under general anesthetics , decreased hearing sensitivity is common in both animal research and clinical settings .
several studies have demonstrated that different anesthetics increase auditory brainstem response thresholds [ 13 ] and depress neural excitability in the auditory midbrain [ 4 , 5 ] and cortex [ 68 ] .
the sensitivity of the auditory system could be changed by anesthetics at two levels : the cochlea and the auditory neurons . because any change in cochlear function may influence the response of central auditory neurons , the effects on the cochlea are essential for the anesthetic - induced reduction in hearing sensitivity .
however , the majority of studies have focused on the neural responses , whereas few have examined cochlear function .
urethane has been widely used in animal research for more than a century because it exerts minimal effects on the cardiovascular and respiratory systems .
although urethane has been reported to depress the sound - evoked activity of the auditory system [ 46 ] , its direct effect on the cochlea , particularly sensory hair cells , remains unknown .
sensory hair cells in the cochlea not only translate sound vibration into electrical impulses but also amplify the signals of low - level sound .
the latter process , defined as cochlear amplification , confers incredible sensitivity on mammalian hearing in a tremendous intensity range .
cochlear amplification in mammals is attributed to outer hair cells ( ohcs ) , which can alter their somatic length on the order of micrometers in response to membrane potential changes .
this electromotility is powered by the unique motor protein , prestin , on the ohc lateral membrane .
the voltage - dependent structural conformation of prestin drives ohc somatic motility , which regulates cochlear amplification [ 11 , 12 ]
. however , the voltage - to - length change conversion function ( l - v ) of ohcs is nonlinear and asymmetric : depolarization produces larger cell length changes than comparable hyperpolarization [ 1315 ] .
therefore , the changes in the membrane potential may alter the operating point on the l - v function and influence the overall level of cochlear amplification . because their electromechanical conversion occurs via a feedback mechanism , ohcs
ohcs are innervated by efferent fibers that originate in the superior olivary complex [ 16 , 17 ] .
these efferent fibers form synapses at the base of the ohcs and use acetylcholine ( ach ) as their primary neurotransmitter [ 18 , 19 ] .
nicotinic ach receptors ( achr ) have been identified on ohcs [ 18 , 20 , 21 ] , and ach hyperpolarizes the membrane potential of isolated ohcs . the efferent activity of the olivocochlear nerve bundle during electrical stimulation has been shown to be inhibitory , thereby reducing the gain of the cochlear amplifier and providing protection to the ear against overstimulation . a pharmacological study has indicated that urethane enhances the function of nicotinic achrs while inhibiting the responses of nmda and ampa receptors .
we hypothesize that urethane influences the micromechanics of the organ of corti via the ohcs and , in turn , the cochlear amplification process .
if so , urethane anesthesia provides an alternative strategy to modulate cochlear amplification . by comparing the cochlear microphonic ( cm ) responses between awake and urethane - anesthetized rats
we also measured the membrane potential and current as well as prestin activity in isolated ohcs in the presence of urethane .
our results indicate that urethane hyperpolarizes the ohc membrane potential , which is at least partially mediated by the achr .
all experimental preparations , surgeries , and protocols used in this study were approved by the animal care and use committee of southern medical university of china .
healthy young sprague dawley rats of either sex ( 2128 days old , body weight 4070  g ) exhibiting normal hearing were used for the experiments .
the cm measurements and whole - cell patch recordings were performed as previously described [ 26 , 27 ] . these methods are briefly described as follows .
the scalp was removed , and a metal screw was mounted on the skull using glass ionomer cement .
the animals were subcutaneously injected with 0.1  mg / kg buprenorphine and returned to their home cages to recover . during the recovery period ,
the animals were trained to become accustomed to being head - fixed in the recording setup . to fix the head , the screw was tightly clamped to a metal post .
the rat was able to run freely on a plastic plate rotating around its center as described in our recent study . on the day of recording
a small incision was made via a dorsolateral approach to the pinna to expose the acoustic bulla .
a silver wire recording electrode ( tip diameter , ~500  m ) was placed near the round window membrane through an opening of 3  mm in diameter on the acoustic bulla ( figure 1(a ) ) .
the animal was allowed to recover from isoflurane anesthesia for at least 30  min . to acquire the cm under awake conditions ,
then , the animal was intraperitoneally injected with 1  g / kg urethane using a pipette to examine the effects of this anesthetic .
