Temporal Response Of Auditory Neurons To Frequency And Amplitude Of Acoustic Stimulus In Inferior Colliculus Of Mice

The auditory system of mammals has many unbelievable abilities.It integrates perfectly the high sensitivity,high accuracy,quick responsibility within milliseconds or even less,and 10⁶ level of dynamic range of detectable sound amplitude. Nevertheless,the mechanisms of signal processing of auditory system,which should have given rise to or at least maintained these seemingly unconcomitant abilities,are still unclear,especially the basic coding mechanism of auditory system.This uncertainty is demonstrated by the numerous studies focused on the representing of acoustic parameters by spike count and / or spike time,no consistent criteria of which one is the dominant coding mechanism,or which parameter is coded by which one of these two candidates.Furthermore,both SC and FSL were affected by various acoustic parameters such as frequency,amplitude,rise-fall time,duration,phase,etc. So,whether there are any relationships within these parameters,what's the possible mechanisms underling the coding or representing of different parameters within one mechanism,these studies are also scarce.So the purpose of our study is that,first,to compare systematically the representation of acoustic amplitude and frequency by SC and FSL,to determine which one is more precise and stable to be the possible coding of acoustic stimulation parameter,and second,to further study the possible mechanism underling the temporal coding by investigating the relationship between amplitude and frequency represented by FSL. Spike counts(SC) or,spike rate and fast spike latency(FSL),are both used to evaluate the responses of neurons to amplitudes and frequencies of acoustic stimuli. The spike count - frequency functions across a population of auditory neurons can be divided into three types:monotonic(the SC increases monotonically as the amplitude of the acoustic stimulus increases),non-monotonic(the firing of non-monotonic neurons first increases and then decreases with further increases in amplitude),and saturated(the SC-amplitude function reaches a plateau with increasing amplitude). These three types are found at almost every central auditory level,e.g.,the cochlear nucleus,superior olivary complex,inferior colliculus,medial geniculate body,and auditory cortex.FSL generally shortens as amplitude increases.However,paradoxical latency shifts involving an increase in FSL with increasing amplitude,and constant latency cells that show a much smaller latency change with signal intensity or an independence of FSL with changing amplitude have also been observed.The SC of a neuron,as a function of acoustic frequency,takes the form of an "upside down V" shape for single peaked tuning curves.The acoustic frequency that evokes the highest response is defined as the best frequency(BF).Both the response-frequency curve and BF are relatively variable,especially at high amplitude, and BF does not generally correspond with the characteristic frequency(CF,the most sensitive frequency or frequency at minimum threshold),particularly when the amplitudes of acoustic stimuli are more than 10 dB above the threshold.In contrast to SC,the FSL- frequency function takes the reverse form to the spike count-frequency function resembling a "V shape",in which the shortest FSL is always at,or near the CF,and FSL lengthens with the increasing difference between the frequency of acoustic stimulus and the CF.However,it is unclear which one of SC and FSL is more suitable as a parameter for evaluating the responses of neurons to acoustic amplitudes and frequencies,since systematic comparisons between SC and FSL tuned to different amplitudes and frequencies,are scarce.Besides,both SC and FSL can be affected by many other acoustic parameters.In our study,a 1.5-cm-long nail was glued onto the dorsal surface of each mouse's skull with dental cement.The animal was held in a polyethylene-foam body-mold with an elastic band hung over a stereotaxic apparatus,which was fixed on an anti-vibration table in a soundproof room.The animal's head was immobilized by fixing the nail to a small metal rod with screws.A 2x2 mm² area on one side of the IC was exposed by opening the skull and dura above the IC.Pure tone bursts were used as acoustic stimuli and were generated and delivered using a Tucker-Davis Technologies System 3.The tone burst signals lasting 50 ms each with a 5 ms rise-fall time.Pure tone bursts were played back using a computer with BrainWare software which controlled the frequency and amplitude of pure tone bursts either manually or automatically.Extracellular recordings were made with glass micropipettes,and data were acquired and processed online with TDT 3.The CF and minimum threshold(MT, defined as the amplitude of tone bursts at CF required to elicit a spike firing probability of 0.1) were first measured approximately by manually varying the frequency and amplitude of tone bursts after an IC neuron was isolated.A frequency and amplitude(F-A) scan was then performed in which frequencies were varied in the range,CF±5 kHz,in 1 or 0.5 kHz steps.The waveforms,numbers and timings of spikes evoked by acoustic stimuli were collected and stored as data sets.The data were monitored with respect to SC-level,frequency function,post-stimulus time histograms(PSTH),spike shapes and feature space window using Brain Ware.Based on these data,spike counts or first spike latencies,and their respective standard deviations(SD) and coefficients of variation(CV) were plotted as functions of amplitude and frequency in offline processing.Totally 163 inferior colliculus neurons were recorded and the systematical comparison of the precision and stability(i.e.,the resolution and the coefficient variation,CV) of SC- and FSL-function as frequencies and amplitudes in the inferior colliculus of mice were made.The results showed that:1.the SC-amplitude functions were of diverse shape.Of 163 neurons,21(13%) were monotonic,47(29%) were saturated,and 95(58%) were non-monotonic.Nevertheless,the FSL-amplitude functions were in close registration,in which FSL decreased with the increase of amplitude and no paradoxical(an increase in FSL with increasing amplitude) or constant(an independence of FSL on amplitude) neuron was observed;2.We examined the discriminability(resolution) of differences in amplitude based on FSL and SC,that is,whether the responses to tones of two neighboring amplitude were significantly different,by one-way ANOVA.For