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Responses to species-specific vocalizations in the auditory cortex of awake and anesthetized guinea pigs
Hearing Research 206 (2005) 177–184 www.elsevier.com/locate/heares
Responses to species-speciWc vocalizations in the auditory cortex of awake and anesthetized guinea pigs Josef Syka a
a,¤
, Daniel Kuta
a,b
, Jilí Popelál
a
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videkská 1083, 142 20 Prague, Czech Republic b 3rd Faculty of Medicine, Charles University, Prague, Czech Republic Received 22 November 2004; accepted 14 January 2005 Available online 12 April 2005
Abstract Species-speciWc vocalizations represent an important acoustical signal that must be decoded in the auditory system of the listener. We were interested in examining to what extent anesthesia may change the process of signal decoding in neurons of the auditory cortex in the guinea pig. With this aim, the multiple-unit activity, either spontaneous or acoustically evoked, was recorded in the auditory cortex of guinea pigs, at Wrst in the awake state and then after the injection of anesthetics (33 mg/kg ketamine with 6.6 mg/kg xylazine). Acoustical stimuli, presented in free-Weld conditions, consisted of four typical guinea pig calls (purr, chutter, chirp and whistle), a time-reversed version of the whistle and a broad-band noise burst. The administration of anesthesia typically resulted in a decrease in the level of spontaneous activity and in changes in the strength of the neuronal response to acoustical stimuli. The eVect of anesthesia was mostly, but not exclusively, suppressive. Diversity in the eVects of anesthesia led in some recordings to an enhanced response to one call accompanied by a suppressed response to another call. The temporal pattern of the response to vocalizations was changed in some cases under anesthesia, which may indicate a change in the synaptic input of the recorded neurons. In summary, our results suggest that anesthesia must be considered as an important factor when investigating the processing of complex sounds such as species-speciWc vocalizations in the auditory cortex. 2005 Elsevier B.V. All rights reserved. Keywords: Anesthesia; Ketamine; Vocalization; Guinea pig; Auditory cortex; Multiple-unit activity
1. Introduction Most information about the function of the mammalian sensory systems (including the auditory system) has been accumulated in electrophysiological studies performed on anesthetized animals. Anesthesia, similarly as a state of vigilance (Edeline et al., 2001), can aVect sensory processing, therefore the investigator must be aware of the inXuences of anesthetics on neural processing and Abbreviations: AC, auditory cortex; DE, drug eVect; MU, multipleunit; PSTH, peri-stimulus time histogram ¤ Corresponding author. Tel.: +420 24106 2700; fax: +420 24106 2787. E-mail address: [email protected] (J. Syka). 0378-5955/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2005.01.013
the relevancy of the obtained data when interpreting results in a non-anesthetized animal. The Wrst studies of unit activity in the auditory cortex already demonstrated strong eVects of anesthesia. Anesthesia was found to reduce the number of units encountered by a micro-electrode (Katsuki et al., 1959) and to reduce the capacity of units to respond to auditory stimuli (Thomas, 1952; Erulkar et al., 1956). Several studies reported a mainly suppressive eVect of various anesthetics on spontaneous activity in diVerent subcortical nuclei (e.g., pentobarbital, chloralose, and halothane, Evans and Nelson, 1973; pentobarbital, Kuwada et al., 1989; ketamine and pentobarbital, Zurita et al., 1994), but less is known about the impact of anesthetics on sound-evoked activity in the auditory system and signal processing in neuronal
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circuits. Kisley and Gerstein (1999) reported that the variability of stimulus-evoked responses in the cortex is modulated by the depth of ketamine anesthesia. The authors showed that trial-to-trial variability was usually lowest under light anesthesia and highest under moderate anesthesia. Gaese and Ostwald (2001) found a loss of tuning in some neurons and a sharpening of the frequency response areas in other neurons in the auditory cortex (AC) of the rat after pentobarbital/chloral hydrate anesthesia. The eVect of anesthesia on the processing of acoustical information has been studied not only at the cortical level, but also at subcortical levels of the auditory system such as the cochlear nucleus (Anderson and Young, 2004), the inferior colliculus (Astl et al., 1996; Torterolo et al., 2002) and the medial geniculate body (Massaux et al., 2004). The eVects of anesthesia seem to be even more important when we attempt to understand the processing of sounds such as species-speciWc vocalizations. These calls are typically complex sounds characterized by timevarying amplitudes and spectral features (Syka et al., 1997; Kuta et al., 2003). It is possible that the processing of such sounds in the auditory cortex depends on the level of vigilance of the animal, especially in the case of calls with a high behavioral impact. The aim of this study was therefore to investigate the eVects of ketamine–xylazine anesthesia on the responses of neurons in the auditory cortex of the guinea pig to a set of spectrally and temporally diVerent complex sounds – guinea pig calls. The responses of multiple units in the auditory cortex of the guinea pig were recorded Wrst in an awake and weakly restrained animal and then after the injection of the anesthetic.