tone bursts ( 50  ms duration , 5  ms rise / fall time ) of various frequencies ( 2 , 4 , 8 , 16 , or 32  khz ) and intensities ( 070  db spl at 5  db intervals ) were presented using a calibrated tdt es1 speaker located 50  cm away from the recorded ear .
the frequency - amplitude scan was computer controlled ( tdt system 3 , tucker - davis technologies ) and was delivered in a randomized sequence .
the cm responses to the tone bursts were amplified , filtered , and recorded using an a / d converter ( 1440a/700b system , molecular devices ) .
customized matlab software was used for offline data processing , such as response averaging and response amplitude extraction .
the animals were anesthetized ( co2 inhalation ) and decapitated , and the inner ears were rapidly removed from the temporal bones and placed in leibovitz 's l-15 media ( invitrogen , carlsbad , ca ) .
the organ of corti was isolated from the middle and apical turns of the cochlea .
after mild enzymatic digestion for 5  min ( 2  mg / ml collagenase iv , sigma , st .
louis , mo ) and gentle pipetting , the cells were transferred to a small plastic chamber filled with enzyme - free culture medium ( ~1.5  ml ) .
the standard medium was leibovitz 's l-15 , supplemented with 10  mm hepes ( invitrogen , carlsbad , ca ) and adjusted to ph 7.35 and 300  mosm .
then , the chamber containing the cells was placed on the stage of an inverted microscope ( nikon , eclipse fn1 ) equipped with a video camera .
healthy - appearing solitary ohcs were selected for the electrophysiological experiments if they displayed no obvious signs of shrinkage , swelling , damage , or deterioration such as granularity or translocation of the nucleus .
these experiments were performed at room temperature ( 22  4c ) under video monitoring .
the ohcs were bathed in l-15 medium buffered with 10  mm hepes ( ph 7.35 , 300  mosm ) .
the patch electrodes were pulled from 1.5  mm glass capillary tubes at resistances between 3 and 6  m using a horizontal micropipette puller ( model p-97 , sutter ) .
the electrodes were back - filled with a solution containing ( in mm ) 145 kcl , 2 mgcl2 , and 10 hepes .
the access resistance typically ranged from 10 to 17  m when the whole - cell recording configuration was established .
in most of our recordings , the whole - cell currents were less than 3  na . under computer control , hyperpolarizing and depolarizing voltage steps ( 250  ms duration and ranging from 140 to + 94  mv in 13  mv increments )
the low - pass - filtered currents ( corner frequency of 5  khz ) were amplified using an axopatch 200b amplifier ( axon instruments ) .
the urethane - evoked current responses were recorded in voltage - clamp mode . to obtain large urethane - evoked outward currents ,
the whole - cell currents and evoked current responses were acquired using pclamp 10 software ( molecular devices ) on a computer connected to an a / d converter ( digidata 1322a , axon instruments ) .
the data were analyzed using the pclamp software package . for nonlinear capacitance ( nlc ) measurements ,
the membrane capacitance was measured using a two - sine - wave voltage stimulus protocol ( 10  mv peak at both 390.6  hz and 781.2  hz ) with subsequent fast fourier transform - based admittance analysis   at a holding potential of 0  mv .
the data were acquired using jclamp software ( scisoft , new haven , ct ) and were analyzed using originpro software ( originlab corporation , northampton , ma ) .
the nlc can be described as the first derivative of a two - state boltzmann function of nonlinear charge movement to voltage .
the capacitance function is described as(1)cm = clin+qmaxexpvmv1/21+expvmv1/22.four parameters ( qmax , v1/2 , clin , and z ) from the equation were obtained : qmax is the maximum charge transfer ; v1/2 is the peak of the voltage - dependent capacitance ; clin is the linear capacitance ; and 
= ze / kt is the slope of the voltage dependence of the charge transfer .
furthermore , k is the boltzmann constant , t is absolute temperature , z is the valence of the charge movement , and e is the electron charge .
clin is the linear capacitance representing the surface area of the membrane ( i.e. , the cell size ) . to compare the magnitude of the nlc and qmax obtained from different cells of varying size , we normalized the nlc and qmax to clin .
