Postembryonic DeveloPment of ElectroPhysiological Characteristic of Primordial HiPPocamPus in Bufo bufo gargarizans

Wei WANG，Xinyang DENG，Chuancheng LlANG，Yong WANG，Liwen WANG*

1.College of ChemistrY and Life Science，ShenYang Normal UniversitY，ShenYang 110034，China；

2.Experiment Center，ShenYang Normal UniversitY，ShenYang 110034，China

Postembryonic DeveloPment of ElectroPhysiological Characteristic of Primordial HiPPocamPus in Bufo bufo gargarizans

Wei WANG1，Xinyang DENG1，Chuancheng LlANG1，Yong WANG2，Liwen WANG1*

1.College of ChemistrY and Life Science，ShenYang Normal UniversitY，ShenYang 110034，China；

2.Experiment Center，ShenYang Normal UniversitY，ShenYang 110034，China

［Objective］The aim was to probe into developing changes of spontaneous electrical activities of primordial hippocampus in Bufo bufo gargarizans in postembrYonic phase.［Method］Nicroelectrode technique was used to record extracellularlY spontaneous electrical activities of neurons in primordial hippocampus of B.bufo gargarizans in postembrYonic phase.［Result］Single，consecutive single，burst，consecutive burst and anomalous spontaneous firing were recorded in primordial hippocampus，in which fourkinds ofspontaneousfiringswere recorded in forepartof postembrYonic development except the anomalous spontaneous firing，and the burst spontaneous firing and consecutive burst spontaneous firing were predominant.The five kinds of spontaneous firings mentioned were recorded in anaphase of postembrYonic development and the single and burst spontaneous firing were predominant. Following with the development of primordial hippocampus，the amplitudes of action potential of single firing and consecutive firing enhanced graduallY，and it reaches the maximum in the metamorphosis phase；the differences of the frequencies of consecutive single firing and consecutive burst firing were not significant.［Conclusion］Following with the development of primordial hippocampus，the neuronal electrical activities showed diversification，the developmental degree of the primordial hippocampus in the metamorphosis phase was close to the subadult and adult.

Bufo bufo gargarizans；Primordial hippocampus；Spontaneous electrical activities；PostembrYonic phase

B ufo bufo gargarizans，is the genus toad，the familY Bufonidae，member of the order Anura and the class of Amphibia.lt is widelY distributed in China and its population is high［1］.lt is an important in struction animal for environment.At present，the development of telencephalon and its proximal species were studied from the perspectives of morphologY and histologY［2-5］.The postembrYonic development of spon taneous electrical activities of the pri mordial pallium and olfactorY bulb in B.gargarizans was reported in the earlY stage［6-7］.The primordial hippocampus was a more developed part in the telencephalon of the amphibian［8］，but its postembrYonic development of electrophYsiological characteristics was not reported Yet.With the aid of microelectrode electrophYsiological technique，the research conducted an electrophYsiologicalresearch on six tYpical periods of primordial pallium after embrYo of B.gargarizans to discuss developmental characteristics of electrical activitY in each phase of primordial hippocampus in B.gargarizans，and to enrich the data of development of the nervous sYstem of Anura.

Materials and Methods

ExPerimental animals

Experimental larvaes of B.gargarizans were collected from suburb of ShenYang， Liaoning Province. Subadult and adult were purchased from ShenYang Nedical College.Six tYpical phases were chosen and divided bY Wang et al.［9］into embrYonic de-velopment-operculum completion phase，hammer limb bud phase，anal canal degradation phase，metamorphosis phase，subadult phase and adult phase.Five animals were used in the experiment for each developmental phase.

Measurement of sPontaneous electrical activities

Nicroelectrode electrophYsiological technique was used to record extracellularlY spontaneous electrical activities of neurons in primordial hippocampus of B. gargarizans in postembrYonic phase.Conventional operation methods were used to expose different periods of telencephalon in B.gargarizans and peel off the toad skull membrane.Glass microelectrode（diameter less-than 1 μm，filled with 3mol KCl）with 5-20 NΩ impedance was inserted into primordialhippocampus with the aid of microelectrode thruster（Northwest Optical lnstrument FactorY，tYpe WF-2）.Discharge signals went through a highimpedance microelectrode amplifier（Chengdu lnstrumentFactorY，tYpe WF-lB）into the signal acquisition and processing sYstem （Chengdu lnstrument FactorY，tYpe RN6240）.Electrical activities of the primordialhippocampusneuronswereobserved and recorded on the computer.

Statistic analysis

SPSS16.0 software was used to make statistics，analYsis and map for experiment data.One WaY ANOVA and TukeY’s test were used to compare the differences between the groups.＂Nean±standard deviation＂（mean±SD）was used to demonstrate the data，and P＜0.05 means there exist significant differences.

Results and Analysis

SPontaneous firings of Primordial hiPPocamPus neurons during Postembryonic develoPment Period

Five discharge tYpes were recorded:single spontaneous firing，consecutive single spontaneous firing，burst spontaneous firing，consecutive burstspontaneous firing and anomalous sustained spontaneous firing（Fig.1）.The proportion ofdischarge units was different in each phase of development（Fig.2A）.ln the earlY operculum completion phase， four discharge tYpes were recorded，without anomalous sustained spontaneous firing.A total of 38 discharge units were recorded.Burst spontaneous firing and consecutive burst spontaneous firing were the main tYpes，accounting for 47.37%and 28.95%. From hammer limb bud phase to the adult phase，there were both five discharge tYpes，and 46，33，47，54 and 28 discharge units were recorded respectivelY.The burst spontaneous firing and consecutive burstspontaneous firing were mainlY apparent in hammer limb bud phase，each accounting for 28.26%and 30.43%respectivelY.The consecutive spontaneous firing and burst spontaneous firing were mainlY apparent in anal canal degradation phase，each ac-counting for 27.27%and 30.3%respectivelY.The single spontaneous firing and consecutive burst spontaneous firing were mainlY apparent in metamorphosis phase，each accounting for 27.99%and 23.40%respectivelY.The single spontaneous firing and anomalous sustained spontaneous firing were mainlY apparent in subadult phase，each accounting for 27.78%and 29.63%respectivelY.The burst spontaneous firing and single spontaneous firing were mainlY apparent in adult phase，each accounting for 50%and 35.71%respectivelY.

