Visual Neuroscience Monocular Deprivation Affects Visual Cortex Plasticity Through cPKCγ-Modulated GluR1 Phosphorylation in Mice Yunxia Zhang,1 Tao Fu,2 Song Han,1 Yichao Ding,2 Jing Wang,2 Jiayin Zheng,1 and Junfa Li1 1Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University, Beijing, China 2Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China Correspondence: Tao Fu, Beijing PURPOSE. To determine how visual cortex plasticity changes after monocular deprivation Tongren Eye Center, Capital Medical (MD) in mice and whether conventional protein kinase C gamma (cPKCγ ) plays a role University, Beijing 100730, China; in visual cortex plasticity. [email protected]. Junfa Li, Department of METHODS. cPKCγ membrane translocation levels were quantified by using immunoblot- Neurobiology, School of Basic ting to explore the effects of MD on cPKCγ activation. Electrophysiology was used to Medical Sciences, Capital Medical record field excitatory postsynaptic potential (fEPSP) amplitude with the goal of observ- University, Beijing 100069, China; ing changes in visual cortex plasticity after MD. Immunoblotting was also used to deter- [email protected]. mine the phosphorylation levels of GluR1 at Ser831. Light transmission was analyzed Received: September 6, 2019 using electroretinography to examine the effects of MD and cPKCγ on mouse retinal Accepted: February 22, 2020 function. Published: April 28, 2020 RESULTS. Membrane translocation levels of cPKCγ significantly increased in the contralat- Citation: Zhang Y, Fu T, Han S, et al. eral visual cortex of MD mice compared to wild-type (WT) mice (P < 0.001). In the Monocular deprivation affects visual contralateral visual cortex, long-term potentiation (LTP) and the phosphorylation levels cortex plasticity through of GluR1 at Ser 831 were increased in cPKCγ +/+ mice after MD. Interestingly, these γ cPKC -modulated GluR1 levels could be downregulated by cPKCγ knockout compared to cPKCγ +/++MD mice (P phosphorylation in mice. Invest < 0.001). Compared to the right eyes of WT mice, the amplitudes of a-waves and b-waves Ophthalmol Vis Sci. 2020;61(4):44. < https://doi.org/10.1167/iovs.61.4.44 declined in deprived right eyes of mice after MD (P 0.001). There were no significant differences when comparing cPKCγ +/+ and cPKCγ −/− mice with MD. CONCLUSIONS. cPKCγ participates in the plasticity of the visual cortex after MD, which is characterized by increased LTP in the contralateral visual cortex, which may be a result of cPKCγ -mediated phosphorylation of GluR1 at Ser 831. Keywords: visual cortex plasticity, monocular deprivation, long-term potentiation, cPKCγ ynaptic plasticity is crucial to our responding flexibly to visual cortex plasticity changes after MD is incompletely S various environments by changing synaptic connections understood. between neurons.1 Activity-dependent synaptic remodeling Conventional protein kinase C gamma (cPKCγ ), a is a key mechanism mediating neural circuit adaptation and member of the PKC family of Ser/Thr kinases that regu- brain plasticity.2 When visual experience regulating visual late a series of cellular processes, is specifically expressed cortical circuits was deprived, the plasticity of the visual in neurons, and cPKCγ may play a key role in synaptic cortex changed.3,4 plasticity. In mutant cPKCγ mice, cPKCγ membrane translo- In animals, monocular deprivation (MD), which was cation kinetics are impaired, which may destroy synap- reported in 1963 to induce amblyopia,5 is often used to tic plasticity, synapse pruning, and synaptic transmission.10 study visual cortex plasticity. Studies using mice have found The offspring of male mice exposed to postnatal traumatic that MD induces robust plasticity of the visual cortex during stress have decreased signaling components of cPKCγ in the critical periods through degradation of the extracellular hippocampus which damages synaptic plasticity when they matrix by upregulation of proteases, thus elevating spine reach adulthood.11 cPKCγ mediates post-tetanic potentiation motility.6 Similar studies using P17 mice have shown that (PTP) by increasing the probability of release in the auditory MD strengthens excitatory synaptic connections of layer 47 brainstem12 and maintains the function of Purkinje cells by and induces plasticity in layers 2 and 3 of the deprived regulating the phosphorylation and insertion of GABAAγ 2 cortex.8 However, some studies have shown that, after MD, into the postsynaptic membrane.13 The activation of cPKCγ miniature inhibitory postsynaptic currents (IPSCs) and the may mediate the neuroprotective effects of resveratrol and density of postsynaptic GABAA receptors were increased in epigallocatechin gallate polyphenols on the cytoskeleton, layer 4 of the visual cortex,9 indicating that the plasticity as well as synaptic plasticity.14 In inflammation-induced of the visual cortex was decreased after MD. Thus, how mechanical allodynia, interneurons expressing cPKCγ are Copyright 2020 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution 4.0 International License. Downloaded from iovs.arvojournals.org on 09/29/2021 cPKCγ -Modulation on Visual Cortex Plasticity IOVS | April 2020 | Vol. 61 | No. 4 | Article 44 | 2 involved in strong morphological reorganization via 5-HT2A 100 in 0.1-M PBS; Sigma-Aldrich, St. Louis, MO, USA) for receptor activation.15 However, few studies have investigated 30 minutes and then incubated with 8% goat serum in PBS the roles of cPKCγ in visual cortex plasticity. In our previous (0.1-M) for 1 hour at room temperature. Primary mouse anti- work, cPKCγ knockout significantly changed the dynamic bodies against the neuron-specific marker NeuN (ab104224, expression of P-synapsin-Ia/b at sites Ser603 and Ser9,16 1:300; Abcam, Cambridge, UK) were added at 4°C overnight. which may play a role in the synaptic plasticity of the visual Following washing with PBS six times for 10 minutes each, cortex.17 Here, we further explore whether cPKCγ partici- the slices were incubated in Alexa Fluor 488 goat anti-mouse pates in visual cortex plasticity in MD mice. IgG (A11029, 1:300; Thermo Fisher Scientific, Watham, MA, USA) secondary antibody for 2 hours at room tempera- ture. Finally, 2-(4-amidinophenyl)-6-indolecarbamidine dihy- MATERIALS AND METHODS drochloride containing mounting media was used to mount Animals and Monocular Deprivation slices. Microphotographs were taken using a Leica micro- scope, and images of the visual cortex were taken for + + The C57BL/6J wild-type (WT, cPKCγ / )andcPKCγ knock- each slice. Image J software (National Institutes of Health, − − out (KO, cPKCγ / ) mice were purchased from The Jack- Bethesda, MD, USA) was used to blindly count the number son Laboratory (Bar Harbor, ME, USA). All animals were of NeuN-staining positive cells per image in a blind housed in a barrier system with constant temperature and manner. humidity and a 12-hour light/dark cycle, and they were exposed to food and water. All procedures were performed according to the ARVO Statement for the Use of Animals in Immunoblotting Ophthalmic and Vision Research and adhered to the guide- Brain tissue from all four groups and regions containing lines required by Animal Care and Use Committee of Capital the visual cortex were collected. The mouse visual cortex Medical University. was dissected and rapidly frozen in liquid nitrogen. Based MD was implemented by eyelid suturing at postnatal day on our previous work,18,19 frozen samples were thawed 7 (P7) to imitate amblyopia caused by congenital cataracts and homogenized in Buffer A (50-mM Tris-Cl, pH 7.5; 1- in mice. Animals were anesthetized by intraperitoneal (IP) mM EGTA; 2 mM-EDTA; 100-μM sodium vanadate; 50-nM injection of pentobarbital sodium (0.06 g/kg). Erythromycin okadaic acid; 50-mM potassium fluoride; 5-mM sodium eye ointment (Guangzhou Baiyunshan Pharmaceutical Co., pyrophosphate; and 5 μg/μL each of pepstatin A, chymo- Ltd., Guangzhou, China) was given to MD mice to prevent statin, leupeptin, and aprotinin). Homogenates were then infection. Mice were then housed for P21 days. centrifuged at 100,000g for 30 minutes at 4°C, and the super- Seventy-two male and female mice were randomly natants were collected as the cytosolic fraction. The pellets + + + + divided into four groups: cPKCγ / ,cPKCγ / +MD were resuspended, sonicated, and completely dissolved in + + − − − − (cPKCγ / mice with MD), cPKCγ / ,andcPKCγ / +MD Buffer C (Buffer A containing 2% SDS20) as the membrane − − (cPKCγ / mice with MD). The visual cortexes of mice in fraction. The cytosolic and membrane fractions were used + + the cPKCγ / and MD groups were prepared to determine to investigate membrane translocation of cPKCγ , and the the membrane translocation of cPKCγ after electroretinog- membrane fractions were used to analyze phosphorylated raphy (n = 6 per group). The visual cortexes of mice in GluR1 (pGluR1) at Ser 831 levels. Protein concentrations the four groups was prepared to determine GluR1 phospho- were determined using a bicinchoninic acid kit (Pierce rylation after electroretinography (n = 6 per group). Mice Biotechnology, Rockford, IL, USA). Albumin dissolved in + + + + in the cPKCγ / and cPKCγ / +MD groups were used for Buffer A or C at various concentration was used as the stan- immunofluorescence staining (n = 6 per group). Mice in all dard. four groups were also used for electrophysiology (n = 6per Protein samples (10 μg/lane) were separated using 10% group). SDS-PAGE, and the proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (0.22 μm; Immunofluorescence GE Healthcare, Chicago, IL, USA). The transferred PVDF membrane was blocked in 10% no-fat milk in A Tween- Mice at P21 days were anesthetized using 1% pentobarbital 20 (Sigma-Aldrich)/Tris-buffered salt solution (TTBS; 20- sodium (0.07 g/kg, IP injection) and perfused transcardially mM Tris-Cl, pH7.5; 0.15-M NaCl; and 0.05% Tween-20) with 0.9% NaCl for 1 minute followed by 4% paraformalde- for 1 hour at room temperature. After washing in TTBS hyde in 0.1-M PBS (pH 7.4) for 15 minutes.