the drugs were dissolved in standard medium ( l-15 ) adjusted to ph 7.35 and 300  mosm .
urethane was delivered via pressure ejection from a micropipette with a tip diameter of ~5  m positioned 2050  m from the bottom of the cell .
care was taken to assure that the application of a drug solution did not alter the position of the cell or influence the measurements . in the strychnine coapplication experiments
, the strychnine solution was slowly perfused into the bath ( 1  ml / min ) without disturbing the position of the cells .
all of the drugs were applied to achieve a final concentration until a consistent response was observed and a washout was performed after each application .
a student 's t - test was used to examine the significance of the difference between the responses obtained before and during the drug applications .
excel software and originpro software were used for calculating , data fitting , and plotting .
the inner and outer hair cells , which are the sensory receptor cells of the inner ear , function as a transducer by converting the mechanical movement of the basilar membrane into an alternating electrical voltage .
this alternating voltage is defined as the cm , which mimics the waveform of a sound stimulus .
representative cm recordings are shown in figure 1 , in which the effects of urethane anesthesia are presented for the same rat .
we performed cm recordings on head - fixed awake rats to monitor the receptor potential before and after urethane application ( figure 1(a ) ) . to avoid the middle ear reflex , mild tone bursts ( less than 70  db spl )
were used to elicit the cm responses . as shown in figure 1(b ) , an 8  khz tone ( level at 70  db spl ) evoked a cm at an amplitude of ~424  v .
the amplitude of the cm responses at saturation levels was reduced by 44% to 238  v after the intraperitoneal injection of urethane . the proximity of the recording electrodes to the hair cells may affect the recorded amplitudes . to ensure that our control recordings were not influenced by the activity of the animal
, we averaged at least three evoked cm measurements before the urethane injection . for the cms measured from all five rats ,
urethane induced a significant decrease in the cm of 39.3% on average ( p < 0.01 , student 's t - test ) .
the time course of this urethane effect was examined in five rats that exhibited at least an 80% recovery in the cm amplitude .
the representative changes in the cm over time after urethane injection are shown in figure 1(c ) . the initial decrease in the cm
was observed ~25  min after the urethane injection , reached its lowest value within ~3 hours , and then recovered gradually .
the time of the peak reduction was highly variable between different rats , ranging from 45  min to 3 hours ( 45  min , 75  min , 90  min , 3 hours , and 3.2 hours , resp . ) .
the cm responses to different sound levels were also measured . as shown in figure 1(c ) , the changes in the cm amplitudes at different sound levels followed a similar time course .
we compared the cm thresholds before and after urethane application ( figure 1(d ) ) .
the cm thresholds were defined as the minimum sound level that evoked a detectable cm response .
consistent with the cm magnitude measurements , the cm thresholds were increased by urethane application at all frequencies examined .
the increase of threshold is similar and shows no significance at different sound frequencies ( p > 0.05 , student 's t - test ) .
the cm response is dominated by the ohcs in the organ of corti [ 9 , 31 ] .
as such , the cm reduction in our experiments represents a significant reduction in ohc activity after urethane application . to determine how urethane alters the responses of ohcs ,
whole - cell current- and voltage - clamp recordings were performed from ohcs acutely isolated from the middle and apical turns of the rat cochlea .
isolated ohcs can easily be identified based on visual inspection : the ohcs display a cylindrical morphology with a nucleus located near the base , whereas inner hair cells are flask - shaped with an upper nucleus position .
another indication of ohcs is a functional characteristic : the visible motile responses elicited by the rapid membrane potential changes generated during our patch - clamp measurements [ 22 , 32 ] .
the average zero current membrane potential under whole - cell recording conditions was 54  mv ( sd = 7  mv , n = 19 ) . to determine the influence of urethane , different concentrations of urethane
were applied to the recorded ohc via local perfusion for ~15  s ( as shown in figure 2(a ) ) . as a control ,
recordings were also performed with l-15 medium : no membrane potential change was detected during l-15 perfusion ( 0  mm in figure 2(b ) ) .
the stability of this recording indicates that our measurements were not affected by the perfusion flow rate .
when 100  mm urethane was delivered to an ohc clamped at 0  na , the steady - state membrane potential was hyperpolarized by 28.6  mv and was repolarized shortly after drug application ( figure 2(b ) ) .