ElectroPhysiological characteristics of sPontaneous firings of the Primordial hiPPocamPus neurons during Postembryonic develoPment Period

A statistic analYsis was conducted aboutsingle spontaneous firing and consecutive spontaneous firing amplitudes，consecutive single spontaneous firing and burst spontaneous firing frequencY ofprimordialhippocampus nerons in B.gargarizans during postembrYonic phase（Fig.2B and Fig.2C）.Fig.2B showed that single spontaneous firing amplitudes increased graduallY along with the development and reached the maximum in the abnormal phase.lt showed significant diversitY against to the prior development phase （P＜0.05）.The change of the consecutive spontaneous firing amplitudes was similar to single spontaneous firing，but there was no significant diversitY between the subadult phase and the adult phase （P＞0.05）.Fig.2C showed that the change of the frequencY of consecutive single spontaneous firing during the whole development was small，and the frequencY of consecutive burst spontaneous firing was higher in the initial phase of development.However，the frequencies of consecutive single spontaneous firing and consecutive burst spontaneous firing in the metamorphosis phase，the subadult phase and the adult phase were similar，and the diversitY was not significant（P＞0.05）.

Discussion

The primordial hippocampus was located at the medial wall of lateral ventricle of telencephalon in B.gargarizans，and was extended to the site of the third ventricle［3］.lt was a relativelY developed part of the cerebral hemisphere. The primordial hippocampus of the amphibians received the nerve fibers of olfactorY bulb，but did not form the olfactorY sensation，and responded to the stimulation，and received projections of visceral fibers from the hYpothalamus［8］.Along with the development of B.gargarizans，the firing tYpes of primordial hippocampus neurons increase step bY step and the neuronal electrical activities diversifY graduallY.Along with the development，the proportion of single spontaneous firing and anomalous sustained spontaneous firing graduallY increased，and the excitabilitY of neurons was incremental，which was consistent with the changes of the olfactorY bulb and the primordial pallium［7］.The proportion of the number of single spontaneous firing，consecutive spontaneous firing，anomalous sustained spontaneous firing was similar during the metamorphosis phase and adult phase，and the frequenciesofconsecutive spontaneous firing and consecutive burst spontaneous firing were equivalencY during the metamorphosis phase，the subadult phase and the adult phase. Besides，the diversitY was not significant，the developmental degree of the primordial hippocampus in the metamorphosis phase was close to the subadultphase and adultphase，which was consistent with the histological findings［3］.lt maY be related to the fact that the area of the primordial hippocampus of B.gargarizans reached the apex in the metamorphosis phase，and the number，and the area of the primordial hippocampus reached the maximum.ln addition，there are tYpical metamorphosis phases in the development process of amphibians.With the metamorphosis from a tadpole to a adult，the bodY undergoes significant changes，including change of ecological tYpe and the emergence of adaptive structure，its respiratorY sYstem，circulatorY sYstem，nervous sYstem，excretion sYstem，and metabolic tYpe allhave taken place great changes［10-11］.This maY be one of the causes of the area and densitY of primordial hippocampus relativelY larger during the metamorphosis phase，and its spontaneous electricalactivities were similar to those of the adult.Thedecrease of the frequencY of consecutive burst spontaneous firing maY be related to the apoptosis of neurons in the development process［12-14］.

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ResPonsible editor：Xiaoxue WANG

ResPonsible Proofreader：Xiaoyan WP

中华大蟾蜍原始海马神经元电生理学特性的胚后发育

王维1，邓昕旸1，梁传成1，王勇2，王丽文1*
（1.沈阳师范大学化学与生命科学学院，辽宁沈阳 110034；2.沈阳师范大学实验中心，辽宁沈阳 110034）

［目的］探讨中华大蟾蜍（Bufo bufo gargarizans）原始海马神经元自发电活电生理学特性的胚后发育变化。［方法］应用微电极电生理技术，对中华大蟾蜍胚后原始海马神经元的自发放电活动进行在体胞外记录。［结果］原始海马神经元的自发放电分为单个放电、连续单个放电、簇状放电、连续簇状放电和不规则放电五种形式。胚后发育的早期为4种，以簇状放电和连续单个放电为主，没有记录到不规则放电；胚后发育的后期为5种，以单个和簇状放电为主。单个放电和连续放电的幅度随着发育的进行而增大，在变态期达最大；连续单个放电和连续簇状放电频率在变态期、亚成体期和成体期相近，差异不显著。［结论］随着原始海马的发育，神经元电活动形式逐渐呈现多样化，变态期原始海马的发育程度接近亚成体、成体。

中华蟾蜍；原始大脑皮层；自发电活动；胚后

辽宁省教育厅资助项目（2011196）。

王维（1990-）女，辽宁葫芦岛人，硕士研究生在读，研究方向：发育神经生物学，E-mail：wangwei19900925@163.com。*通讯作者，E-mail：wangliwen1956@163.com。

2015-07-07

Supported bY grants from the Liaoning Provincial Department of Education（2011196）.

*Corresponding author.E-mail:wangliwen1956@163.com

Received:JulY 7，2015 Accepted:August 16，2015

修回日期 2015-08-16