2. Methods 2.1. Animal preparation Experiments were performed on 12 adult, healthy, pigmented male guinea pigs, 3–9 months old (mean age 6 § 1.7 months), weighing 300–500 g. The care and use of animals reported in this study were approved by the Ethics Committee of the Institute of Experimental Medicine and followed the guidelines of the Declaration of Helsinki. 2.2. Recording of neuronal activity in the AC Neuronal activity in the AC was recorded by either of two procedures. In the Wrst procedure, four platinum– iridium electrodes (Bionic Technologies, impedance 0.5– 2 M) were implanted into the AC. Animals were anesthetized with an intramuscular injection of a mixture of 33 mg/kg ketamine (Narkamon 5%, Spofa) and
6.6 mg/kg xylazine (Sedazine 2%, Fort Dodge). The skin and underlying muscles on the skull were retracted to expose the dorsal cranium between points bregma and lambda. A small hole (diameter 5 mm) was made by a trephine in one side of the skull above the AC, and the electrode array was introduced into the AC through the dura mater and Wxed to the skull by acrylic resin. A small connector was Wxed to the dorsal skull by two screws, electrodes were soldered to the pins and the connector was secured to screws by acrylic resin. The exposed tissue was treated with an antibiotic (Framykoin, Spofa) to prevent inXammation, and the wound was sutured. The recording of neuronal activity was performed at least 10 days after the surgery. In the second procedure, a hole (diameter 5 mm) was made in the skull above the AC by the same procedure as described above. A small plastic tube was Wxed by two screws above the hole as a support. The wound was treated with antibiotic, covered by the tissue, and the support Wlled with isotonic solution and plugged. A few days after the surgery a miniature mechanical electrode driver was Wxed on the support to insert the electrode array (four epoxylite insulated tungsten electrodes, impedance 0.4–2 M) into the AC. Both types of recording resulted in the same responses of multiple units, therefore the results of the recordings are presented together. Some animals were used in two or three experimental sessions. In both types of experiments the neuronal activity was recorded in guinea pigs placed in a plastic box, securing their heads by a sliding ring over the nose. This type of Wxation enabled the animal’s head to be free for electrode penetration and for free-Weld acoustical stimulation. The neuronal responses were recorded Wrst in an awake and weakly restrained animal and then after the intramuscular injection of the anesthetic. At the beginning of the experiment the syringe needle was preWxed into the leg muscle to minimize manipulations with the animal during the later anesthetic injection. A DC-powered electric heating pad maintained body temperature at 37–38 °C. The signal from the electrodes was ampliWed by a custom made four-channel diVerential ampliWer and bandpass Wltered in the range of 300 Hz to 10 kHz (Wlter slopes 12 dB/octave). The signal was transmitted via a Cambridge Electronic Design (CED 1401plus) interface into a PC computer running the Spike2 program, where the activity was saved and later analyzed. The neuronal activity was recorded simultaneously from one or more microelectrodes in the form of multiple-unit (MU) activity. 2.3. Acoustic stimulation Electrophysiological recordings were made in a soundproof anechoic room. The walls and ceiling inside the room were covered by cones from phono-absorbent material; the attenuation was 55 dB at 250 Hz and 60–70
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dB for frequencies above 500 Hz. Acoustical stimuli were delivered in free-Weld conditions via a two-way loudspeaker system (Tesla ARN 5614 woofer and Motorola KSN-1005 tweeter) placed 70 cm in front of the animal’s head. The acoustic system was calibrated with a B&K 4133 microphone, placed in the position of the animal’s head and facing the speakers. The set of applied stimuli consisted of a broadband noise (BBN) burst (duration 100 ms with 3 ms rise/fall times), four typical guinea pig calls (purr, chutter, chirp and whistle) and time-reversed whistle. Vocalization calls were chosen from the large variety of guinea pig natural calls (11 distinct calls according to Harper, 1976). Calls were previously tape recorded from spontaneously vocalizing female guinea pigs (age 2–24 months) in a sound-attenuated room. The temporal and spectral parameters of the calls and their variability have been described in Syka et al. (1997) and Kuta et al. (2003). Purr consists of a series of regular low-frequency impulses (fundamental frequency around 300 Hz). The most complex sound is whistle, which is a long-lasting frequency- and amplitude-modulated sound consisting of many harmonics over a wide frequency range. Chutter is a sequence of irregular noise bursts, and chirp is an isolated brief acoustic impulse with a harmonic structure. The time-reversed whistle was generated by reversing the time course of the natural whistle call. All other parameters of the call, such as the sound level or the repetition rate, were preserved. Sounds were presented in a pseudorandom order, once every 2.9 s at a maximal eVective sound level of 75 dB SPL from a TDT System3 setup including an RP2 signal processing unit (sampling rate 50 kHz) and PA5 attenuator. Such stimulation helps to eliminate the eVect of habituation and ensures better response stability over time compared to using individual stimuli step by step. 2.4. Data analysis The neuronal responses were recorded Wrst in an awake animal, then the anesthetic was injected intramuscularly (ketamine 33 mg/kg and xylazine 6.6 mg/kg). The next series of neuronal responses was recorded at least 5 min after the injection. The Wnal evaluation of neuronal responses were processed with Matlab software. The following parameters of the response were evaluated: The level of the spontaneous Wring rate calculated as the total number of spikes normalized per 1 s of each neuron and evaluated from 500 ms periods preceding the onset of each stimulus. The peri-stimulus time histograms (PSTH) were computed separately for each type of stimulus with a 5-ms bin width. The response strength was expressed as the driven Wring rate, which was calculated as the total number of spikes over the stimulus duration, shifted by the
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response latency and normalized per 1 stimulus and per 1 s with the spontaneous Wring rate subtracted. The eVect of the drug application DE (drug eVect) was evaluated for each type of stimulus as DE D (Ranest. ¡ Rawake)/(Ranest. + Rawake), where Ranest. is the response strength in an anesthetized animal and Rawake is the response strength in an awake animal. The relative DE index was calculated for each type of stimulus as the value of the DE minus the mean DE of all six sounds for a particular MU. All statistical tests were performed using GraphPad Prism 4 software. 2.5. Histological control At the end of the experiments, the guinea pigs were sacriWced with an overdose of pentobarbital (Pentobarbital, Spofa, 200 mg/kg) and perfused with 10% formaldehyde. The brains were sectioned in the frontal plane on a freezing microtome (slice thickness 40 m) and stained with cresyl violet. The subsequent reconstruction of individual electrode positions within the AC from histological sections conWrmed that all recorded neurons were located at a depth of 1000–1500 m in the primary auditory cortex AI.
3. Results The results are based on 20 multiple-unit (MU) recordings in 12 guinea pigs for which reliable records were obtained in both states, i.e., awake as well as anesthetized. The spontaneous activity under anesthesia was positively correlated with the level of spontaneous activity in awake animals (R2 D 0.64). Ketamine–xylazine anesthesia reduced spontaneous neural activity on average to 79% of the activity level present in an awake animal (means of 16.4 spikes/s in awake animals vs. 13.0 spikes/s in anesthesia, medians of 12.8 vs. 9.7 spikes/s) (Fig. 1). The slope of the linear regression line is signiWcantly less than 1 corresponding to the diagonal (p < 0.01). SigniWcant diVerences (p < 0.01, Mann–Whitney test) were found in 8 MUs; in seven of them, less spontaneous activity was observed under anesthesia while in one case less activity was present in the awake animal. Fig. 2 shows examples of the peri-stimulus histograms (PSTHs) of responses in 1 MU to individual stimuli before and after the administration of anesthesia. Although there was almost no response to whistle (Fig. 2A) or to reversed whistle (Fig. 2B) in the awake animal (left plots), strong responses appeared under anesthesia (right plots) to both of these stimuli. The diVerence in response strength was expressed using the parameter DE, where positive values of DE correspond to a stronger response under anesthesia, zero or near zero values of DE are present when the response remains stable
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Fig. 1. Comparison of the spontaneous activity in awake (abscissa) and anesthetized (ordinate) animals. Each dot represents 1 MU (n D 20).
