Developmental Changes Of Kv Expression Affect The Function Of Bushy Cells In Rat Anteroventral Cochlear Nucleus

BACKGROUND&OBJECTIVEHearing is one of important source of information for human perception and feeling the surrounding environment.Audible signal begins cochlea,and then by the auditory nerve conduction into the central nervous system,via signal processing by different nuclei and after we perceive.Specifically:the sound of the outside world in the form of a mechanical wave propagation in the elastic medium through the outer ear,the middle ear to the inner ear auditory receptor-cochlea.Hair cells within the cochlea convert the mechanical sound signal to chemical electrical signal,and then through the auditory nerve fibers uploaded into the central auditory system.Afferent nerve which in type I nerve fibers mainly afferent the auditory information to the first leg of the auditory central nervous system-cochlear nucleus.After carry hearing information,the auditory nerve fibers into the cochlear nucleus start projection to three different partitions:the anterior ventral cochlear nuclei(AVCN),posterior ventral cochlear nuclei(PVCN)and dorsal cochlear nuclei(DCN)and and form specific tonotipic.Acoustic signal has been coded initially in the three partitions of cochlear nucleus and then uploaded to the more advanced nuclei.Where in the anterior ventral cochlear nucleus directly accept a large auditory nerve fibers projection,it is the center of the initial stage of auditory information processing.Comparative Analysis on the Status and developmental differences of anterior Ventral cochlear nucleus primary neurons has great significance in the understanding of the auditory signal transduction and processing center.The primary neurons of anterior Ventral cochlear nucleus are mainly consists of two types:bushy cell and stellate cell which have large differences in morphology and function.In morphology,bushy cell have larger,more rounded cell body with one or two main dendrites,which are generally stretch along to two different directions and branch is shorter,more limited scope.Stellate cells,also known as multi-polar cells,smaller cell body,and there are three or more primary dendrites which relative to the bushy cells is not so dense and long,and some can also reach across the midline contralateral.In function,bushy and stellate cells form two parallel pathways that enhance different features of their auditory nerve input.Bushy cells preserve the fine temporal structure of their inputs and project to the superior olivary comples,whereas stellate cells encode the amplitude envelope of sound in the band of frequencies to which they are tuned,cues that are know to be critical for the understanding of speech and project to the inferior colliculus.Functional difference stems from differences on the electrophysiological properties.Mature bushy cells have an important feature:the instantaneous discharge mode.That is,by phase locking feature or high frequency sound envelope formed faithfully reflect the instantaneous sound characteristics to ensure accurate and effective transmitted the time code information to postsynaptic neurons.However,when encoded pectral characteristics of complex signals the stellate cells have an important feature:a linear encoder.That is,discharge rate and intensity of neurons have good follow nature.The differernt morphology and electrophysiological characteristics between busy cells and stellate cells led to they played a completely different role in the function of processing an auditory signal.The instantaneous discharge mode of bushy cells in AVCN is an important feature which ensure accurate and effective transmitted the time code information to postsynaptic neurons.Moreover,the AVCN tonotopic map is the other feature essential to its function.Afferent auditory nerve which mainly in type I nerve fibers through the anterior ventral cochlear nucleus into the cochlear nucleus.Low-fiber from the top of the cochlea mainly distributed in the front ventral cochlear nucleus closer to the ventral portion and the high frequency fiber from the bottom of the cochlea mainly distributed in the section near the back side.This constitutes the tonotopic map in the anterior ventral cochlear nucleus that the ventral portion of AVCN low-frequency to the dorsal portion of AVCN high-frequency.In cats,comparative analysis the relationship between the AVCN tonotopic map and bushy cells soma area found that the soma surface area of bushy cells in the ventral portion,low-frequancey of AVCN is large,the soma surface area of bushy cells in the dorsall portion,high-frequancey of AVCN is small and,the cell surface area becomes gradually smaller along the low to high frequency.Moreover,Osen divided the bushy cells in AVCN into two categories:large spherical bushy cells and small spherical bushy cells.large spherical bushy cells mainly distributed in the ventral portion of AVCN and receiving low-fiber projection from the top of the cochlea,small spherical bushy cells mainly distributed in the dorsal portion of AVCN and receiving high-fiber projection from the bottom of the cochlea.In the other hand,the traditional view that the primary auditory(brainstem)nuclei audio tonotipic map has been established early in development,rarely affect by the activity dependent signal input.Recently,however,this traditional picture of a developmentally predetermined and‘hard-wired'auditory brainstem has undergone a substantial revision.It is becoming increasingly apparent that auditory synapses in the brainstem can express activity-dependent synaptic plasticityl4-16 and that auditory brainstem circuits undergo an unexpected degree of synaptic reorganization.Moreover,the functional activity signal input will cause the number of the target nucleus neurons significant changes.In the auditory system,deprivation of auditory information input before the auditory critical period(P10-P12)will cause in the number of cells in anterior ventral cochlear nucleus was significantly deaths(25%-61%),while the deprivation in the mature stage without significant changes the number of cells.This suggests the functional activity signal input of cells during development plays an important role.In summary,the tonotopic map and electrophysiological properties of the bushy cells in AVCN have undergone significant changes associated with functional activity during the critical period of development.To analysis the linear correlation between surface area of bushy cells and tonotopic map in development already have established or in the critical period of auditory when the number of cells change significantly.In the development of the nervous sciences,performing specific functions of neurons gradually mature and form a precise and orderly neural network to perform its functions is essential to the process.In this paper,we explore the function changes of bushy cells in the AVCN during development.We analyzed in two parts:First.Based on the electrophysiological properties of single bushy cell to explain the changes of instantaneous discharge characteristics during the development and the internal mechanism.Second.Based on the distribution properties of bushy cells in the AVCN to explain the distribution changes during developmental and their impact on its function.METHODS(1)Electrophysiology recording in vitro:Developmental ages for the current study were postnatal days from 7 to 25.The whole-cell current-clamp was performed to recording the action potentials firing of cells.Comparative Analysis of the changes of cells firing patterns and electrophysiological characteristics during development process.