this reversible hyperpolarization was detected in all five ohcs measured ( mean  sd = 27.0  3.9 ) . to rule out the effects of urethane on the ohc membrane potential
urethane at a concentration as low as 0.1  mm , which is ~1/100 of the dose typically used to anesthetize animals , elicited a detectable membrane hyperpolarization .   figure
the smooth curve represents a fit according to the following form of the hill equation : vuret = 100/[(kd/[uret ] ) + 1 ] . a fifty percent reduction in the membrane potential
was detected at 15.4  mm ( kd ) , and the slope ( n ) of the membrane potential change to the urethane concentration was 0.91 .
subsequent experiments used 100  mm urethane because this concentration consistently evoked an apparent response .
we also examined the effects of urethane on the membrane current by using voltage - clamp recordings .
a representative example of the whole - cell current recorded from an isolated ohc is shown in figure 3(a ) . when the membrane potential was clamped from 140 to + 94  mv , the cell currents changed from 445  pa ( inward ) to + 1480  pa ( outward ) , resulting in a dynamic range of 1925  pa .
the response measured under control conditions is consistent with results previously published for guinea pig [ 22 , 33 ] and gerbil ohcs .
the local perfusion of 100  mm urethane significantly increased the current magnitudes , especially at potentials greater than 50  mv .  
figure 3(b ) shows the current - voltage ( i - v ) curve derived from the steady - state responses shown in figure 3(a ) .
the mean change of i - v curves recorded from 11 ohcs was shown in figure 3(c ) .
therefore , it is unlikely that the effects of urethane are mediated by a specific urethane receptor .
the apparent changes in the ohc current response in the voltage - clamp experiments are most likely due to the effects of urethane on existent ion channels .
acetylcholine is the primary efferent neurotransmitter in the cochlea and is released from the efferent chemical synapses at the base of the ohcs [ 19 , 24 ] .
the 9 subunit of the nicotinic achr family , which was identified from a rat genomic library , has been demonstrated to play an important role in the ach - induced responses of ohcs [ 18 , 20 , 21 ] . to determine whether the urethane - induced response occurs via this achr
, we examined the effect of strychnine ( a potent antagonist of the 9 achr subunit ) on the urethane - induced responses . because the membrane current is directly related to nicotinic achr activity and is easy to record
, we measured the urethane - induced current as an indicator of nicotinic achr activity . to obtain the urethane - induced current ,
the top trace in figure 4(a ) shows a 280  pa upward change of membrane current that correlated in time to the perfusion of 100  mm urethane onto this cell ( ~20  s ) . as indicated in figure 3(b ) , urethane increased the outward current at a membrane potential of 0  mv .
therefore , the magnitude of the observed current change reflects the amplitude of the outward current elicited by urethane .
coapplication of 0.01  m strychnine reduced the amplitude of the urethane - induced current to 114  pa at saturation level ( figure 4(a ) , middle trace ) .
these data suggest that the urethane - induced response is mediated , at least in part , via achr assembled from 9 subunits .
then , a higher concentration of strychnine ( 0.1  m ) was coapplied to examine whether the urethane response was blocked in a dose - dependent manner . to minimize desensitization of the receptor , 3  min washout was set between two concentrations . as shown in the bottom trace ( figure 4(a ) ) , the urethane - induced outward current was further reduced to 48  pa in the presence of 0.1  m strychnine . in the seven ohcs measured ( figure 4(b ) ) , the magnitude of the urethane - induced response was significantly reduced by the coapplication of both 0.01  m ( by 54  12% on average ) and 0.1  m strychnine ( by 79  10% on average ) ( both p < 0.001 , student 's t - test ) .
mammalian ohcs contract or elongate at acoustic frequencies depending on the membrane potential of the cell [ 10 , 35 , 36 ] .
this process , defined as electromotility , is necessary for cochlear amplification [ 3739 ] .
prestin , a unique voltage - dependent motor protein found in the membrane of ohcs , mediates the electromotility of ohcs [ 11 , 40 ] .
the voltage - sensing and motor functions of mammalian prestin manifest as two characteristics : the nlc and electromotility . the nlc and electromotility are fully coupled in mammals [ 30 , 41 , 42 ] and can be characterized using a simple two - state boltzmann function .
because the nlc can be easily and accurately measured experimentally , we measured the nlc to evaluate the effects of urethane on prestin function . 