irrespective of the vigilance state, and negative values means a suppression of the response after the administration of the anesthesia. For Figs. 2A and B, the DE values were 0.85 and 0.74, respectively. Interestingly, exactly the opposite trend was observed in the same MU for purr (Fig. 2D) and chutter (Fig. 2E). The responses in the awake animal were much stronger in comparison with those obtained under anesthesia, DEs were ¡0.45 and ¡0.34, respectively. Fig. 3 summarizes the results for all MUs. Symbols represent the DE values for diVerent types of acoustical stimuli. The DE values for individual stimuli measured in each MU are displayed in one row (ordinate), the MUs are arranged according to the mean DE calculated from all six stimuli. The majority of negative values (73% of all DEs) demonstrates that typically, but not always, a weaker response was observed in anesthetized animals than in awake animals. The DEs are qualitatively divided into three distinct groups: near zero (with limits of ¡0.25 and 0.25), clearly negative (below the near-zero zone) and clearly positive (above the near-zero zone). All MUs demonstrated a strong modulation of the response (resulting in an DE value outside the near-zero interval) for at least one sound. Nineteen MUs, i.e., all but one, displayed a clearly negative DE for one or more stimuli while 9 MUs produced a clearly positive DE for at least one type of stimulus. A schematic classiWcation on the basis of the distribution of DEs in individual MUs is shown in Table 1. The DEs ranged from near-zero to clearly positive in 1 MU, from near-zero to clearly negative in 8 MUs, they were only clearly negative in 3 MUs, and in 8 MUs the DEs ranged from clearly negative to clearly positive (an example of the last case is illustrated in Fig. 2). As regards the eVect of anesthesia on the responses to individual sounds, the average DE for all sounds was
Fig. 2. Comparison of responses to whistle (A), reversed whistle (B), chirp (C), purr (D), and chutter (E) in an awake animal (left side) and an anesthetized animal (right side). The value of the DE is shown for each type of stimulus. The sound waveform is shown below each PSTH.
negative, but for all types of sound stimuli positive DEs were also observed: in the case of chirp this was seen in 8 MUs and for the remaining stimuli in 3–5 MUs (Table 2). The responses to chirp were the least inXuenced by anes-
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Fig. 3. Summary of the DEs for all stimuli in all MU. DE values for individual stimuli are shown for each MU – each row shows data from 1 MU. To highlight the range of observed DEs in each MU, the symbols representing DE for individual calls are connected with a dotted line. The numbers indicate CF values (in kHz) for each MU; thresholds at the CF were 10–30 dB SPL for all MUs. Individual MUs are arranged according to the mean DE for all six stimuli.
Table 1 ClassiWcation of individual MUs based on the interval of DEs as obtained for individual calls
The horizontal lines are a schematic illustration of the DE ranges and their classiWcation into individual separate categories: clearly negative, near zero and clearly positive.
thesia with the lowest average DE (mean ¡0.09, median ¡0.18), the most negative average DE value (i.e., the greatest suppression by the anesthesia) was observed for purr (mean ¡0.46, median ¡0.41). When the relative DE index was calculated for each stimulus and each MU by subtracting the mean DE for a particular MU, the chirp value was positive (0.20) while the lowest average relative DE value was again for purr (¡0.10). A change in the response to acoustical stimuli induced by anesthesia may appear not only in the response strength, but also in the temporal response pattern. This phenomenon is illustrated in Fig. 2C, where the precise timing in the response in an awake animal has been
Fig. 4. Examples of changes in the temporal response pattern illustrated by PSTHs in 4 MU. The left plot presents data in an awake animal; the right one in an anesthetized animal. The sound waveform is shown below each PSTH.
changed into a much broader PSTH when the animal was anesthetized. Four other examples measured in three animals are shown in Fig. 4. Fig. 4A demonstrates a case in
Table 2 DE statistics for individual sound stimuli
Whistle Reversed whistle Chirp Purr Chutter BBN
# of negative DEs
# of positive DEs
Mean DE
Mean relative DE
16 (80%) 17(85%) 12 (60%) 15 (75%) 16 (80%) 16 (80%)
4 (20%) 3 (15%) 8 (40%) 5 (25%) 4 (20%) 4 (20%)
¡0.25 ¡0.29 ¡0.09 ¡0.46 ¡0.32 ¡0.23
0.01 ¡0.06 0.20 ¡0.10 ¡0.04 ¡0.01
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Fig. 5. Comparison of responses (PSTHs) to chirp in an awake animal (A), during anesthesia (B) and a repeated recording in the awake state two days later (C) in 1 MU.
which a sustained type of response to whistle seen in an awake animal (left plot) changed to a more complex response in the anesthetized animal, with decreased activity in the central part (»0.3 s) of the PSTH and enhanced oVset activity (>0.6 s) (right panel). Similarly, the response to reversed whistle in the same MU (Fig. 4B), represented by sustained activity during the entire duration of the stimulus in the awake animal, was transformed into a more complex pattern consisting of a sequence of three bursts of activity in the PSTH in the anesthetized guinea pig. Fig. 4C shows an example of a response to broadband noise stimuli in which the administration of anesthesia changed a sustained response to a predominantly oVset response. Finally, a change of an on–oV response to chirp to a sustained burst of activity under anesthesia is illustrated in Fig. 4D. DiVerences in the temporal response patterns between PSTHs recorded in awake animals and those recorded after the injection of anesthetics were observed in approximately two-thirds of cases. The stability of the neuronal activity was controlled during the entire experiment. First, the spike shapes were evaluated by the template procedure in the Spike2 program to conWrm that the units recorded before and after anesthesia injection were the same. Second, the consistency of single responses was analyzed to ensure that there was not any response instability due to adaptation, habituation, etc. The regression lines calculated for individual responses recorded during the preinjection or postinjection period did not diVer from a horizontal line. Finally, the long-term stability of the recording was conWrmed in two animals with implanted electrodes by repeated recordings over the course of several days. An example in Fig. 5 shows a change in the response to chirp when the animal was anesthetized (A – awake state vs. B – anesthetized state), and C illustrates the response obtained 2 days later.
