ABSTRACT
Background Plasticity of visual system is one of the mechanisms of deprivation amblyopia.Our previous study showed that synapsin plays a role during visual developmental plasticity,and conventional protein kinase C-γ (cPKC-γ) probably is one of upstream kinases of synapsin.However,whether or how the cPKC-γ plays its effects on visual developmental plasticity is below understood.Objective This study was to investigate the dynamic expression of cPKC-γ in visual cortex of normal mice and explore the effects of abnormal visual experience on cPKC-γ expression.Methods The bilateral visual cortex tissues were obtained from 36 clean C57BL/6 mice at postnatal (P) 7,14,21,28,35,42 days respectively and 6 mice for each for the researching of cPKC-γ dynamical expression in visual cortex over aging.Other 24 C57BL/6 mice were randomized into developmental phase group and adult phase group,12 for each group.The monocular deprived (MD) models were established by suturing the upper and inferior eyelides in P14 mice for 14 days in 6 mice in the developmental phase group and 6 healthy mice served as controls,and MD models were established in the same way in 6 P60 mice in the adult phase group,and the same aged mice (6 mice) were used as controls.The mice were sacrificed and bilateral visual cortexes were obtained.The expression of cPKC-γ protein in the visual cortex was quantitatively detected using Western blot assay.The study protocol was approved by Ethic Committee of Tongren Eye Hospital.The use and care of the experimental mice followed the ARVO Statement.Results The cPKC-γ protein was faintly expressed in visual cortex in normal P7 mice,with the related expressing level of (39.74± 11.22)％ and (40.78± 10.37)％ in the left and right cortex,respectively.The expressing level of cPKC-γ protein was gradually increased over aging,with the peak value of (138.68±15.73)％ and (138.47±23.48)％ in P21 mice.A significant difference was found in the expression of cPKC-γ protein in various ages of mice (Fage =57.174,P =0.000),and the expression of cPKC-γ protein was not significantly different between the left and right visual cortexses (Flateral =0.059,P =0.809).No significant differences were found in the expression of cPKC-γ protein on bilateral visual cortexes among the mice of the developmental phase group and adult phase group (Fage =1.798,P =0.159) or among the MD group and normal control group (Fgroup =0.104,P=0.749).Conclusions The dynamic expression of cPKC-γ in the visual cortex of normal mice presents a consistant tend with the aging and development of visual critical period.MD does not affect the expression of cPKC-γ protein in bilateral visual cortexes.Further researches should be performed in the activity of cPKC-γ protein in MD mice.
ABSTRACT
Background The visual development is completed during the critical period in human and mammals.However,the critical period is not the initial of receiving visual experience.It is known that before the onset of critical period in mammals,such as mouse,there is an earlier stage for visual development,the pre-critical period.The research of response characteristics of the visual cortical neurons and the synaptic plasticity in the pre-critical period is still in the exploratory stage.Objective The study aimed to preliminarily investigate the response properties of neurons and synaptic plasticity in mouse visual cortex during the pre-critical period.Methods Fortyeight postnatal day 13-17 C57BL/6J mice were used for in vivo whole-cell recordings and in vitro brain slice wholecell recordings.In vivo whole-cell recordings were done in anesthetized mice.Moving bars in different directions were produced and controlled by a Matlab program.Cell recordings were obtained at the depth of layer Ⅳ of visual cortex.Step current stimuli under current clamp were given to measure the membrane response properties of neurons.Optimal visual stimuli were given to measure the in vivo largest responses of membrane potentials.In vitro experiments were performed after in vivo experiments.All cells were given current step stimuli to measure the membrane response properties of neurons.Different intensities of white-matter-to-layer-Ⅳpathway stimulation were given to measure the evoked response properties.All cells from 48 mice were randomized into 4 groups according to different stimulus training modes,including low frequency stimulation (LFS),high frequency theta-burst stimulation (TBS),pre-post synaptic timing stimulation (pre-post TS) and post-pre synaptic timing stimulation (post-pre TS).Under the voltage clamp of-70 mV,excitatory postsynaptic currents (EPSCs) before and after training were recorded to measure the plastic changes of excitatory synaptic connections.pClamp 10 was used for the pre-analysis of data and Matlab 2008a was used for statistical analysis.The use and care of the animals followed the Statement for the Use of Animals in Ophthalmic and Vision Research.Results Thirty-nine cells and 48 cells were successfully recorded in the in vivo and in vitro experiments,respectively.The steady-state average number of action potentials (APs) were (1.01 ± 0.03)/sweep and (1.01 ±0.05)/sweep,the AP thresholds were (-40.2 ± 3.2) mV and (-39.6 ±2.0) mV,and the threshold step current levels were (126.7 ± 17.4) pA and (129.6 ± 17.5) pA in the in vivo and in vitro recordings,respectively,with no significant differences between them (APs:t =0.512,P =0.610;AP thresholds:t =-1.074,P =0.286;current levels:t =-0.776,P =0.440).Under the optimal visual or pathway stimulation,the average peak response of membrane potentials was (7.3 ±4.3)mV and (6.4±2.8)mV with rarely evoked APs in the in vivo and in vitro experiments,respectively,with no significant difference between them (t =1.234,P =0.221).Under the in vitro recording,the EPSCs before LFS were [(138.1 ±51.9)pA],which was significantly higher than that after LFS [(76.1 ± 34.8)pA] (t=4.437,P=0.001),but no significant differences were seen in EPSCs before and after TBS (t=-0.756,P=0.466).The EPSCs before and after pre-post TS were (122.4±62.2)pA and (78.5±46.7)pA,and those before and after post-pre TS were (131.9 ±48.0) pA and (74.3 ± 30.7) pA,showing significant differences between them (pre-post TS:t =3.558,P =0.004;post-pre TS:t =4.283,P =0.001).Conclusions The construction of fundamental neural circuits in layer Ⅳ of mouse visual cortex is completed during pre-critical period.However,the membrane responsive capability of neurons and the synaptic connections are in an immature state,and the evoked responses to visual pathway inputs are basically subthreshold.The strength of synaptic connections is depressed with low frequency stimulation or pre-post/post-pre synaptic timing stimulation,and kept unchanged with high frequency stimulation.The development of visual neural system of PSP in mouse presents different characteristics from CP.