figure 5(a ) shows an example of the nlc obtained from an isolated ohc under the whole - cell patch - clamp configuration . as shown in the control ( open circles and gray line ) treatment before urethane application , the nlc is characterized by a bell - shaped dependence on the membrane potential and a peak at 54.8  mv for this cell .
no clear change was detected in response to application of 100  mm urethane ( filled circles and black line ) .
we examined the nlc from a total of 10 ohcs in response to urethane treatment .  
figure 5(b ) presents the mean and sd of the normalized nlc from these cells .
four parameters ( qmax , clin , v1/2 , and z ) were obtained from a curve fit of the nlc response using the first derivative of the boltzmann function ( heavy lines in figures 5(a ) and 5(b ) ) .
the normalized mean values and sds of the four parameters from the 10 ohcs are plotted in figure 5(c ) .
no statistically significant difference was found in response to urethane treatment for all parameters ( p > 0.05 , student 's t - test ) .
we have shown for the first time that urethane affects the electrical response properties of isolated ohcs .
our voltage - clamp data shows that when ohcs were depolarized , the outward current was significantly increased by urethane ( figure 3 ) .
this effect was voltage - dependent : it was more pronounced at membrane potentials higher than 50  mv .
in mature ohcs , two currents are primarily involved in this process : ( 1 ) the voltage activated outward k current and ( 2 ) the ca - activated k current .
the voltage - dependent k current is activated at membrane potentials from 90  mv to 50  mv , displaying half activation at 80  mv . because the urethane - induced current change was clearly detected at membrane potentials > 50  mv ,
the ca - activated k channel was first reported by ashmore and meech . at membrane potentials
> 35  mv , this k channel is opened by an influx of ca , leading to k efflux . under physiological conditions ,
we assumed that the effect of urethane on ohcs involves this process based on our strychnine experiment .
the 9 subunit of the nicotinic achr family has been demonstrated to be the primary nicotinic achr subunit in ohcs .
the 9 subunit displays unique pharmacological properties similar to those detected in cochlear hair cells .
we found that strychnine , a potent antagonist of 9-containing achrs , significantly blocks the urethane - induced outward current ( figure 4 ) .
then , this influx of ca leads to the opening of ca - activated k channels and subsequent k efflux , resulting in hyperpolarization of the ohcs .
consistent with our results , a study in xenopus oocytes indicates that urethane enhances the response of nach receptor .
our data is also supported by the urethane effects on cochlear function by measuring dpoae .
urethane decreases the efferent influence from medial olivocochlear terminus to ohcs via the 9 receptor .
the cylindrically shaped ohcs alter their cell length in response to membrane potential changes , exhibited as either a somatic elongation ( upon hyperpolarization ) or contraction ( upon depolarization ) .
this somatic motility of ohcs is responsible for cochlear amplification , which contributes to the exquisite frequency selectivity and sensitivity in mammals [ 10 , 37 ] .
however , this change in length is asymmetric : the magnitude of contraction is much larger than that of elongation [ 1315 ] .
this potential change moves the operating point of the voltage - to - length change conversion function toward lower slope , thereby reducing the overall augmentation of ohc electromotility .
based on the voltage - to - length change conversion function for ohcs in the guinea pig , a 30  mv hyperpolarization reduces the total motility magnitude by approximately 2535% .
however , in vitro acetylcholine application evokes an increase of electromotile responses of isolated ohcs that develops on a time scale of several seconds .
it would increase the driving force for the mechanotransduction current , causing the increase of cochlear microphonic potential .
it seems a mismatch between our in vitro data and in vivo cm results . for in vitro experiment , a high concentration urethane was applied to isolated ohcs directly .
in addition to a change in the membrane potential , mechanical properties of ohcs could also influence the cochlear amplifier .
it is the consensus that normal cell morphology and somatic stiffness of ohcs are essential for cochlear amplification [ 12 , 47 ] .
acetylcholine decreases the axial stiffness of the ohcs and reduces the overall mechanical load of the aggregate ohc , resulting in the increase of motile magnitude .