4. Discussion The results of our experiments demonstrate a signiWcant inXuence of ketamine–xylazine anesthesia on the responses of neurons in the auditory cortex of the guinea
pig to complex acoustical stimuli such as species-speciWc vocalizations. The strength of the response and the temporal pattern of the response are modiWed in many AC neurons. The eVect is, however, not uniform since the administration of anesthesia may increase the response to some types of stimuli and suppress the response to others. The suppressive eVect of anesthesia is, however, more frequently seen than augmentation. Neuronal activity in the AC was recorded either by four implanted platinum–iridium electrodes or, in acute experiments, by an electrode array introduced into the brain with a miniature mechanical electrode driver. Both methods have advantages and disadvantages. Implanted electrodes enabled long-term repeated recordings with a relatively stable neuronal activity during the recording. The recorded activity usually consisted of spikes of relatively small amplitude originating from several (more than 3) neurons. However, the signal quality deteriorated during several weeks of testing, probably caused by the creation of small lesions around the electrode tips. The second method based on the introduction of an electrode array with a miniature mechanical electrode driver in acute experiments made it possible to determine an electrode position with a good signal. In this case, two or three separate spike types were possible to discriminate reliably. However, the time of recording was limited to several tens of minutes, allowing the recording of only pre-injection and immediate post-injection responses. The average level of spontaneous neural activity was reduced by ketamine–xylazine anesthesia to 79% in comparison with the pre-injection level. A similar suppression of spontaneous neural activity by ketamine was also demonstrated in the rat somatosensory cortex (Patel and Chapin, 1990). It has been shown previously that Equithesin (pentobarbital and chloral hydrate) reduces the spontaneous neural activity in the rat auditory cortex to 20.5% of the awake activity level (Gaese and Ostwald, 2001). A pronounced suppression of spontaneous activity in the inferior colliculus of the cat after the injection of pentobarbital was described by Bock and Webster (1974). A larger suppressive eVect of pentobarbital in comparison with ketamine–xylazine anesthesia was also demonstrated by Astl et al. (1996). The authors found signiWcantly
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higher number of spontaneously active neurons in the guinea pig inferior colliculus under ketamine–xylazine anesthesia than in guinea pigs under pentobarbital anesthesia. The diVerences in the degree of suppression between ketamine and pentobarbital can be caused by diVerences in the action mechanisms of both drugs. Whereas pentobarbital is a -aminobutyric acid (GABA) agonist (Willow and Johnston, 1983), ketamine is a dissociative anesthetic belonging to a group of non-competitive N-methyl-D-aspartate (NMDA) sub-type glutamate receptor antagonists (Thomson et al., 1985; Franks and Lieb, 1994). The diversity of response changes at one recording site can be associated with a change in synaptic inputs, which can alter the spectro-temporal response Weld and so modify responses to complex stimuli such as the calls used in this study. A wide variety of response patterns to acoustical stimuli was described in the Wrst experiments in which single units were recorded in the auditory cortex of non-anesthetized cats (Gerstein and Kiang, 1964; Evans and WhitWeld, 1964). Elhilali et al. (2002) studied spectro-temporal response Welds in awake and ketamineanesthetized ferrets using dynamic, broadband stimuli. They found more complex receptive Weld shapes, more complex spectral processing and increased selectivity in the direction of frequency modulation in awake animals. Among classical narrow V-shaped tuning curves, mostly found in anaesthetized preparations, complex forms of frequency response areas with several separate subregions in many cortical neurons were detected in the auditory cortex of awake rats (Gaese and Ostwald, 2003). These studies reported a higher spike rate in awake animals than under anesthesia, which corresponds with the results of our present study. Most suppressed in our experiments were responses to purr, i.e., to a burst of short low-frequency impulses. It is possible that purr has a strong behavioral impact that is recognizable only in a fully awake state. Already in 1959, Hubel et al. described “attention” units in the auditory cortex of unrestrained and unanesthetized cats that appear to be sensitive to auditory stimuli only if the cat “pays attention” to the sound source. However, it is more likely that ketamine– xylazine anesthesia reduces the responsiveness to a series of acoustical impulses (purr) and inXuences to a lesser extent isolated impulses (chirp). Patel and Chapin (1990) suggested two separable eVects of ketamine in the somatosensory cortex: (i) a strong inhibition of all somatosensory responsiveness and (ii) a tonic excitatory inXuence expressed heterogeneously on a subgroup of neurons. This coexistence of excitation and suppression in the same cortical region can contribute to the diversity of response changes in individual cortical neurons. Cortical neurons perform the Wnal analysis of sound stimuli using complex neural circuits. Kanwal et al. (2002) suggested that there are dual functions within individual cortical neurons for vocal communication
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and also for echolocation in the mustached bat. Anesthesia could unbalance the complex synaptic inputs in the cortex, which can result in dramatic changes in the response strength and/or temporal response pattern as found in this study. Zurita et al. (1994) observed in the auditory thalamus and cortex of the cat after the injection of ketamine an increase in tonal selectivity due to a decrease in the response bandwidth. In complex sounds such as animal vocalizations, a decrease in the response bandwidth may result in a decrease in neuronal responsiveness as found in the present study. The eVect of anesthesia involves the processing of acoustical information not only at the cortical level, but also in the subcortical stages of the auditory system such as the cochlear nucleus (Anderson and Young, 2004), inferior colliculus (Kuwada et al., 1989; Astl et al., 1996; Torterolo et al., 2002) or medial geniculate body (Cotillon-Williams and Edeline, 2003; Massaux et al., 2004). Therefore, the modiWcation of cortical activity can result not only from the direct eVect of an anesthetic agent on intracortical processing, but also from modiWed subcortical activity or, most likely, from a combination of subcortical and intracortical eVects. Anesthesia is an important factor in experimental neuroscience that must be considered in the interpretation of results and the evaluation of their relevancy. The signiWcant eVects of anesthesia demonstrated in this study argue for the use of awake and unrestrained animals, which, while respecting all ethical aspects of experimental work, seems to be an important step in adequately addressing key issues such as the neural representation of complex acoustical signals (Wang, 2000).
Acknowledgments The study was supported by the Grant Agency of the Czech Republic (GA CR No. 309/04/1074) and the Grant Agency of the Ministry of Health of the Czech Republic (NR 8113-4).
