato et al. 1987; Sillito and Kemp 1983). Therefore the effect of ACh on these sensory cortices are the same as that on the AC (Figs. 4A and 5B). In the AC of cats, ACh evokes facilitation or suppression of auditory responses depending on frequencies of stimulus tones (McKenna et al. 1989; Metherate and Weinberger 1989, 1990). The data obtained from the AC of cats appear to be different from those obtained from the somatosensory and visual cortices. However, this is probably not the case. In our present experiments, augmentation of cortical auditory responses to tone bursts disappears 150 min after an ACh application (Fig. 4A). However, the cortical BF shift lasts much longer than 150 min (Fig. 8A). The BF shift (i.e., the shift in frequency-tuning curve) is evoked by a decrease in the response at the BF and an increase in the responses at other frequencies (Fig. 2B). Therefore our data are not contradictory at all to the data obtained from the cat's AC. In other words, augmentation of a response evoked by ACh and a shift in receptive field evoked by a sensory stimulus have different time courses from each other. Therefore no difference in ACh effect will be found between sensory cortices if the changes in response magnitude and receptive field are further studied as a function of time after an ACh application and a conditioning stimulus.

Cholinergic basal forebrain and cortical plasticity

It has been well established that the cholinergic basal forebrain releases ACh in the cerebral cortex, facilitates cortical responses to sensory stimuli, and causes cortical plasticity (review by Rasmusson 2000). This conclusion is based on a number of findings, enumerated in the following text. Cholinergic neurons in the basal forebrain project to the cerebral cortex (Semba and Fibiger 1989). Electrical stimulation of the basal forebrain elicits ACh release in the cortex (Casamenti et al. 1986; Kurosawa et al. 1989). Electrical stimulation of the basal forebrain evokes long-lasting facilitation of cutaneous responses in the somatosensory cortex (Howard and Simmons 1994; Rasmusson and Dykes 1988; Tremblay et al. 1990a,b; Webster et al. 1991) and auditory responses in the rat's AC (Edeline et al. 1994; Hars et al. 1993). It was also demonstrated that electric stimulation of the basal forebrain together with tone burst stimulation evokes over-representation of the frequency of the tone burst in the rat's AC (Kilgard and Merzenich 1998).

In the big brown bat, Ma and Suga (2000) found that cortical and collicular plasticities evoked by focal electric stimulation of the AC are augmented by electric stimulation of the basal forebrain. In our present paper, we demonstrated that subthreshold cortical and collicular plasticities caused by a 15-min-long auditory conditioning are augmented by ACh applied to the AC (Figs. 6B and 8A). Therefore our data further support the hypothesis that the cholinergic basal forebrain plays an important role in cortical plasticity by increasing cortical ACh level.


    ACKNOWLEDGMENTS

We thank N. Laleman for editing the manuscript.

This work was supported by a research grant from the National Institute on Deafness and Other Communication Disorders (DC-00175).

Present address of E. Gao: Dept. of Neurology, General Hospital of the Navy, Beijing 100037, P. R. China.


    FOOTNOTES

Address for reprint requests: N. Suga, Dept. of Biology, Washington University, One Brookings Dr., St. Louis, MO 63130 (E-mail: suga{at}biology.wustl.edu).

Received 6 November 2000; accepted in final form 27 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society