(2)Morphometric analysis:To verify cell indentity(To observe the morphology of cells).0.25%biocytin was added to the intracellular solution and fully exchange with cell after 15 min recording.Observed the overall morphogy of AVCN under the light microscope and positioning caculated the location of each recording pipettes along the dorsal-ventral axis of the AVCN to standardized determine the cells location.The soma of bushy cells was assumed to be the solid figure produced by rotating an ellipse along its major axis.(3)Western blotting:To quantify analysis changes of protein expression in different development process by Western blotting.(4)Immunofluorescence technique:Immunofluorescence double staining specifically labeled the protein and neurons.Comparative Analysis the changes of Kv1.1 protein in AVCN during development process.RESULTSIn the experimental results,we can found that:morphological and electrophysiological properties of the bushy cells and stellate cells in the anterior ventral cochlear nucleus have significantly changed during development.Specifically:in morphology,the dendrites of bushy cells wider distribution,more slender in young,then gradually shorter,more concentrated with age.The dendrites of stellate cells become more diffused,elongated with age.In electrophysiological properties,The firing patterns of bushy cells is not always just a single action potential firing in younger cells which often found firing multiple action potentials,but with age becoming only firing a single action potentials.The electrophysiological firing patterns of stellate cell change little during development.However,when analysis the relationship between the number of spiking and mutual stimulation intensity found that the characteristic linear encoder of stellate cells is gradually increased during the development.Analyzing and comparing different developmental stages bushy cells firing mode changes influence the auditory information processing function of encode time accuracy,we introduced two indexes to analyze developmental changes of bushy cell function:Latency and Jitter.We found that the value of Latency and Jitter in bushy cells are significantly reduced during the developmen,suggesting that with the development of its function improved significantly.This also led us to study the underlying mechanisms of electrophysiological properties and functional characteristics change in bushy cells during development is of great significance.We Specifical analyze the bushy cells upthreshold action potential characteristics and subthreshold membrane properties developmental changes found that during development,the bushy cells upthreshold action potential characteristics(threshold current and half-width)and subthreshold membrane properties(input resistance and time constant)have undergone significant changes.In mature neurons,since the issuance of action potentials substantially the same in terms of basic composition.thus the waveform and amplitude of action potential does not have significance in information coding.Therefore busy cells encoding time information accuracy characteristics vary with age and development to improve may be mainly due to the cell membrane characteristic change and the value of Latency and the input resistance,the time constant have a good linear correlation.By adding the appropriate blocking agent confirmed that the low voltage-sensitive potassium channels in bushy cells play an important role in encode time accuary.At the same time,we have adopted western blot and immunohistochemistry confirmed the expression of low-voltage-sensitive potassium channels(Kvl.1)protein have a significant increase during development.we comparative analysis the correlation between surface area of bushy cells and its location in different developmental stages found that surface area of bushy cells and location have a significant linear relationship.However,the bushy cells scattered distribution along the axis of ventral to dorsal position,no significant cell surface area distribution in early development.We also compared the relationship between surface area of stellate cells and its location in development found that the stellate cells scattered distribution,no significant cell surface area distribution during the development.During development,the bushy cells in the anterior ventral cochlear nucleus not alway firing a single action potential(onset firing).In P7,P14,we still also can record the bushy cells that firing multiple spiking(pause firing).When statistics the ratio of pause firing bushy cells during development,we can found that the number of pause firing bushy cells gradually become less during the development,to P21 basically not found.When comparing different firing patterns bushy cells in P14,we can found that onset firing bushy cells were mainly distributed near the ventral portion of the AVCN,pause firing bushy cells were mainly distributed near the dorsal portion of the AVCN and onset firing bushy cells have relatively large surface area.pause firing bushy cells have relatively small surface area.Moreover,when comparing with the electrophysiological properties of two different firing patterns bushy cells find that their input resistance and time constant has a large difference.The input resistance and time constant of pause firing bushy cells is larger than the onset firing bushy cells.This may indicate that the different firing patterns,location,surface area bushy cells may play a different role in the information processing.In summary,bushy cells and stellate cells in the anterior ventral cochlear nucleus played a different,important role respectively when processing audio information.This article focuses on comparative analysis of relations between bushy cells improve the encoding time information accuracy and electrophysiological properties during the development.Morever,we confirmed Kv1.1 developing elevated expression in bushy cells improved the encoding time information accuracy.Additionally,the distribution of the soma area of bushy cells in the AVCN occur significant changes during development.In early development,the distribution of the soma area of bushy cells in the AVCN shows no obvious regularity.With the development of increasingly mature,the distribution tended to showing the big bushy cells locate in the ventral of AVCN and the small bushy cells locate in the dorsal site.Moreover.In the critical period,the development of bushy cells in different parts exhibit the phenomenon of sync which the ventral bushy cells develop faster than the dosal portion of AVCN.CONCLUSION1.Bushy cells in AVCN improve the encoding time information accuracy during the development.Kv1.1 developing elevated expression in bushy cells improved the encoding time information accuracy.2.The distribution characteristics of bushy cells in AVCN were Gradual established during the auditory critical period.Moreover,In the critical period,the development of bushy cells in different parts exhibit the phenomenon of sync which the ventral bushy cells develop faster than the dosal portion of AVCN.