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Responses to species-speciWc vocalizations in the auditory cortex of awake and anesthetized guinea pigs Josef Syka a
a,¤
, Daniel Kuta
a,b
, Jilí Popelál
a
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videkská 1083, 142 20 Prague, Czech Republic b 3rd Faculty of Medicine, Charles University, Prague, Czech Republic Received 22 November 2004; accepted 14 January 2005 Available online 12 April 2005
Abstract Species-speciWc vocalizations represent an important acoustical signal that must be decoded in the auditory system of the listener. We were interested in examining to what extent anesthesia may change the process of signal decoding in neurons of the auditory cortex in the guinea pig. With this aim, the multiple-unit activity, either spontaneous or acoustically evoked, was recorded in the auditory cortex of guinea pigs, at Wrst in the awake state and then after the injection of anesthetics (33 mg/kg ketamine with 6.6 mg/kg xylazine). Acoustical stimuli, presented in free-Weld conditions, consisted of four typical guinea pig calls (purr, chutter, chirp and whistle), a time-reversed version of the whistle and a broad-band noise burst. The administration of anesthesia typically resulted in a decrease in the level of spontaneous activity and in changes in the strength of the neuronal response to acoustical stimuli. The eVect of anesthesia was mostly, but not exclusively, suppressive. Diversity in the eVects of anesthesia led in some recordings to an enhanced response to one call accompanied by a suppressed response to another call. The temporal pattern of the response to vocalizations was changed in some cases under anesthesia, which may indicate a change in the synaptic input of the recorded neurons. In summary, our results suggest that anesthesia must be considered as an important factor when investigating the processing of complex sounds such as species-speciWc vocalizations in the auditory cortex. 2005 Elsevier B.V. All rights reserved. Keywords: Anesthesia; Ketamine; Vocalization; Guinea pig; Auditory cortex; Multiple-unit activity
1. Introduction Most information about the function of the mammalian sensory systems (including the auditory system) has been accumulated in electrophysiological studies performed on anesthetized animals. Anesthesia, similarly as a state of vigilance (Edeline et al., 2001), can aVect sensory processing, therefore the investigator must be aware of the inXuences of anesthetics on neural processing and Abbreviations: AC, auditory cortex; DE, drug eVect; MU, multipleunit; PSTH, peri-stimulus time histogram ¤ Corresponding author. Tel.: +420 24106 2700; fax: +420 24106 2787. E-mail address: [email protected] (J. Syka). 0378-5955/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2005.01.013
the relevancy of the obtained data when interpreting results in a non-anesthetized animal. The Wrst studies of unit activity in the auditory cortex already demonstrated strong eVects of anesthesia. Anesthesia was found to reduce the number of units encountered by a micro-electrode (Katsuki et al., 1959) and to reduce the capacity of units to respond to auditory stimuli (Thomas, 1952; Erulkar et al., 1956). Several studies reported a mainly suppressive eVect of various anesthetics on spontaneous activity in diVerent subcortical nuclei (e.g., pentobarbital, chloralose, and halothane, Evans and Nelson, 1973; pentobarbital, Kuwada et al., 1989; ketamine and pentobarbital, Zurita et al., 1994), but less is known about the impact of anesthetics on sound-evoked activity in the auditory system and signal processing in neuronal
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circuits. Kisley and Gerstein (1999) reported that the variability of stimulus-evoked responses in the cortex is modulated by the depth of ketamine anesthesia. The authors showed that trial-to-trial variability was usually lowest under light anesthesia and highest under moderate anesthesia. Gaese and Ostwald (2001) found a loss of tuning in some neurons and a sharpening of the frequency response areas in other neurons in the auditory cortex (AC) of the rat after pentobarbital/chloral hydrate anesthesia. The eVect of anesthesia on the processing of acoustical information has been studied not only at the cortical level, but also at subcortical levels of the auditory system such as the cochlear nucleus (Anderson and Young, 2004), the inferior colliculus (Astl et al., 1996; Torterolo et al., 2002) and the medial geniculate body (Massaux et al., 2004). The eVects of anesthesia seem to be even more important when we attempt to understand the processing of sounds such as species-speciWc vocalizations. These calls are typically complex sounds characterized by timevarying amplitudes and spectral features (Syka et al., 1997; Kuta et al., 2003). It is possible that the processing of such sounds in the auditory cortex depends on the level of vigilance of the animal, especially in the case of calls with a high behavioral impact. The aim of this study was therefore to investigate the eVects of ketamine–xylazine anesthesia on the responses of neurons in the auditory cortex of the guinea pig to a set of spectrally and temporally diVerent complex sounds – guinea pig calls. The responses of multiple units in the auditory cortex of the guinea pig were recorded Wrst in an awake and weakly restrained animal and then after the injection of the anesthetic.
2. Methods 2.1. Animal preparation Experiments were performed on 12 adult, healthy, pigmented male guinea pigs, 3–9 months old (mean age 6 § 1.7 months), weighing 300–500 g. The care and use of animals reported in this study were approved by the Ethics Committee of the Institute of Experimental Medicine and followed the guidelines of the Declaration of Helsinki. 2.2. Recording of neuronal activity in the AC Neuronal activity in the AC was recorded by either of two procedures. In the Wrst procedure, four platinum– iridium electrodes (Bionic Technologies, impedance 0.5– 2 M) were implanted into the AC. Animals were anesthetized with an intramuscular injection of a mixture of 33 mg/kg ketamine (Narkamon 5%, Spofa) and
6.6 mg/kg xylazine (Sedazine 2%, Fort Dodge). The skin and underlying muscles on the skull were retracted to expose the dorsal cranium between points bregma and lambda. A small hole (diameter 5 mm) was made by a trephine in one side of the skull above the AC, and the electrode array was introduced into the AC through the dura mater and Wxed to the skull by acrylic resin. A small connector was Wxed to the dorsal skull by two screws, electrodes were soldered to the pins and the connector was secured to screws by acrylic resin. The exposed tissue was treated with an antibiotic (Framykoin, Spofa) to prevent inXammation, and the wound was sutured. The recording of neuronal activity was performed at least 10 days after the surgery. In the second procedure, a hole (diameter 5 mm) was made in the skull above the AC by the same procedure as described above. A small plastic tube was Wxed by two screws above the hole as a support. The wound was treated with antibiotic, covered by the tissue, and the support Wlled with isotonic solution and plugged. A few days after the surgery a miniature mechanical electrode driver was Wxed on the support to insert the electrode array (four epoxylite insulated tungsten electrodes, impedance 0.4–2 M) into the AC. Both types of recording resulted in the same responses of multiple units, therefore the results of the recordings are presented together. Some animals were used in two or three experimental sessions. In both types of experiments the neuronal activity was recorded in guinea pigs placed in a plastic box, securing their heads by a sliding ring over the nose. This type of Wxation enabled the animal’s head to be free for electrode penetration and for free-Weld acoustical stimulation. The neuronal responses were recorded Wrst in an awake and weakly restrained animal and then after the intramuscular injection of the anesthetic. At the beginning of the experiment the syringe needle was preWxed into the leg muscle to minimize manipulations with the animal during the later anesthetic injection. A DC-powered electric heating pad maintained body temperature at 37–38 °C. The signal from the electrodes was ampliWed by a custom made four-channel diVerential ampliWer and bandpass Wltered in the range of 300 Hz to 10 kHz (Wlter slopes 12 dB/octave). The signal was transmitted via a Cambridge Electronic Design (CED 1401plus) interface into a PC computer running the Spike2 program, where the activity was saved and later analyzed. The neuronal activity was recorded simultaneously from one or more microelectrodes in the form of multiple-unit (MU) activity. 2.3. Acoustic stimulation Electrophysiological recordings were made in a soundproof anechoic room. The walls and ceiling inside the room were covered by cones from phono-absorbent material; the attenuation was 55 dB at 250 Hz and 60–70
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dB for frequencies above 500 Hz. Acoustical stimuli were delivered in free-Weld conditions via a two-way loudspeaker system (Tesla ARN 5614 woofer and Motorola KSN-1005 tweeter) placed 70 cm in front of the animal’s head. The acoustic system was calibrated with a B&K 4133 microphone, placed in the position of the animal’s head and facing the speakers. The set of applied stimuli consisted of a broadband noise (BBN) burst (duration 100 ms with 3 ms rise/fall times), four typical guinea pig calls (purr, chutter, chirp and whistle) and time-reversed whistle. Vocalization calls were chosen from the large variety of guinea pig natural calls (11 distinct calls according to Harper, 1976). Calls were previously tape recorded from spontaneously vocalizing female guinea pigs (age 2–24 months) in a sound-attenuated room. The temporal and spectral parameters of the calls and their variability have been described in Syka et al. (1997) and Kuta et al. (2003). Purr consists of a series of regular low-frequency impulses (fundamental frequency around 300 Hz). The most complex sound is whistle, which is a long-lasting frequency- and amplitude-modulated sound consisting of many harmonics over a wide frequency range. Chutter is a sequence of irregular noise bursts, and chirp is an isolated brief acoustic impulse with a harmonic structure. The time-reversed whistle was generated by reversing the time course of the natural whistle call. All other parameters of the call, such as the sound level or the repetition rate, were preserved. Sounds were presented in a pseudorandom order, once every 2.9 s at a maximal eVective sound level of 75 dB SPL from a TDT System3 setup including an RP2 signal processing unit (sampling rate 50 kHz) and PA5 attenuator. Such stimulation helps to eliminate the eVect of habituation and ensures better response stability over time compared to using individual stimuli step by step. 2.4. Data analysis The neuronal responses were recorded Wrst in an awake animal, then the anesthetic was injected intramuscularly (ketamine 33 mg/kg and xylazine 6.6 mg/kg). The next series of neuronal responses was recorded at least 5 min after the injection. The Wnal evaluation of neuronal responses were processed with Matlab software. The following parameters of the response were evaluated: The level of the spontaneous Wring rate calculated as the total number of spikes normalized per 1 s of each neuron and evaluated from 500 ms periods preceding the onset of each stimulus. The peri-stimulus time histograms (PSTH) were computed separately for each type of stimulus with a 5-ms bin width. The response strength was expressed as the driven Wring rate, which was calculated as the total number of spikes over the stimulus duration, shifted by the
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response latency and normalized per 1 stimulus and per 1 s with the spontaneous Wring rate subtracted. The eVect of the drug application DE (drug eVect) was evaluated for each type of stimulus as DE D (Ranest. ¡ Rawake)/(Ranest. + Rawake), where Ranest. is the response strength in an anesthetized animal and Rawake is the response strength in an awake animal. The relative DE index was calculated for each type of stimulus as the value of the DE minus the mean DE of all six sounds for a particular MU. All statistical tests were performed using GraphPad Prism 4 software. 2.5. Histological control At the end of the experiments, the guinea pigs were sacriWced with an overdose of pentobarbital (Pentobarbital, Spofa, 200 mg/kg) and perfused with 10% formaldehyde. The brains were sectioned in the frontal plane on a freezing microtome (slice thickness 40 m) and stained with cresyl violet. The subsequent reconstruction of individual electrode positions within the AC from histological sections conWrmed that all recorded neurons were located at a depth of 1000–1500 m in the primary auditory cortex AI.
3. Results The results are based on 20 multiple-unit (MU) recordings in 12 guinea pigs for which reliable records were obtained in both states, i.e., awake as well as anesthetized. The spontaneous activity under anesthesia was positively correlated with the level of spontaneous activity in awake animals (R2 D 0.64). Ketamine–xylazine anesthesia reduced spontaneous neural activity on average to 79% of the activity level present in an awake animal (means of 16.4 spikes/s in awake animals vs. 13.0 spikes/s in anesthesia, medians of 12.8 vs. 9.7 spikes/s) (Fig. 1). The slope of the linear regression line is signiWcantly less than 1 corresponding to the diagonal (p < 0.01). SigniWcant diVerences (p < 0.01, Mann–Whitney test) were found in 8 MUs; in seven of them, less spontaneous activity was observed under anesthesia while in one case less activity was present in the awake animal. Fig. 2 shows examples of the peri-stimulus histograms (PSTHs) of responses in 1 MU to individual stimuli before and after the administration of anesthesia. Although there was almost no response to whistle (Fig. 2A) or to reversed whistle (Fig. 2B) in the awake animal (left plots), strong responses appeared under anesthesia (right plots) to both of these stimuli. The diVerence in response strength was expressed using the parameter DE, where positive values of DE correspond to a stronger response under anesthesia, zero or near zero values of DE are present when the response remains stable
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Fig. 1. Comparison of the spontaneous activity in awake (abscissa) and anesthetized (ordinate) animals. Each dot represents 1 MU (n D 20).
irrespective of the vigilance state, and negative values means a suppression of the response after the administration of the anesthesia. For Figs. 2A and B, the DE values were 0.85 and 0.74, respectively. Interestingly, exactly the opposite trend was observed in the same MU for purr (Fig. 2D) and chutter (Fig. 2E). The responses in the awake animal were much stronger in comparison with those obtained under anesthesia, DEs were ¡0.45 and ¡0.34, respectively. Fig. 3 summarizes the results for all MUs. Symbols represent the DE values for diVerent types of acoustical stimuli. The DE values for individual stimuli measured in each MU are displayed in one row (ordinate), the MUs are arranged according to the mean DE calculated from all six stimuli. The majority of negative values (73% of all DEs) demonstrates that typically, but not always, a weaker response was observed in anesthetized animals than in awake animals. The DEs are qualitatively divided into three distinct groups: near zero (with limits of ¡0.25 and 0.25), clearly negative (below the near-zero zone) and clearly positive (above the near-zero zone). All MUs demonstrated a strong modulation of the response (resulting in an DE value outside the near-zero interval) for at least one sound. Nineteen MUs, i.e., all but one, displayed a clearly negative DE for one or more stimuli while 9 MUs produced a clearly positive DE for at least one type of stimulus. A schematic classiWcation on the basis of the distribution of DEs in individual MUs is shown in Table 1. The DEs ranged from near-zero to clearly positive in 1 MU, from near-zero to clearly negative in 8 MUs, they were only clearly negative in 3 MUs, and in 8 MUs the DEs ranged from clearly negative to clearly positive (an example of the last case is illustrated in Fig. 2). As regards the eVect of anesthesia on the responses to individual sounds, the average DE for all sounds was
Fig. 2. Comparison of responses to whistle (A), reversed whistle (B), chirp (C), purr (D), and chutter (E) in an awake animal (left side) and an anesthetized animal (right side). The value of the DE is shown for each type of stimulus. The sound waveform is shown below each PSTH.
negative, but for all types of sound stimuli positive DEs were also observed: in the case of chirp this was seen in 8 MUs and for the remaining stimuli in 3–5 MUs (Table 2). The responses to chirp were the least inXuenced by anes-
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Fig. 3. Summary of the DEs for all stimuli in all MU. DE values for individual stimuli are shown for each MU – each row shows data from 1 MU. To highlight the range of observed DEs in each MU, the symbols representing DE for individual calls are connected with a dotted line. The numbers indicate CF values (in kHz) for each MU; thresholds at the CF were 10–30 dB SPL for all MUs. Individual MUs are arranged according to the mean DE for all six stimuli.
Table 1 ClassiWcation of individual MUs based on the interval of DEs as obtained for individual calls
The horizontal lines are a schematic illustration of the DE ranges and their classiWcation into individual separate categories: clearly negative, near zero and clearly positive.
thesia with the lowest average DE (mean ¡0.09, median ¡0.18), the most negative average DE value (i.e., the greatest suppression by the anesthesia) was observed for purr (mean ¡0.46, median ¡0.41). When the relative DE index was calculated for each stimulus and each MU by subtracting the mean DE for a particular MU, the chirp value was positive (0.20) while the lowest average relative DE value was again for purr (¡0.10). A change in the response to acoustical stimuli induced by anesthesia may appear not only in the response strength, but also in the temporal response pattern. This phenomenon is illustrated in Fig. 2C, where the precise timing in the response in an awake animal has been
Fig. 4. Examples of changes in the temporal response pattern illustrated by PSTHs in 4 MU. The left plot presents data in an awake animal; the right one in an anesthetized animal. The sound waveform is shown below each PSTH.
changed into a much broader PSTH when the animal was anesthetized. Four other examples measured in three animals are shown in Fig. 4. Fig. 4A demonstrates a case in
Table 2 DE statistics for individual sound stimuli
Whistle Reversed whistle Chirp Purr Chutter BBN
# of negative DEs
# of positive DEs
Mean DE
Mean relative DE
16 (80%) 17(85%) 12 (60%) 15 (75%) 16 (80%) 16 (80%)
4 (20%) 3 (15%) 8 (40%) 5 (25%) 4 (20%) 4 (20%)
¡0.25 ¡0.29 ¡0.09 ¡0.46 ¡0.32 ¡0.23
0.01 ¡0.06 0.20 ¡0.10 ¡0.04 ¡0.01
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Fig. 5. Comparison of responses (PSTHs) to chirp in an awake animal (A), during anesthesia (B) and a repeated recording in the awake state two days later (C) in 1 MU.
which a sustained type of response to whistle seen in an awake animal (left plot) changed to a more complex response in the anesthetized animal, with decreased activity in the central part (»0.3 s) of the PSTH and enhanced oVset activity (>0.6 s) (right panel). Similarly, the response to reversed whistle in the same MU (Fig. 4B), represented by sustained activity during the entire duration of the stimulus in the awake animal, was transformed into a more complex pattern consisting of a sequence of three bursts of activity in the PSTH in the anesthetized guinea pig. Fig. 4C shows an example of a response to broadband noise stimuli in which the administration of anesthesia changed a sustained response to a predominantly oVset response. Finally, a change of an on–oV response to chirp to a sustained burst of activity under anesthesia is illustrated in Fig. 4D. DiVerences in the temporal response patterns between PSTHs recorded in awake animals and those recorded after the injection of anesthetics were observed in approximately two-thirds of cases. The stability of the neuronal activity was controlled during the entire experiment. First, the spike shapes were evaluated by the template procedure in the Spike2 program to conWrm that the units recorded before and after anesthesia injection were the same. Second, the consistency of single responses was analyzed to ensure that there was not any response instability due to adaptation, habituation, etc. The regression lines calculated for individual responses recorded during the preinjection or postinjection period did not diVer from a horizontal line. Finally, the long-term stability of the recording was conWrmed in two animals with implanted electrodes by repeated recordings over the course of several days. An example in Fig. 5 shows a change in the response to chirp when the animal was anesthetized (A – awake state vs. B – anesthetized state), and C illustrates the response obtained 2 days later.
4. Discussion The results of our experiments demonstrate a signiWcant inXuence of ketamine–xylazine anesthesia on the responses of neurons in the auditory cortex of the guinea
pig to complex acoustical stimuli such as species-speciWc vocalizations. The strength of the response and the temporal pattern of the response are modiWed in many AC neurons. The eVect is, however, not uniform since the administration of anesthesia may increase the response to some types of stimuli and suppress the response to others. The suppressive eVect of anesthesia is, however, more frequently seen than augmentation. Neuronal activity in the AC was recorded either by four implanted platinum–iridium electrodes or, in acute experiments, by an electrode array introduced into the brain with a miniature mechanical electrode driver. Both methods have advantages and disadvantages. Implanted electrodes enabled long-term repeated recordings with a relatively stable neuronal activity during the recording. The recorded activity usually consisted of spikes of relatively small amplitude originating from several (more than 3) neurons. However, the signal quality deteriorated during several weeks of testing, probably caused by the creation of small lesions around the electrode tips. The second method based on the introduction of an electrode array with a miniature mechanical electrode driver in acute experiments made it possible to determine an electrode position with a good signal. In this case, two or three separate spike types were possible to discriminate reliably. However, the time of recording was limited to several tens of minutes, allowing the recording of only pre-injection and immediate post-injection responses. The average level of spontaneous neural activity was reduced by ketamine–xylazine anesthesia to 79% in comparison with the pre-injection level. A similar suppression of spontaneous neural activity by ketamine was also demonstrated in the rat somatosensory cortex (Patel and Chapin, 1990). It has been shown previously that Equithesin (pentobarbital and chloral hydrate) reduces the spontaneous neural activity in the rat auditory cortex to 20.5% of the awake activity level (Gaese and Ostwald, 2001). A pronounced suppression of spontaneous activity in the inferior colliculus of the cat after the injection of pentobarbital was described by Bock and Webster (1974). A larger suppressive eVect of pentobarbital in comparison with ketamine–xylazine anesthesia was also demonstrated by Astl et al. (1996). The authors found signiWcantly
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higher number of spontaneously active neurons in the guinea pig inferior colliculus under ketamine–xylazine anesthesia than in guinea pigs under pentobarbital anesthesia. The diVerences in the degree of suppression between ketamine and pentobarbital can be caused by diVerences in the action mechanisms of both drugs. Whereas pentobarbital is a -aminobutyric acid (GABA) agonist (Willow and Johnston, 1983), ketamine is a dissociative anesthetic belonging to a group of non-competitive N-methyl-D-aspartate (NMDA) sub-type glutamate receptor antagonists (Thomson et al., 1985; Franks and Lieb, 1994). The diversity of response changes at one recording site can be associated with a change in synaptic inputs, which can alter the spectro-temporal response Weld and so modify responses to complex stimuli such as the calls used in this study. A wide variety of response patterns to acoustical stimuli was described in the Wrst experiments in which single units were recorded in the auditory cortex of non-anesthetized cats (Gerstein and Kiang, 1964; Evans and WhitWeld, 1964). Elhilali et al. (2002) studied spectro-temporal response Welds in awake and ketamineanesthetized ferrets using dynamic, broadband stimuli. They found more complex receptive Weld shapes, more complex spectral processing and increased selectivity in the direction of frequency modulation in awake animals. Among classical narrow V-shaped tuning curves, mostly found in anaesthetized preparations, complex forms of frequency response areas with several separate subregions in many cortical neurons were detected in the auditory cortex of awake rats (Gaese and Ostwald, 2003). These studies reported a higher spike rate in awake animals than under anesthesia, which corresponds with the results of our present study. Most suppressed in our experiments were responses to purr, i.e., to a burst of short low-frequency impulses. It is possible that purr has a strong behavioral impact that is recognizable only in a fully awake state. Already in 1959, Hubel et al. described “attention” units in the auditory cortex of unrestrained and unanesthetized cats that appear to be sensitive to auditory stimuli only if the cat “pays attention” to the sound source. However, it is more likely that ketamine– xylazine anesthesia reduces the responsiveness to a series of acoustical impulses (purr) and inXuences to a lesser extent isolated impulses (chirp). Patel and Chapin (1990) suggested two separable eVects of ketamine in the somatosensory cortex: (i) a strong inhibition of all somatosensory responsiveness and (ii) a tonic excitatory inXuence expressed heterogeneously on a subgroup of neurons. This coexistence of excitation and suppression in the same cortical region can contribute to the diversity of response changes in individual cortical neurons. Cortical neurons perform the Wnal analysis of sound stimuli using complex neural circuits. Kanwal et al. (2002) suggested that there are dual functions within individual cortical neurons for vocal communication
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and also for echolocation in the mustached bat. Anesthesia could unbalance the complex synaptic inputs in the cortex, which can result in dramatic changes in the response strength and/or temporal response pattern as found in this study. Zurita et al. (1994) observed in the auditory thalamus and cortex of the cat after the injection of ketamine an increase in tonal selectivity due to a decrease in the response bandwidth. In complex sounds such as animal vocalizations, a decrease in the response bandwidth may result in a decrease in neuronal responsiveness as found in the present study. The eVect of anesthesia involves the processing of acoustical information not only at the cortical level, but also in the subcortical stages of the auditory system such as the cochlear nucleus (Anderson and Young, 2004), inferior colliculus (Kuwada et al., 1989; Astl et al., 1996; Torterolo et al., 2002) or medial geniculate body (Cotillon-Williams and Edeline, 2003; Massaux et al., 2004). Therefore, the modiWcation of cortical activity can result not only from the direct eVect of an anesthetic agent on intracortical processing, but also from modiWed subcortical activity or, most likely, from a combination of subcortical and intracortical eVects. Anesthesia is an important factor in experimental neuroscience that must be considered in the interpretation of results and the evaluation of their relevancy. The signiWcant eVects of anesthesia demonstrated in this study argue for the use of awake and unrestrained animals, which, while respecting all ethical aspects of experimental work, seems to be an important step in adequately addressing key issues such as the neural representation of complex acoustical signals (Wang, 2000).
Acknowledgments The study was supported by the Grant Agency of the Czech Republic (GA CR No. 309/04/1074) and the Grant Agency of the Ministry of Health of the Czech Republic (NR 8113-4).
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