Comparison of tonal response properties of primary auditory cortex neurons of adult rats under urethane and ketamine anesthesia

doi 10.3969/j.issn.1673-4254.2013.06.02 785 Original Article Comparison of tonal response properties of primary auditory cortex neurons of adult rats under urethane and ketamine anesthesia HUANG Lingyue, BAI Lin, ZHAO Yan, XIAO Zhongju* Department of Physiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China Abstract: Objective To compare tonal response properties of neurons in the primary auditory cortex of Sprague-Dawley rats anesthetized with urethane and ketamine-xylazine. Methods Forty-five female Sprague-Dawley rats (200-250 g) were randomized into two groups and anesthetized with urethane or ketamine-xylazine. Tone pips were chosen as the stimuli to obtain the action potentials of the single neurons by in vivo cell-attached recording. The features of the action potentials were extracted with Matlab software to comparatively analyze the acoustic response properties of the neurons between the two anesthetic groups. Results The Q values and the characteristic frequencies were independent of the types of anesthetic agents, but with urethane anesthesia, the neurons tended to have higher minimum thresholds, lower spontaneous firing rates, longer response latencies, and more frequent occurrence of tuning with stronger inhibition compared to those in ketamine-xylazine group. Conclusion Urethane and ketamine might have no obvious impact on the transmission pathway of frequency tuning from the periphery to the auditory cortex, but neurons from rats with urethane anesthesia receive enhanced inhibition mediated by the interneurons or have a lower intrinsic excitability. Key words: urethane; ketamine; primary auditory cortex; cell-attached recording INTRODUCTION Anesthetized animals are widely used in scientific experiments for their stability and controllability. However, anesthetic agents can influence the functionality and responses of the body, especially in the sensory and nervous systems. Many previous studies have been conducted to explore the changes of auditory processing in anesthetic condition. A functional magnetic resonance imaging study has proved that propofol dose-dependently attenuate responses of the [1] auditory cortex to acoustic stimulation. Electrophysiological experiments have also shown the changes of response characteristics (such as the spontaneous rate, response latency, tuning sharpness, and minimum threshold) of individual auditory neurons under different anesthesia protocols [2-3]. Non-volatile anesthetics, such as pentobarbital sodium [2-6], urethane [7-11], and ketamine [12-15], are commonly used in auditory experiments. Pentobarbital sodium has been demonstrated to produce hyperpolarizing effect by activating gammaaminobutyric acid (GABA), and reduces the firing rates of neurons in the inferior colliculus (IC) and the auditory Received: 2013-04-22 Accepted: 2013-05-09 Supported by National Natural Science Foundation of China (31171059). HUANG Lingyue and BAI Lin contributed equally to this work. *Corresponding author: XIAO Zhongju, Professor, E-mail: xiaozj@fimmu.com cortex (AC) [4, 16]. In addition, the response patterns and receptive areas of AC in most neurons also change under anesthesia with barbiturates [5]. Thus pentobarbital does not seem to be an ideal anesthetic for studies on AC. Ketamine is a rapid-acting anesthetic and used primarily for the induction and maintenance of general anesthesia, usually in combination with a sedative such as xylazine or medetomidine. As a noncompetitive N-methyl-d-aspartate (NMDA) receptors antagonist, ketamine produces an anesthetic state termed dissociative anesthesia [17], because it appears to selectively block signal transmission within the association pathway of the brain. Electroencephalography (EEG) showed that ketamine, in contrast to barbiturates, depressed the recruiting response when thalamic-neocortical activation was minimally affected [18], suggesting the less impact of ketamine on the response properties of the AC neurons. The disadvantage of ketamine is its short-term anesthetic effect to require additional doses for maintenance in long-term recording in vivo [19]. Urethane, with a long-lasting effect and less influence on breathing, is suitable for prolonged studies [7-9]. The molecular and cellular mechanisms of urethane are intricate and still poorly understood. Earlier studies have shown that urethane can enhance the inhibitory currents (γ-aminobutyric acid type A, GABA A and glycine currents) and the function of neuronal acetylcholine (nach) receptors, and simultaneously inhibit current responses of the excitatory receptors

786 (NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, AMPA receptors) [11, 20]. The dual effects of urethane on excitatory and inhibitory inputs may consequentially influence the integration of neural outputs. Previous extracellular study has also demonstrated the same effects of urethane on neurons in the IC [19]. The aforementioned electrophysiological studies of anesthetics for their impact on the auditory responses were conducted using mostly extracellular recording methods. The characteristic properties of individual neurons should be analyzed by sorting single unit responses from multiple units, through the shape features of action potentials [21]. The influence by the neighboring neurons can not be totally eliminated using this approach. In recent years, the development of in vivo patch-clamp techniques provides a more direct and accurate means to obtain the activities of single cells. By forming a high seal resistance (10 MΩ to >5 GΩ) between the pipette tip and the cell membrane, the method of cell-attached recording overcomes the disadvantage of extracellular sorting, and maintains a stable condition for the attached cell within several hours [22]. So far few studies have been reported to address the varying effects of different anesthetics on the auditory responses of single neurons using in vivo cell-attached recording techniques. We therefore conducted this study to investigate the diversities in the frequency-intensity receptive areas (FIRAs) and the response patterns of individual neurons in the primary auditory cortex (A1) to tone bursts in rats anesthetized with urethane or ketamine-xylazine (the mixture of ketamine and xylazine, K-X). The characteristic frequency (CF), minimum response threshold (MT), bandwidth (BW) and Q values of FIRA, spontaneous rates and response latencies were compared between the two anesthesia conditions, which may provide evidence for choice of anesthetics in further electrophysiological studies of the auditory system. MATERIALS AND METHODS Animals Forty-five healthy adult female Sprague-Dawley rats (200-250 g) were purchased from the Animal Experiment Center of Southern Medical University. All the surgical procedures were approved by the Animal Care and Use Committee of Southern Medical University. Surgical procedures The rats were randomized into urethane group and K-X group. Atropine sulfate (1.2 mg/kg) was administered intraperitoneally before the surgery to reduce bronchial secretions in both groups. In urethane group, the rats received an intraperitoneal dose of urethane (1.2-1.5 mg/kg) followed by tracheotomy and a cisternal drainage of the cerebrospinal fluid to minimize brain edema. The rats were then fixed by a head-holder device, and a rectal probe was deployed to record the body temperature (which was maintained around 37.3 using a homeothermic blanket system). Craniotomy was performed over the right auditory cortex (a 4 4 mm 2 window over A1 was carefully exposed under a surgical microscope) with the dura dissected. A tiny hole about 0.2-0.3 mm 2 was drilled in the right parietal bone to place the reference electrode. The right ear canal was plugged with agarose (30 g/l). Heart rate, respiratory rate, and corneal reflex were monitored to ensure that a moderately deep anesthetic state was maintained during the whole recording procedure. Artificial cerebrospinal fluid (ACSF) was administered periodically to prevent desiccation of the pial surface throughout the experiment. Low melting point paraffin was provided to cover the surgical sites to reduce the fluctuation of the brain during the recording. The rats in the K-X group were anesthetized with the mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg) and anesthesia was maintained with supplemental doses at half of the initial doses every 1.5 h. The other operations were identical to those in the urethane group. In vivo cell-attached recording procedures All the following procedures were conducted in an electromagnetic shielding, shockproof, no reverberation, and sound-insulated chamber at controlled temperatures (24-28 ). Cell-attached recording electrodes were prepared from thick-walled borosilicate glass caplillaries using a micropipette puller (Sutter, P-97) and filled with ACSF (containing, in mmol/l, 132 NaCl, 20 NaHCO 3, 2.5 KCl, 1.2 NaH 2PO 4, 1.1 MgSO 4, 2 CaCl 2, 3 HEPES and 15 D-glucose) with 5-8 MΩ resistances. Blind recording was used throughout the experiment. The microelectrode (within 3 Psi positive pressures to avoid potential electrode tip blockage when penetrating the pia mater) was placed orthogonally above a selected point on the surface of A1 under the surgical microscope, and was advanced into the cortex with a remote-controlled micromanipulator (Narishige, SM-21). After electrode penetration to 100 μm below the pial surface, liquid paraffin was administered to prevent cortical pulsation, and the positive pressure within the electrode was reduced to 0.3 Psi. White noise was burst out to detect the acoustic response neurons during the electrode penetration. Once a neuron was probed, the positive pressure within the electrode was revoked, and 0.3 Psi suction was applied; the seal resistance between the recording pipette and the cell membrane was increased to 20 MΩ-0.5 GΩ to allow recording of the spikes only from the patched cell. After a few minutes for the patched cell to stabilize, the stimulus was changed into tone pips at a delivery rate of 2/s, and recording was initiated under voltage-clamp mode without an input current. Stimulus generation and delivery The acoustic signals were programmed, generated, and delivered with a Tucker-Davis Technologies System

787 3 apparatus (TDT 3, Tucker-Davis Technologies). An array of tones (2-64 khz at 0.1 octave intervals, 50 ms duration, 5 ms raised ramp; 0-70 db SPL, 10 db SPL step) and white noise (70 db, 50 ms duration, and 5 ms ramps) were used as stimuli in this study. Acoustic stimuli were synthesized using a real-time processor (RP2.1) and a customized program (written with RPvdsEx software) and loaded into a programmable attenuator (PA5). The signals recorded were amplified by an electrostatic speaker driver (ED1) and delivered to the rat via a free-field loudspeaker (ES1, 2-110 khz) placed 10 cm to the contralateral (left) ear. Acoustic calibration was performed with 1/8 and 1/4 inch microphones (Brüel and Kjaer 4138, 4135, Naerum, Denmark) and an amplifier (Brüel and Kjaer 2610, Naerum, Denmark) at the beginning of the experiment to ensure a flat frequency response (± 2 db SPL) of the loudspeaker's output. Signals collected by a low-noise data acquisition system (Axon, Digidata 1440A) were amplified (2000-10, 000 ), band-pass filtered at 0.3-5 khz, sampled at 20 khz with a digital amplifier (Axon, Multiclamp 700B), recorded and displayed with BrainWare software (which was also used to control the signal parameters, either manually or within the programming parameters). Data acquisition and analysis The acquired data were processed online using the TDT 3 system and BrainWare software. Offline analysis was done with Matlab 7.0 software, SPSS 13.0 statistical software, Photoshop 7.0, Origin 8.0 and Canvas 9.0 for different purposes. The relevant parameters of FIRAs, including CF, MT, BW, Q 10 and Q 30 (a CF divided by the BW at 10- and 30- db SPL above MT) as well as such parameters of the post-stimulus time histograms (PSTHs) as spontaneous firing rate, minimum response latency (L min), and peak latency (L peak) were analyzed. CF is the most sensitive frequency of an auditory neuron, which reflects the frequency response properties. In the current study, MT was defined as the lowest sound level at which responses in CF condition could be evoked. Combined with CF, MT represents the sensitivity of a neuron to acoustic stimuli. FIRAs of each neuron were obtained from 5 randomly selected presentations of the pure tone pips. Neural responses with a signal-noise ratio greater than 4: 1 were selected as action potential data for analysis. The pcolors (the color indicating the amount of responses to a given tone) of FIRAs were drawn by Matlab software, and the envelope curves of each neuron with obvious FIRA were outlined at the level of 30% of the neuron's largest spike count by Matlab 7.0. The simulative curves were taken as the frequency-threshold curves. The horizontal and ordinate values of the lowest point of each curve were defined as CF and MT of the neuron, respectively. BW 10dB and BW 30dB were the bandwidth 10- and 30- db SPL above MT of the curve, respectively, as shown in Fig.1A. PSTHs were generated from the same tone pips above, and with the 2 ms time bin. The X-axis represented the recording time window of a given tone, which began at 10 ms before acoustical stimuli bursting and ended at 200 ms after starting of stimuli. The Y-axis represented the amount of responses in each time bin to 5 presentations of tone pips. The spontaneous rate of each neuron was calculated from both the first 10 ms (stimuli not presented) and the last 40 ms. There was no significant difference between these two measurement methods (P=0.156 by paired t-test). L min was defined as the first bin over 2 standard deviations above the mean of spontaneous rate of each neuron, and L peak was the highest bin of PSTH. Statistical analysis The data were shown as Mean±SE. Mann-Whitney test was applied for comparing the distribution of different FIRA patterns and the response patterns between the two groups. Independent-sample t-test was used for comparing CF distributions between the two groups. Two-way ANOVA was used to compare the differences in MT and Q values, and Kruskal-Wallis test to compare spontaneous firing rate and L min between the two groups. The significance level of all hypothesis testing was set at α=0.05. RESULTS General description Complete data were obtained from 117 wellisolated single neurons collected from 45 rats. Among these neurons, 62 were recorded in 25 rats in urethane group and 55 in 20 rats in K-X group. Recording sites distributed over a small area about 4-6 mm posterior to the bregma and 1.5-2.5 mm below the edge of the temporal bone, known as the A1 area. Scatter plots of the recording sites showed no regional selectivity between the two groups (data not shown). The recording depth was between 200 and 1200 μm below the pial surface with the seal resistances ranged from 20 MΩ to 250 MΩ (48.9±3.5 MΩ), and only the signals from the target neuron were recorded [23]. The ensemble and mean of action potential waveforms recorded in an example neuron was displayed in Fig.1G. Note the uniformity of the waveforms, which confirmed the homogeneity of the action potentials. FIRA patterns in the two anesthesia conditions FIRA reveals the tonal receptive field of individual neuron according to the responses of the neuron to tone stimuli with different frequencies and intensities. In this study, the FIRAs were represented by pcolor maps, with the color indicating the amount of responses to a given tone. Five different FIRA shapes were recorded from the rat A1: U/V-shaped, multi-peak, closed, atypical and untuned (examples are shown in Fig.1). According to the customized standard of group, neurons with FIRA resembling a single-peak "V" or "U" were sorted into V/ U-shaped type (Fig.1A). Multi-peak neurons (Fig.1B) had FIRAs with a closed bottom and an open top to form

788 a multi-peak shape. Neurons showing a non-monotonic response to sound intensity, with a closed FIRA shape, were defined as the closed type (Fig.1C). Atypical neurons (Fig.1D) exhibited a visible inhibitory region among a wider excitatory area, referred to also as inhibitory type in other studies. Neurons which responded to the broadband stimuli but showed no selectivity to any frequency/intensity combination were classified as Untuned (Fig.1E). The distribution of the FIRA patterns of A1 neurons showed significant difference between the two anesthesia conditions (Fig.1F, P<0.05 by Mann-Whitney test). The urethane group contained a greater proportion of V/U-shaped (45/ 62, 72.58%), closed (5/62, 8.06%) and untuned types (7/ 62, 11.29% ) compared with the K-X group (31/55, 53.4% ; 2/55, 3.6% ; 4/55, 7.3%, respectively). Conversely, multi-peak (14/65, 25.4% ) and atypical neurons (4/55, 7.3%) appeared more frequently in K-X group than urethane group (4/62, 6.45% ; 1/62, 1.61%, respectively). A B C D E F Fig.1 Examples of FIRA patterns and their distribution in the two groups. A: FIRA pcolor of a typical V/ U-shape neuron. Red profile indicates the simulative envelope curve of the FIRA. Color bar represents the amount of responses to a given tone with 5 repeats. The horizon and ordinate values of the lowest point of the curve separately display the CFand MT of the neurons (except for atypical and untuned types). Three dash lines from the bottom up indicate the MT, 10- and 30- db SPL above MT, respectively. Other FIRA patterns are shown in B (multi-peak), C (closed), D (atypical) and E (untuned) in the same manner. F: Distribution of different FIRA patterns in the two groups (white for urethane and black for K-X groups). Asterisks indicate a significant difference (P<0.05, Mann-Whitney test). G: The ensemble and mean of action potential waveforms recorded by in vivo cell-attached recording in an A1 neuron are displayed by blue lines and the red line separately. Influence of two anesthetics on CF and MT As shown in Fig.1A, the coordinate values of the lowest point in the envelop curve indicated CF (29.8 khz) and MT (26.5 db SPL) of the neuron. No accurate values of CF and MT were obtainable from atypical and

789 untuned neurons, therefore 54 of 62 neurons in the urethane group and 47 of 55 in the K-X group were analyzed. As shown by open circles in Fig.2, under urethane anesthesia condition, CFs of A1 neurons ranged from 6.5- to 48.5- khz (28.0±1.5 khz) while CFs ranged from 7.0- to 55.7- khz (28.1 ± 1.6 khz) in the K-X group. There was no significant difference in CFs distributions between the two groups (P=0.972, independent-sample t-test), suggesting that neither of the anesthetics obviously disturbed the auditory cortical neurons' frequency selectivity inherited from periphery auditory system. Meanwhile, the distributions and means of MTs were significantly different between the two conditions (urethane: 0- to 60.2- db SPL, 22.4±2.7 db SPL vs K-X: 0- to 53.5- db SPL, 13.8±2.8 db SPL). To distinguish the diversity of MT between the two groups precisely, the FIRA patterns considered as a fixed factor. The results showed that FIRAs of A1 neurons were turned to higher tone levels by urethane anesthesia than by K-X (P=0.007, Two-Way ANOVA), indicating that A1 neurons tended to have a lower excitability under urethane anesthesia. Influence of the two anesthetics on Q values For a given CF, Q values (Q=CF/BW) are dependent on BW. We compared the influence of the two different anesthetics on Q values (Q 10 and Q 30). In urethane group, Q 10 and Q 30 were both calculated in 46 neurons whose CF ranged from 6.5 to 48.6 khz (29.0± 1.6 khz); in K-X group, 45 neurons were analyzed with CF ranged from 7.0 to 55.7 khz (28.5 ± 1.6 khz). No MT (db SPL) 70 60 50 40 30 20 10 Urethane K-X Mean of urethane Mean of K-X 0 0 20 40 60 CF (khz) Fig.2 Distribution of MT with corresponding CF of each neuron recorded from A1 in the two groups. Open circles indicate 54 neurons in the urethane group, and solid circles represent 47 neurons in K-X group. Open and solid pentagram marks indicate the means under urethane and K-X anesthesia conditions, respectively. The diversity of MT between two groups shows significant difference (P=0.007, Two-Way ANOVA). significant difference was found in CF distribution between the two groups (P=0.841, independent-sample t-test), so that the the influence of the diversity of CF on Q values could be eliminated in different anesthesia conditions. As shown in Fig.3, although the neurons in the urethane group showed lower Q values, neither Q 10 (urethane: 2.7±0.3 khz/db vs K-X: 3.1±0.3 khz/db) nor Q 30 (urethane: 1.9±0.2 khz/db vs K-X=2.1±0.2 khz/db) displayed any remarkable difference between the two groups (P>0.05, Two-way ANOVA). Q10 (khz/db SPL) 9 6 3 A B Urethane 6 K-X Mean of urethane Mean of K-X Q10 (khz/db SPL) 4 2 0 0 20 40 60 CF (khz) 0 0 20 40 60 CF (khz) Fig.3 Distribution of Q10 and Q30 with corresponding CF of individual neurons recorded from A1 in different anesthetic conditions. A: Distribution of Q10. Open circles represent 46 neurons in urethane group, and solid circles represent 45 neurons in K-X group. The open and the solid pentagram marks indicate the means under urethane and K-X anesthesia, respectively. B: Distribution of Q30. No significant difference was found in Q values between the two groups (P>0.05, Two-way ANOVN). Temporal response properties of neurons under the two narcotics Spontaneous rate of a neuron could reflect excitability of the neuron. While, anesthetic conditions do have some impact on the excitability. The first 10 ms discharges before the tone stimuli presentation were chosen as the time window to evaluate the diversity of spontaneous rate of each neuron between the two groups (arrows in Fig.4B). The mean of spontaneous rate was 2.5±0.6 spikes/s in urethane group, and 5.6±1.2 spikes/ s in K-X group. Apparently, neurons in urethane anesthetized animals tended to have lower spontaneous rate (P=0.028, Kruskal-Wallis test) and therefore lower

790 excitability. Response latency to acoustic stimulus of an auditory neuron can reflect the integrated outputs of both excitatory and inhibitory inputs of the neuron. First, we calculated L min of each neuron in the two groups (Fig.4A). L min in the urethane group was 19.7 ± 2.3 ms, significantly longer than that in K-X group (12.7 ± 1.2 ms, P=0.007, Kruskal-Wallis test). The response temporal pattern of the A1 neurons was sorted into different types according to L min and L peak. In principle, in the urethane group, auditory neurons in A1 could be divided into 4 groups: (1) Onset type (34/ 62, 54.8%, Fig.4B) with L min shorter than 30 ms ; (2) Long-latency type (7/62, 11.3%, Fig.4C) in which the neurons responded later than 30 ms after the tone burst, with a L peak shorter than 80 ms; (3) Rebound type (6/62, 9.7%, Fig.4D) in which the neurons displayed a postinhibitory rebound only with L min longer than 30 ms and L peak longer than 80 ms; (4) Onset combined with rebound type (Onset-Rebound; 15/62, 24.2%) with onset responses followed by a postinhibitory rebound (Fig.4E). Neurons recorded in the K-X group also exhibited onset type (53/55, 96.4% ), rebound type (1/55, 1.8% ), onset-rebound type (1/55, 1.8% ), but no long-latency type. Most of the neurons revealed onset responses within 30 ms after tone burst (79.0% in urethane group, and 98.2% in K-X group regardless of the rebound composition occurrence), and comparison of L min of onset compositions showed no obvious difference between urethane and K-X groups (12.0±0.6 ms vs 11.6± 0.4 ms, P=0.534 by Kruskal-Wallis test). However, as shown in Fig.4F, the distribution of the response patterns under the two conditions displayed a distinct diversity (P<0.05, Mann-Whitney test). Neurons recorded under K-X anesthesia contained a greater proportion of onset type, and the other types appeared sparse compared to those of the urethane group. Both of the anesthetics appeared to affect the temporal patterns of responses for A1 neurons to tone stimuli. The A1 neurons under urethane anesthesia seemed to receive more depressed excitatory inputs or inhibitory inputs than those under K-X anesthesia, resulting in greater probabilities of later responses or postinhibitory rebound. DISCUSSION The data we obtained demonstrate that some response parameters of A1 neurons to tone stimuli may differ in rats treated with different anesthetics. The V/ U-shaped FIRA represents the dominant response pattern of both anesthetics, consistent with the previous reports [24-25]. The CFs of FIRAs showed no statistical difference between the two groups. However, the probabilities of the 5 FIRA patterns varied significantly between the two groups: the V/U-shaped and closedtype neurons appeared more frequently with urethane anesthesia, and the multi-peak type more frequently with K-X anesthesia. Since the FIRA of an auditory neuron is mainly drawn by thalamocortical excitatory inputs and sharpened by cortical lateral inhibition [26-27], the V/U-shaped and the closed types, compared with the multi-peak type, seem to be a result of stronger lateral inhibition [23]. This suggests that these two anesthetics produce no obvious impact on the transmission pathway of frequency tuning from the periphery to the auditory cortex, but the neurons with urethane anesthesia received enhanced lateral inhibition mediated by activating GABA receptors of the interneurons, leading to more sharpening FIRA. The higher MT in the urethane group could further confirm this presumption. The Q value is quantitative descriptor of sharpness of FIRA. The higher Q values, the more sharply tuned the neurons are when CFs keep consistent [28]. The BW of the auditory nerve fiber was reported to be approximately consistent with that of psychophysics [26], and at the IC level, this consistency elevates to a great extent [29]. In this study, although the Q values were slightly higher in urethane group than in K-X group, no statistical correlation was found between the Q values and the anesthetics. These results differ from the viewpoint mentioned above, for U/V-shaped and closed types were more common with urethane and multi-peak type more frequent with K-X. This discrepancy arose, probably, from the relatively small sample size in our study. PSTH principally represented the integration of excitatory and the inhibitory inputs. Generally, neurons sensitive to acoustic stimuli are highly spontaneously active [30]. The lower spontaneous rate in urethaneanesthetized rats further verifies that urethane more strongly reduces the sensitivity to sound stimuli than K-X. With constant stimuli parameters (stimulus amplitude, rise time, and spectrum at the excitatory ear), the transmission pathway originating from the periphery and extending to the cortex is the determining factor of response latency [30]. Our data showed that the onset-type neurons in K-X-anesthetized rats were more numerous than those in urethane-anesthetized rats, but the longlatency neurons, observed in the IC in both group [19], appeared to be more frequent in rats anesthetized by urethane. It is assumed that urethane can enhance the inhibitory input in the auditory pathway. Scholfield pointed out that ketamine and urethane could double the inhibition postsynaptic potentials (IPSP) duration [31], and previous studies have shown that enhancement of inhibition could change the response pattern [32], such as lengthened response latency [33], which were supported by our data. The frequent occurrence of long-latency neurons in urethane-treated rats might involve the cellular and synaptic mechanisms of the two anesthetics. Hara and Harris demonstrated that urethane significantly potentiated the currents of the inhibitory receptors (GABA A receptors and glycine receptors), inhibited the current responses of the excitatory receptors (NMDA receptors and AMPA receptors) in a reversible and concentration-dependent manner in Xenopus oocytes transfected with several recombinant neurotransmistter receptors, and non-selectively affected the excitatory and inhibitory currents [11, 20]. In addition, Schreiner et al maintained that urethane's action on neocortical pyramidal cells in rat visual cortex could be thought of as a tonic reduction of intrinsic excitability through a

791 A B C D E F Fig.4 Temporal response properties of neurons recorded from A1 and distribution of temporal response patterns under different anesthesia. A: Histograms of Lmin of each neuron in urethane (white) and K-X (black) groups; B: PSTH of a typical onset type neuron with a 2 ms time bin. The X-axis represents the recording time window of a given tone, and the Y-axis the amount of responses in each time bin to 5 presentations of tone pips. Red dash line indicates the 2 standard deviations above mean of spontaneous rate of the neuron, and the time window (arrows) was the first 10 ms discharges before the tone stimuli presentation. The bar under PSTH marks the 50 ms duration of tones. Three other types, namely long-latency (C), rebound (D), and onset-rebound (E) are listed in sequence. F: Distribution of different types in the two groups. Asterisks indicate significant differences (P<0.05, Mann-Whitney test). specific K + back-ground leak conductance [34]. Based on the data of this study, we might assumed that urethane can both enhance the inhibitory input of acoustic response neurons and depress the neurons' intrinsic excitability. Ketamine is reported to inhibit the channel function of NMDA receptors and nach receptors [17, 35], and prolong polysynaptic excitation [36-37]. Presumably, urethane, compared with ketamine-xylazine, has a wider range of inhibition to contribute to the greater frequency of long-latency neurons. Scholl et al emphasized that the excitatory and inhibitory currents were evoked by tone onset, and the occurrence of rebound was not correlated with the inhibitory currents [38]. Our finding that rebound and onset-rebound types, common in rats dosed with urethane, can confirm to some degree the assumption that urethane produces stronger inhibition on auditory pathway. Besides, some in vivo electrophysiological experiments demonstrated that both urethane and ketamine could depress the responsiveness of neurons in the visual cortex [34, 39-40], motor cortex and sensory cortex [41-43], indicating the nonspecific inhibitory effect of these two

792 anesthetics on the auditory cortex. In conclusion, FIRA patterns, Q values and CFs of the auditory neurons in A1 exhibit no obvious diversities in rats dosed with the two anesthetics, demonstrating that urethane and ketamine do not obviously affect the transmission pathway of frequency tuning from the periphery to the auditory cortex. Urethane produces stronger inhibition on the neurons than K-X in view of lower spontaneous rate and higher MT. These two anesthetics have nonspecific inhibitory effect on the auditory cortex. Taken together, our data indicate that when investigating acoustic response characteristics of the neurons in the auditory pathway, at least in A1, anesthetic factors should be carefully evaluated. REFERENCES [1] Dueck MH, Petzke F, Gerbershagen HJ, et al. Propofol attenuates responses of the auditory cortex to acoustic stimulation in a dose-dependent manner: a FMRI study[j]. Acta Anaesthesiol Scand, 2005, 49(6): 784-91. [2] Cohen MS, Britt RH. 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793 乌拉坦和氯胺酮麻醉条件下大鼠初级听皮层听觉神经元对纯音 反应特性的比较 反应特性的比 较 黄玲玥 柏 林 赵 岩 肖中举 南方医科大学基础医学院生理教研室 广东 广州 510515 摘要 目的 用在体细胞贴附式记录的方式 比较乌拉坦和氯胺酮麻醉条件下初级听皮层听觉神经元对纯音的反应特性 探查两 种麻醉剂对初级听皮层单个神经元纯音编码机制的影响 方法 选取 45 只体质量 200~250 g 的 Sprague-Dawley 雌性健康大鼠 将其随机分为两组 分别用乌拉坦和氯胺酮作为基础麻醉剂 手术暴露初级听皮层 选取短纯音作为刺激条件 用在体细胞贴 附式记录的方式获得大鼠初级听皮层的单个听觉神经元动作电位发放情况 并用Matlab软件提取动作电位的相关参数 分析两 种麻醉剂对听神经元的声反应特性的影响 结果 除了特征频率和 Q 值 特征频率与频率带宽的比值 反应听觉神经元的频率 调谐特性 以外 最小阈强度 自发放水平 声反应的潜伏期 刺激时间直方图以及频率-强度调谐曲线的类型均受到麻醉剂类型 的影响 在乌拉坦麻醉条件下 声反应神经元对短声刺激表现出较高的阈强度 较低的自发放水平 较长的潜伏期 且接受较强 抑制性频率-强度调谐曲线出现较频繁 结论 乌拉坦和氯胺酮对声音频率信息在听觉通路中的传递无明显影响 但在乌拉坦 麻醉条件下 初级听皮层神经元受到更强的抑制 这可能与乌拉坦能加强中间抑制性神经元的作用或抑制听神经元本身的兴奋 性有关 关键词 乌拉坦 氯胺酮 初级听皮层 在体细胞贴附式记录 收稿日期 2013-04-22 基金项目 国家自然科学基金 31171059 作者简介 黄玲玥 在读硕士研究生 E-mail: huanglingyueliyan@yahoo.com.cn 柏 林 讲师 在读博士研究生 E-mail: bolinwu@126.com 黄玲玥 柏林 共同为第一作者 通信作者 肖中举 教授 博士生导师 E-mail: xiaozj@fimmu.com