SC only 2 or 3 data points were significantly different(Fig.3 A1,B1 and C1,the asterisks for the open circles).For FSL,however,most of the data points showed differences(Fig.3 A1,B1 and C1,the asterisks for the filled circles).Same analysis was applied to the discriminability of frequency based on FSL and SC,and similar results were acquired.Based on these analyses,we concluded that the discriminability(resolution) of differences in amplitude and frequency based on FSL are higher than those based on SC;3.To compare CV_SC and CV_FSL,we plotted them against each other for single neurons.The CV index of FSL(CVI_FSL),defined as the ratio of the number of the points at which CV_FSL is smaller than CV_SC to all the observed points on the amplitude or frequency functions within an individual neuron,was measured for all studied neurons.Out of 163 recorded neurons,respectively 151(93%) neurons responding to varying amplitudes at the neurons' CFs,or responding to varying frequencies at the amplitude of which the CV(SC)- and CV(FSL)-amplitude curves intersected,showed DI_FSL greater than 0.5.In other words,for more than haif of the observed points within one neuron, CV_FSL was lower than CV_SC.The CVs of FSL for low amplitude stimuli were smaller than those of SC;4.the fraction of neurons for which best frequency(BF) = characteristic frequency(CF)(within±500 Hz) obtained from FSL was higher than that from SC at any amplitude of sound.The BF_SC of 85%(138 of 163) of neurons matched their CF at 5 dB above MT,but the fraction decreased sharply to about 50% as the amplitude increased to over 10 dB above MT,and to 12%at 60 dB above MT. The BF_FSL matched well with CF within 20dB above MT(with 99%,89%and 85% neurons at 5 dB,10 dB and 20 dB above MT,respectively) and also gradually decreased as the amplitude increased,reaching a minimum of 21%at 60 dB above MT.Therefore,systematical comparison of the representation of acoustic amplitude and frequency by SC and FSL showed that,SC and FSL may vary,independent from each other and represent different parameters of an acoustic stimulus,but FSL with its precision and stability appears to be a better parameter than SC in evaluation of the response of a neuron to frequency and amplitude in mouse inferior colliculus.The peripheral auditory system(cochlea) behaves as if it contains a bank of overlapping bandpass filters,which is finely arrange on the basilar membrane,with higher frequencies near the base of the cochlea and lower frequencies on the apex. This frequency selectivity distribution is the so called cochleaotopographic (tonotopical) organization of basilar membrane.Paralleled to the tonotopical feature of basilar membrane,neurons at each level of auditory pathway also demonstrate tonotopically-organized arrangement according to their CFs or BFs.Several basic principles in the signal processing of central nervous system is suggested by previous works.One of these principles is the rank-order processing hypothesis,i.e.,lower levels of central nervous system can only perform simple signal processing,while the more complicated works are left to the higher nuclei. Another one is the parallel signal processing hypothesis,suggesting that there are many parallel tonotopic structures on different levels of the signal transferring pathway,which allows the neurons at different levels to abstract and process different characters of the signal.Signal processing in the auditory system is also hypothesized parallel-hierarchical,and the tonotopic structures on different levels of auditory pathway are thought to be the structural basis.But,no substantial evidence of the hypothesis is directly observed.To evidence it,filtering characteristic of a neuron should be found and relayed from basilar membrane,i.e.,the response of a neuron to any frequency acoustic stimulus is due to frequency-amplitude trading relative to CF or the resonant frequency(RF) acoustic stimulus.And also,this neuronal filtering characteristic would be relayed from BM.However,both of them are unexplored.According to the above comparisons of the representation of sound frequency and amplitude by FSL andSC,FSL is better than SC when representing the frequency and amplitude,so it was used as the response parameter to calculate the relationship between the response for neuronal CF and other frequencies.The materials and methods such as the surgical operation,acoustic stimuli,data acquisition are mostly the same as above.After the single peak auditory neuron was isolated and its CF and MT were measured,F-A scan were then performed with the range of CF±5 kHz and step of 1 or 0.5 kHz.Based on these data,MT,CF,spike counts,spike rates,first spike latency and successive spike latency were calculated and plotted as functions of frequency and amplitude,and the furthermore processing such as slope of functions, slopes distribution over CF,MT and population neurons.57 single peak neurons were recorded and the results of analysis showed that,1. FSL can accurately represent the stimulus frequency and amplitude within a neuron with the exponential first decay function when other parameters such as rise time, phase and so on are fixed,and can be fitted well by the function of Pieron's empirical law(the range of R² was between 0.917 and 0.998).The FSL-amplitude functions for non-CF frequency are the same as that for CF frequency;2.Single peak inferior colliculus neurons show filter characteristic in that the FSL - amplitude functions at different frequencies are similar to each other.When FSL is plotted by the effective amplitude normalized by CF(i.e.,the effective amplitude at other frequency equals the amplitude at CF subtracted by a fixed valueâ–³x),then all the FSL - amplitude curves at different frequencies will overlap with each other,which is resemble the feature of a single filter.Thus,the filter characteristic of the inferior colliculus neurons is relayed from a single point of basilar membrane;3.The FSL transferring character can be described by the exponential constant(constant "c or b"),which was bigger for higher threshold neuron regardless of CF(when doing the linear regression against CF,R² = 0.0042).Our results suggested that,high level neurons in auditory system showed a filter characteristic relayed from BM,and substantially supplied evidences that signal processing in the auditory system is parallel-hierarchical.Furthermore,when other parameters such as rise time and phase are fixed,the latency of neuron firing can represent amplitude and frequency simultaneously.