it has been suggested that the changes of the ohcs stiffness are mediated by ca - dependent phosphorylation of the ohc cortical cytoskeleton [ 22 , 48 ] . since urethane
would also activate 9-containing achrs and induce a ca influx , a similar decrease of ohcs axial stiffness and a reduction of global cochlear activity may be induced by urethane application in vivo .
the electromotility of ohcs is presumably attributed to the voltage - dependent activity of the motor protein prestin [ 11 , 40 ] .
a generally accepted model for prestin function is that intracellular anions ( in most cases cl ) move toward the extracellular surface upon hyperpolarization and toward the cytoplasmic side in response to depolarization .
this anion translocation produces a nonlinear change in the membrane capacitance and , subsequently , triggers a conformational change in prestin , which ultimately alters the somatic length of the ohc . according to this model , any changes in this voltage - dependent activity of prestin may alter the motility of ohcs and affect the overall level of cochlear amplification . for all measured parameters reflecting the properties of prestin function
, we did not observe any significant change in response to urethane in our experiments ( figure 5 ) .
therefore , we assumed that the influence of urethane was not the alteration of the motile activity of prestin .
nonetheless , anion transfer may be significantly reduced by urethane - induced hyperpolarization . in mammalian ohcs , this charge movement is represented as the nlc , which is characterized by a bell - shaped dependence on the membrane potential that peaks between 70 and 20  mv ( 50  mv in our results ) [ 30 , 41 ] .
the urethane - induced hyperpolarization shifts the operating range of the membrane potential away from this peak , thus reducing the charge movement and the activity of prestin .
this effect is a likely mechanism by which urethane reduces ohc motility in vivo . because the electromotility of ohcs feeds a cycle - by - cycle force to the organ of corti so that the sound vibration is amplified , it is conceivable that even a modest reduction in the motility of ohcs could reduce the overall level of cochlear amplification .
the results from isolated ohcs are consistent with our in vivo experimental evidence ( figure 1 ) .
although the time courses of the urethane - induced effects were different , all of the rats examined exhibited a similar reduction in the cm magnitude . despite the expanding research to awake animal models of monkeys [ 49 , 50 ] , bats [ 51 , 52 ] , mice , and rats ,
the frequency selectivity of auditory neurons is composed of two elements : the frequency tuning of the cochlea and the refinement of the auditory neural system .
the detected anesthesia - induced changes in neural responses involve the effects of the anesthetic on not only the neurons themselves but also the peripheral receptors ( hair cells ) .
therefore , it is critical to identify the influence over hair cells from the integrity .
our data show that urethane elicited a ~10  db cm threshold lifting ( figure 1(d ) ) , which indicates that the depression of ohcs leads to a reduction in hearing sensitivity .
furthermore , this depression is identical at all sound frequencies , suggesting that urethane affects ohcs along the entire basilar membrane .
these results are similar to those of isoflurane and ketamine , which have been assessed using distortion product otoacoustic emissions .
the present study found that urethane hyperpolarizes outer hair cells , resulting in a reduction in hearing sensitivity without affecting frequency selectivity .
our findings indicate that anesthetics directly affect cochlear hair cells and provide an alternative strategy to modulate cochlear functions .
 the cochlea converts sound vibration into electrical impulses and amplifies the low - level sound signal . 
 urethane , a widely used anesthetic in animal research , has been shown to reduce the neural responses to auditory stimuli . 
 however , the effects of urethane on cochlea , especially on the function of outer hair cells , remain largely unknown . in the present study , we compared the cochlear microphonic responses between awake and urethane - anesthetized rats . 
 the results revealed that the amplitude of the cochlear microphonic was decreased by urethane , resulting in an increase in the threshold at all of the sound frequencies examined . to deduce the possible mechanism 
 underlying the urethane - induced decrease in cochlear sensitivity , we examined the electrical response properties of isolated outer hair cells using whole - cell patch - clamp recording . 
 we found that urethane hyperpolarizes the outer hair cell membrane potential in a dose - dependent manner and elicits larger outward current . 
 this urethane - induced outward current was blocked by strychnine , an antagonist of the 9 subunit of the nicotinic acetylcholine receptor . 
 meanwhile , the function of the outer hair cell motor protein , prestin , was not affected . 
 these results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells , whereas the frequency selectivity of cochlea remains unchanged . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions