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Conserved Cellular Function and Stress-Mediated Regulation Among Members of the Proteolipid Protein

Conserved Cellular Function and

Stress-Mediated Regulation Among Members of the Proteolipid Protein Family

Mar?′a E.Ferna′ndez,*Julieta Alfonso,Marcela A.Brocco,and Alberto C.Frasch IIB-INTECH-CONICET-UNSAM,San Martin,Buenos Aires,Argentina

Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews,including atrophy of CA3dendrites and loss of synapses.The molecular mechanisms underlying these structural changes remain largely unknown.We have previously identi?ed M6a as a stress responsive gene and shown that M6a is involved in?lopodium/spine outgrowth and, likely,synapse formation.M6a belongs to the proteoli-pid protein(PLP)family,all of their members having four transmembrane domains that allow their localization at the plasma membrane.In the present work,we ana-lyzed other members of this family,the closely related M6b as well as PLP and its splice variant DM20.We found that chronic restraint stress in mice reduces M6b and DM20,but not PLP,mRNA levels in the hippocam-pus.In addition,M6b and DM20,but again not PLP, induce?lopodium formation in primary cultures of hip-pocampal neurons.Several M6b protein isoforms were studied,all of them having similar effects except for the one lacking the transmembrane domains.Our results reveal a conserved cellular function and a stress-medi-ated regulation among members of the proteolipid pro-tein family,suggesting an involvement of proteolipid proteins in the stress response.V C2009Wiley-Liss,Inc. Key words:proteolipid proteins;stress;hippocampus;?lopodium formation;mouse

Prolonged exposure to stressful situations can bring severe consequences for the brain,conferring susceptibil-ity to certain psychiatric disorders.In fact,chronic stress is one of the main factors known to trigger depression in humans(Kendler et al.,1999).The hippocampal formation,which possesses a remarkable degree of plas-ticity,is particularly sensitive to stress.Morphological alterations provoked by chronic stress have been well documented in the hippocampus of rodents and tree shrews.Stressed animals display a reduction of apical dendritic branching and total dendritic length in CA3 pyramidal neurons as well as reorganization within the mossy?ber terminals(MFT;for review see McEwen et al.,2002).Chronic stress has been also found to induce retraction of thorny excrescences and loss of syn-apses in CA3neurons(Sandi et al.,2003;Stewart et al., 2005).It is worth noting that antidepressant treatment can prevent most of the described stress effects(McEwen et al.,2002).Although there has been important progress regarding the understanding of the stress response(for review see Ising and Holsboer,2006;McClung and Nestler,2008),the molecular pathways underlying these plastic alterations remain largely unknown.

We have previously identi?ed M6a as a stress responsive gene.M6a expression was found to be down-regulated in the hippocampus of both socially and physically stressed animals,and this effect could be coun-teracted by antidepressant administration(Alfonso et al., 2004,2006).We have also studied possible biological functions for the glycoprotein M6a and have found that it is involved in neurite outgrowth and?lopodium/spine formation(Alfonso et al.,2005).

M6a belongs to the myelin proteolipid protein (PLP)family.In mammals,other members of this family include the closely related M6b and the founder PLP, with its splice variant DM20.M6b was identi?ed several years ago and was found to be expressed both in neurons and in glia(Yan et al.,1993,1996).Several isoforms have been described(Werner et al.,2001);however,the cellular function of the protein remains unknown.PLP and DM20constitute the most abundant proteins in the

Mar?′a E.Ferna′ndez’s current address is Biozentrum,University of Basel, Klingelbergstrasse50/70(CH-4056)Basel,Switzerland.

Julieta Alfonso’s current address is Department of Molecular Neuro-science,Max-Planck-Institute for Medical Research,69120Heidelberg, Germany.

Contract grant sponsor:International Research Scholars Grant from the Howard Hughes Medical Institute;Contract grant sponsor:Agencia Nacional de Promocio′n Cient?′?ca y Tecnolo′gica(ANPCyT);Contract grant sponsor:National Research Council(CONICET)of Argentina(to M.E.F.,J.A.,A.C.F.).

*Correspondence to:Mar?′a E.Ferna′ndez,Universidad Nacional de San Mart?′n,Av.Gral Paz5445,INTI Edi?cio24,1650San Martin,Buenos Aires,Argentina.E-mail:mefernan@https://www.wendangku.net/doc/cc16834356.html,.ar

Additional Supporting Information may be found in the online version of this article.

Received7May2009;Revised9September2009;Accepted14 September2009

Published online23November2009in Wiley InterScience(www. https://www.wendangku.net/doc/cc16834356.html,).DOI:

10.1002/jnr.22298

Journal of Neuroscience Research88:1298–1308(2010) '2009Wiley-Liss,Inc.

CNS myelin(Mikoshiba et al.,1991).PLP was identi-?ed over50years ago(Folch et al.,1951),and in the mouse brain its expression is con?ned to oligodendro-cytes(Yan et al.,1996).It is generally accepted that one of the biological functions of PLP/DM20is to maintain the structural integrity of the myelin membrane(Duncan et al.,1987;Boison and Stoffel,1994;Boison et al., 1995).PLP and DM20have also been involved in sur-vival and differentiation of oligodendrocytes(Yang and Skoff,1997;Nadon and West,1998).Given the results previously obtained with M6a,the aim of this work was to study the effect of chronic stress on the expression of other members of this protein family,namely,M6b and PLP/DM20,in the hippocampal formation.We also assessed their capacity to induce?lopodium formation.

MATERIALS AND METHODS

Animals

Inbred male C57BL/6mice were acquired from the Instituto de Investigaciones Biotecnolo′gicas(IIB,San Mart?′n, Argentina).Animals were between2and3months old.The experimental procedures reported here were approved by the Animal Care Committee of the Instituto de Investigaciones Biotecnolo′gicas of the National Council for Research of Ar-gentina and were carried out in accordance with the guide-lines laid down by the U.S.National Institutes of Health.

Stress Procedure

All the animals were housed in groups and maintained on a12/12-hr light/dark cycle under controlled temperatures between188C and228C.Food and water were available ad libitum.Stressed animals(n59)were restrained daily(from 11AM to3PM)during21days in well-ventilated polypropyl-ene tubes(2.8cm diameter311.5cm length)without access to food and water.Animals were not physically compressed and did not experience pain.Animals from the control group (n59)were housed under normal conditions.Consistently with previous data(Magarinos and McEwen,1995a),animals subjected to this stress procedure showed a signi?cant reduc-tion in body weight gain after3weeks of treatment(control: 108.4462.6,stress:95.8461.7expressed as a percentage of initial body weight;P<0.01,determined with Student’s t-test).

Hippocampal mRNA Isolation and cDNA Synthesis One day after the last stress session,animals were killed by cervical dislocation.Left and right hippocampi were surgi-cally removed and stored in liquid nitrogen.Tissues were ho-mogenized in Trizol reagent(Life Technologies,Rockville, NY),and total RNA was isolated following the manufac-turer’s instructions.Poly-A1mRNA was isolated from total RNA using the PolyATract mRNA Isolation System (Promega,Madison,WI).Complementary DNA was synthe-sized by retrotranscription using oligo-dT and SuperScriptTM II Reverse Transcriptase enzyme(Life Technologies)accord-ing to the manufacturer’s instructions.Quantitative Real-Time Reverse

Transcription-Polymerase Chain Reaction

Real-time polymerase chain reactions(PCRs)were car-ried out in a GeneAmp5700Sequence Detection System (Applied Biosystems,Foster City,CA).cDNA amounts pres-ent in each sample were determined by using the SYBR Green PCR Core Reagents kit(Applied Biosystems).Each reverse transcription-PCR(RT-PCR)quanti?cation experiment was performed in duplicate.Primer sequences were designed in Primer Express software(Applied Biosystems).Each SYBR Green reaction contained4l l of diluted cDNA as template. The?nal concentrations of the reagents were:13SYBR Green Reaction Buffer,3mM MgCl,1mM dATP,1mM dUTP,1mM dCTP,1mM dGTP,0.3l M of each primer, 0.01U/ml UNG-Enzyme,and0.025U/ml Taq Gold DNA Polymerase(SYBR Green PCR Core Reagents;PE Biosys-tems)The reactions were incubated at508C for2min to acti-vate the uracil N0-glycosylase and then for10min at958C to inactivate this enzyme and activate the Amplitaq Gold poly-merase,followed by40cycles at958C for15sec(denaturation) and at608C for1min(annealing and extension).After the ?nal cycle of the PCR,the reactions were subjected to a heat dissociation protocol(heat denatured over a358C temperature gradient at0.038C/sec from60–958C),to verify that the SYBR Green dye detected only one PCR product.Oligonu-cleotide sequences used were:50AAA TAA TGA TGT AGC CTG ACA AGA AAT TT30and50AAT GCA CTT ACA CTG AAG GAG GAA T30,forward and reverse primers for M6a;50AGC AGT GGA GGT GCT GTT AAG AGT30and 50GGC TTT AGA CAC CGC CTC TTC T30,forward and reverse primers for M6b30UTR;50AAT TGT TTA TCT CTT GTT TGG AGT TGT ATC30and50AGA ATC ATC CTC CAG GTC TTT CTG30,forward and reverse primers for PLP/DM2030UTR;50GGC CTG AGC GCA ACG GTA30,50GGC CTG AGC GCA ACG TTT G30and50 AGG AGC CAT ACA ACA GTC AG30,forward PLP,for-ward DM20and reverse PLP/DM20respectively;50TCC CGT CAG TCT CCA ACC A30and50CCC AGA CAC TTG ATG CAG CA30,forward and reverse M6b exon Ia;50 TAT GGT CGC CTG CTC CTT G30and50CGC TCT GCC TAC TGG TCC A30,forward and reverse for M6b exon Ib;50AAA TAG ATT CAG GAT GCC ACA CC30 and50AGT TAG CAG TCC CAT CTT CCC A30,forward and reverse for M6b exon III;50AAG CAT ACA GGT CCT GGC ATC T30and50CAT TCA GTC TTG GCA GTG CAG30,forward and reverse primers for cyclophilin,50TGA CCA ATT CCA TAC TCC ATG GT30and50ATT CTA GCT GCT GTG CTT GCC T30,forward and reverse pri-mers for glucose-6-phosphate-dehydrogenase(G6PDH).For data normalization,we?rst measured mRNA levels for the reference genes cyclophilin and G6PDH.Values shown in Results were calculated with cyclophilin as reference gene. Normalizations with G6PDH resulted in almost identical data. Group means were analyzed for statistical signi?cance with Student’s t-test,using the software Analyse-It for Microsoft Excel(Analyse-It Software Ltd.,Leeds,United Kingdom). Relative isoform expression level was calculated as previously described(Pfaf?,2001).

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Hippocampal Cultures and Cell Lines

Dissociated neuronal cultures were prepared from hip-

pocampi of embryonic day19Wistar rats,as described by Brocco et al.(2003).Brie?y,tissue was treated with0.25%

trypsin in Hanks’solution for15min at378C.A single-cell

solution was prepared in MEM containing2mM glutamine, 10l M sodium pyruvate,100units/ml penicillin,and100l g/

ml streptomycin(MEM13l)with10%(vol/vol)horse serum.

Cells were seeded on coverslips coated with0.1mg/ml poly-(L-lysine)hydrobromide(Sigma,St.Louis,MO)and20l g/ l l laminin(Gibco,Grand Island,NY)at a density of20,000 cells/cm2.After2hr,medium was changed to MEM/N2 (MEM13with1g/liter ovalbumin and B27serum-free sup-

plements from Gibco).In B27-supplemented cultures,glial

growth is reduced to less than0.5%of cultured cells(Brewer

et al.,1993).Mouse neuroblastoma2a(N2a)and COS-7cells were cultured in DMEM with10–20%(vol/vol)fetal bovine serum,penicillin,and streptomycin.

Plasmids

GFP::M6b TMD x,GFP::M6b TMD/,GFP::M6b a TMD x,GFP::M6b a TMD/,GFP::M6b ag,GFP::PLP, and GFP::DM20:cDNAs were cloned by RT-PCR using adult mouse hippocampal cDNA as a template.Primers were designed to amplify the complete coding sequences(CDS) from initial ATG to stop codon.M6b forward primers contain the restriction recognition site for XhoI,and reverse primers contain the restriction recognition site for BamHI to allow in-frame cloning into XhoI/BamHI sites present in the multiple cloning sites(MCS)of EGFP-C1vector(Clontech,Logan, UT).PLP and DM20were ampli?ed with XhoI and EcoRI recognition sites at their ends to allow in-frame cloning into XhoI/EcoRI sites present in the MCS of EGFP-C1.Oligo-nucleotide sequences used were:50CTC GAG CC A TG G GTT GCT TCG AAT GCT30M6b forward a,50CTC GAG GT A TG A AGC CAG CCA TGG AAA C30M6b forward b(a domain),50GGA TCC TTA AGT GTA AGA ATT GAG TTG TTC T30M6b reverse x,50GGA TCC TTA GAA CTT AGT GCA GCA GTC TT30M6b reverse /,50GGA TCC TTA GCA GTC CCA TCT TCC CA30 M6b reverse g,50GTC TCG AGC T AT G GG CTT GTT AGA GTG TTG30forward PLP/DM20and50ACG AAT TC T CA G AAC TTG GTG CCT CG30reverse PLP/ DM20.All PCRs were performed with an annealing tempera-ture of558C.GFP::M6a has been described previously (Alfonso et al.,2005).VSVG::GFP:thermosensitive(ts045)G protein of vesicular stomatitis virus(VSVG)cDNA(kindly provided by Dr.Maccioni,Universidad de Co′rdoba,Argen-tina)was cloned in frame into the EcoRI/BamHI sites present in the MCS of EGFP-N1.

Transfections

For transfections,2l g of plasmid DNA was mixed with 1l l or2l l(for neurons or cell lines,respectively)of Lipo-fectamine2000(Invitrogen,La Jolla,CA)and added to each well in a24-well per plate format according to the manufac-turer’s instructions.Media were changed3–5hr later.Immuno?uorescence and Image Analysis

Neurons and neuroblastoma2a(N2a)and COS7cells were?xed in4%paraformaldehyde/4%sucrose in PBS for20 min at room temperature.For F-actin staining,permeabiliza-tion was carried out with0.1%Triton X-100in PBS for2 min.Cultures were blocked with3%BSA in PBS for1–2hr, followed by incubation with the rhodamine phalloidin1/ 1,000(Molecular Probes,Eugene,OR)in3%BSA in PBS at 378C for1hr.Fluorescent images were acquired by using a Nikon E600microscope equipped with epi?uorescence illu-mination(Nikon)or a confocal laser scanning microscope (Olympus FV300)with a360oil-immersion lens.Filopo-dium density(number of protrusions per20l m neurite length measured within50l m from the soma)was quanti?ed in over80neurites of different neurons(one or two neurites per neuron)per group.Neurons were distinguished from glial cells(which almost do not grow under the conditions used) based on morphology observation.For cell lines,the percent-age of cells showing?lopodial protrusions was calculated from over100cells per coverslip.At least six coverslips from multi-ple experiments were used.Group means were then analyzed for overall statistical signi?cance by using Student’s t-test or ANOVA followed by Dunnet’s post hoc test.Calculations were performed with the software Analyse-It for Microsoft Excel.

RESULTS

M6b and DM20Expression is Down-Regulated in the Hippocampus of Chronically Stressed Mice In mammals,the PLP family is constituted by M6a, M6b,and PLP/DM20(Yan et al.,1993).According to prediction models,these proteins share a common struc-ture composed by four hydrophobic transmembrane (TM)domains,separated by three hydrophilic loops, with the N and the C termini facing the cytosol(Popot et al.,1991;Yan et al.,1993).Figure1shows the amino acid identities in the region encompassed by the four membrane spans(gray boxes),the small extracellular loop(EC I),the intracellular loop(IC),and the large extracellular loop(EC II).Upon analysis of the entire region,M6a shares greater identity with M6b than with DM20.PLP,having35extra amino acids encoded by alternative exon3b,shows lower identity with M6a. When each region is analyzed separately,uneven similar-ities are found.The greatest identity is observed in the ?rst TM domain,with values of82%for M6b and67% for PLP and DM20with respect to M6a.Although the IC and the second TM span still share high identity (with values ranging from55%and65%),no signi?cant similarities(NSS)were found in the EC I and the fourth TM region.In the third TM domain,homology is found only between M6a and M6b,whereas,in the EC II,only PLP and DM20show signi?cant similarity to M6a.

To evaluate the effect of stress on the expression of genes of the PLP family in the hippocampus,we used chronic restraint stress in mice.mRNA levels were measured using real-time RT-PCR.As shown in Figure

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2b,we found a reduction in M6b expression level in the hippocampus of chronically stressed mice.In agreement with our previous ?ndings (Alfonso et al.,2006),we also found a reduction in the mRNA levels for M6a (Fig.2a).As mentioned above,several M6b isoforms have been identi?ed (Werner et al.,2001).In our assay (Fig.2b),we measured all M6b isoforms,because the primers used are located at the 30UTR,a region shared by all the M6b mRNAs described.The different M6b mRNA molecules arise from alternative transcription starting site in combination with alternative splicing (Werner et al.,2001).The presence or absence of the a domain at the N terminus of the protein is determined by the transcription starting site used.Alternative splicing is responsible for the C terminal domains x or /.Inclu-sion of alternative exon III gives origin to an mRNA coding for a short protein with no transmembrane domains (TMD)and a g domain at the C terminus (see schemes for the different proteins in Fig.3).To deter-mine the relative contribution of each M6b isoform to the total M6b mRNA level,we measured the relative expression level of several isoforms independently in the hippocampus of control mice.Speci?c primers were designed for exons Ia and Ib (which represent the alter-native transcription starting sites)as well as for exon III,which is present only in mRNAs coding for short pro-teins with no TMD.As shown in Figure 2c,we found similar levels of mRNA molecules with exon Ia and exon Ib,which might indicate similar use of the two al-ternative transcription starting sites.On the other hand,the amount of mRNAs containing exon III was much lower,indicating that M6b isoforms with no TMD are not signi?cantly expressed and cannot,therefore,account for the reduction observed in total M6b expression in the hippocampus of chronically stressed animals.

In the case of PLP and DM20,we ?rst measured mRNA levels with primers at the 30UTR that recognize both mRNA molecules,and no signi?cant differences

between control and stressed animals were observed (Fig.2d).When quantifying the relative expression level of each transcript,we found that PLP expression was about 10times higher than DM20expression (Fig.2e).When each mRNA was assayed separately in control and treated animals,DM20was found to be down-regu-lated in the hippocampus of chronically stressed mice (Fig.2f).On the contrary,no signi?cant differences were found for PLP (Fig.2g),explaining the results obtained when both splice variants were measured to-gether.

Cloning and Expression of M6b and PLP Isoforms

In a previous study,we have shown that one mem-ber of this protein family,the glycoprotein M6a,promotes neurite outgrowth and ?lopodium/spine formation in pri-mary cultures of hippocampal neurons and in different cell lines (Alfonso et al.,2005).To study possible functions of M6b and PLP proteins in ?lopodium induction,we ?rst cloned different isoforms of M6b and PLP and analyzed their cellular localization,as shown in Figure 3.In the case of M6b,from the eight species described in the literature (Werner et al.,2001),?ve were cloned by RT-PCR from mouse hippocampus:M6b TMD x ,M6b TMD /,M6b a TMD x ,M6b a TMD /,and M6b ag .Molecules con-taining the b domain in the N cytosolic region,which is encoded by alternative exon II,failed to be ampli?ed.We also cloned the complete coding sequence for PLP and DM20isoforms.cDNAs coding for the different isoforms were cloned in frame with the green ?uorescent protein (GFP)coding region.To observe the cellular localization of the fusion proteins,cultured hippocampal neurons and neuroblastoma 2a (N2a)cells were transfected with each of the indicated recombinant DNAs.As has been previ-ously described for the endogenous protein (Werner et al.,2001),GFP::M6b TMD x can be observed in intracellular structures and at the cell surface of neurons (Fig.3a,b).

Fig.1.Amino acid identities among members of the PLP family.Schematic representations showing the amino acid identities among the transmembrane domains (gray boxes),extracellular loops (EC),and intracellular loops (IC)of M6b,DM20,and PLP relative to M6a.Amino acid identity in each domain is shown below each dia-gram.Amino acid identity in the entire region is shown between brackets.3b,Region coded by alternative exon 3b;NSS,no signi?-cant similarity;NT,N terminus;CT,C terminus.

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shown in Figure 3c,confocal images of N2a-transfected cells suggest a membrane localization of the fusion protein.GFP::M6b TMD /,GFP::M6b a TMD x ,and GFP::M6b a TMD /showed the same localization (Fig.3d–l).GFP::M6b ag ,lacking the TMD,is found in the cytosol (Fig.3m–o).GFP::PLP and GFP::DM20also localize in accordance to what was previously described for the endogenous proteins (Thomson et al.,1997).Both pro-teins show a similar localization,including some presence in intracellular structures as well as a prominent expression at the cell surface,presumably the cell membrane (Fig.3p–u).VSVG,a protein known to be located at the cell membrane (Vasserman et al.,2006),was used as a con-trol (Fig.3v–x).

M6b and DM20Overexpression Induces

Filopodium Formation in Cultured Hippocampal Neurons and in Cell Lines

To assess the ability of M6b to promote ?lopodium formation,different M6b isoforms were overexpressed in primary cultures of hippocampal neurons.Hippocampal neurons were transfected with GFP,GFP::M6a,or con-structs of GFP fused to different M6b isoforms with TMD.One day after transfection,neurons were ?xed,and F-actin was stained with phalloidin to label ?lopo-dia/spines (Fig.4a).Between 3and 8days in vitro (DIV),dendritic protrusions are mainly ?lopodia,which may later evolve into dendritic spines (Ziv and Smith,1996).As shown in Figure 4a,membrane protrusions contain an F-actin cytoskeleton.We quanti?ed the number of membrane protrusions per 20l m dendritic length.Figure 4b shows that all the M6b isoforms with TMD studied induced ?lopodium formation,similarly to M6a.

Next,we analyzed the capacity of PLP and DM20to modify ?lopodium density using the same approach described above.Hippocampal neurons were transfected with GFP,GFP::M6b ag ,VSVG::GFP,GFP::M6a,GFP::PLP,or GFP::DM20,and the numbers of ?lopo-dia were counted (Fig.5a,b).We found that

DM20

Fig.2.Stress effect on PLPs expression in the hippocampus.Male

C57BL/6mice were either untreated control subjects (n 59,black bars)or physically stressed during 3weeks (n 59,white bars).mRNA samples from hippocampal formation were used for cDNA synthesis and quanti?cation of M6a (a ),M6b (b ),PLP/DM20(d ),DM20(f ),and PLP (g )expression levels by real-time PCR.mRNA values for each individual were normalized with the reference gene cyclophilin.Results show the mean value of the groups (6SEM)as a percentage of the control group.Signi?cant differences were deter-mined by Student’s t -test,two tailed.*P <0.05,**P <0.01.To quantify the relative expression of M6b isoforms (c ),primers coding for alternative exons Ia,Ib,or III were used.Results are expressed as mean value of three control animals for the isoform (6SEM)as a percentage of exon Ib.All M6b protein isoforms described in the lit-erature are included in the ?gure.TMD,transmembrane domains.To quantify the relative expression between PLP and DM20(e ),a common reverse primer recognizing both PLP and DM20was used together with speci?c forward primers for PLP or DM20mRNA molecules.Results are expressed as mean value for the isoform (6SEM)of nine control animals as a percentage of PLP.

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overexpression promoted ?lopodium formation,although ?lopodium density induced by DM20was not as high as that promoted by M6a (Fig.5b).On the other hand,overexpression of PLP did not result in an increase in ?lopodium density.To assess the importance of the membrane localization and/or the TMD,we used M6b ag ,which lacks the hydrophobic transmembrane spans.Overexpression of GFP::M6b ag did not result in ?lo-podium formation,suggesting that M6b subcellular local-ization is crucial for its function.To test whether mem-brane localization alone would lead to ?lopodium for-mation,we utilized VSVG.This control protein did not induce ?lopodium formation,demonstrating a speci?c effect of M6proteins and DM20.

With the aim of studying whether the capacity of M6b and DM20to induce ?lopodium formation is re-stricted to cultured hippocampal neurons,different trans-fected cell lines were analyzed.The neuronal N2a and the nonneuronal COS7cell lines were used (Fig.6;https://www.wendangku.net/doc/cc16834356.html,.Fig.1).As shown in Figure 6b,c,GFP alone did not promote ?lopodium formation (negative con-trol).Conversely,the percentage of cells with

?lopodia

Fig.4.Overexpression of M6b isoforms containing TMD increases ?lopodium density in hippocampal neurons.a :Cultured hippocampal neurons were transfected 7days after plating with GFP,GFP::M6a,GFP::M6b TMD x ,GFP::M6b TMD /,GFP::M6b a TMD x ,or GFP::M6b a TMD /.One day later,neurons were ?xed,and F-actin was stained with phalloidin.b :Filopodium density (number of pro-trusions per 20l m neurite length)as shown in A was quanti?ed.Results are expressed as mean value of the group (6SEM)of at least 80neurites and as a percentage of the control group (GFP).Signi?-cant differences were determined by using one-way ANOVA fol-lowed by Dunnet’s post hoc test.**P <0.01against GFP.No sig-ni?cant differences were found between M6a and M6b isoforms with TMD.Figure 4b shows the results of one assay,representative of two independent experiments.Scale bar 510l

Fig.3.Cellular localization of overexpressed M6b and PLP isoforms.The schematic representations on the left show the different cDNAs that were cloned in frame with the coding region of GFP.Plasmids encoding the fusion proteins were transfected into hippocampal neu-rons and N2a cells.The pictures on the right are confocal images showing the localization of the fusion proteins in neurons (a,d,g,j,m,p,s,v ;enlargements b,e,h,k,n,q,t,w )and N2a cells (c,f,i,l,o,r,u,x ).VSVG,membrane protein used as control;TMD,transmembrane domains;3b,alternative spliced exon present in PLP.Scale bars 510l m.

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was increased in cells overexpressing M6a,M6b isoforms with the TMD,or DM20.In both cells lines,the differ-ent M6b isoforms containing the TMD strongly induced ?lopodium formation,similarly to M6a.In accordance with what was found in hippocampal neurons,DM20weakly induced ?lopodium formation compared with M6a in N2a.On the other hand,in COS7cells (Fig.6c),DM20strongly promoted ?lopodium formation,similarly to M6a.

DISCUSSION

In the present work,we found that chronic stress signi?cantly reduces the hippocampal expression of M6b and DM20,whereas PLP levels remain unaltered.We

also observed that four M6b isoforms differing in their

N and C terminal domains but harboring the hydropho-bic spans promoted ?lopodium outgrowth in hippocam-pal neurons.The level of ?lopodium induction was sim-ilar between the different M6b TMD isoforms and with respect to M6a.Therefore,the distinct N and C termi-nal domains are not the determinants for this cellular function.The cytosolic M6b protein isoform did not augment ?lopodium density,demonstrating that the membrane domain is essential for M6b function.In cul-tured hippocampal neurons,DM20,but not PLP,pro-moted ?lopodium outgrowth.However,the ?lopodium density induced by DM20was not as high as that induced by M6a.M6b and DM20induced ?lopodium formation,not only in hippocampal neurons,but also in the neuronal cell line N2a and the nonneuronal cell line COS7.

Potential Involvement of PLPs in Neuronal

Remodelling in the Hippocampus of Chronically Stressed Animals

Chronic stress induces neuronal remodelling in the hippocampus,which is characterized by a reduction of apical dendritic branching and total dendritic length as well as a marked retraction of thorny excrescences and reduced synaptic density in CA3pyramidal neurons (Watanabe et al.,1992;Magarinos et al.,1996;Sousa et al.,2000;Sandi et al.,2003;Stewart et al.,2005).Structural reorganization in the mossy ?ber terminals (MFT),including a reduction in the plasmalammelal sur-face area and in the number of synapses between MFT and CA3pyramidal neurons,has also been described in the hippocampus of chronically stressed animals (Magar-inos et al.,1997;Sousa et al.,2000).

In previous studies,we found that M6a expression was reduced in the hippocampal formation of animals subjected to chronic stress (Alfonso et al.,2004,2006).Moreover,the number of neurites and ?lopodium/spine-like protrusions,as well as the synaptic cluster den-sity in hippocampal neurons,was found to be modulated by the levels of M6a expression (Alfonso et al.,2005).This led us to the conclusion that reduced expression of M6a could be,at least in part,responsible for the neuro-plastic changes observed in hippocampal neurons of chronically stressed animals.

In the present work,M6b expression was also found to be down-regulated in hippocampal tissue of chronically stressed mice.In addition,M6b isoforms with TMD proved to be inducers of ?lopodium forma-tion in cultured hippocampal neurons.These ?ndings,together with the fact that M6b shows a high level of expression in CA3pyramidal neurons in the hippocam-pus (Yan et al.,1996),suggest that reduced expression of M6b can also contribute to the plastic alterations found in the hippocampus of animals subjected to chronic stress.

A recent paper states that,in the adult rat brain,M6a is localized to distinct sites within the axonal

mem-Fig.5.DM20,but not PLP,increases ?lopodium density in hippo-campal neurons.a :After 3DIV,hippocampal neurons were tran-fected with GFP,GFP::M6b ag ,VSVG::GFP,GFP::M6a,GFP::PLP,or GFP::DM20.Fixation and F-actin staining with phal-loidin was performed 1day later.b :Filopodium density (number of protrusions per 20l m neurite length)as shown in A was quanti?ed.Results show the mean value of the group (6SEM)of at least 80neurites and as a percentage of the control group (GFP).Signi?cant differences were determined using one-way ANOVA followed by Dunnet’s post hoc test.**P <0.01against GFP,#P <0.01against M6a.Figure 5b shows the results of one assay,representative of two independent experiments.Scale bar 510l m.

1304Ferna

′ndez et al.Journal of Neuroscience Research

brane and terminal regions of excitatory neurons (Cooper et al.,2008).Hippocampal mossy?bers are not myelinated(Blackstad and Kjaerheim,1961;Frotscher et al.,2006)and have unique specialized axonal protru-sions(Acsady et al.,1998).Interestingly,M6a seems to be enriched within these?lopodial extensions(Cooper et al.,2008).Therefore,M6a reduced expression in the hippocampus of stressed animals and can be in part re-sponsible for the remodelling observed in the MFT. Afferent glutamaergic input from MFT has been sug-gested to be involved in the atrophy of CA3pyramidal neurons in stressed animals,insofar as damage can be prevented by treatment with phenytoin,a drug that reduces excitatory amino acid release(Watanabe et al., 1992),or NMDA receptor agonists(Magarinos and McEwen,1995b).Through this mechanism,M6a could also be indirectly participating in the plastic alterations observed in CA3pyramidal dendrites through the remodelling of the MFT.

The cellular consequences of DM20reduced expression in the hippocampus of chronically stressed animals remain to be examined.A relationship between DM20expression and the plastic alterations observed in the hippocampal neurons of stressed animals is not straightforward,in that PLP and DM20seem not to be expressed in neuronal populations(Verity and Campag-noni,1988;Yan et al.,1993,1996).

Evolution and Functional Homology

of Proteolipids

Our results reveal that,in addition to M6a,other members of the PLP family,M6b and DM20,promote ?lopodium formation.The functional homology among M6a,M6b,and DM20could be a result of their high amino acid identity and this can be explained in terms of their evolution.Proteolipids are thought to have evolved from a single ancestor(bilaterian)gene,whereas in the vertebrate lineage three proteolipid genes emerged, orthologs to mammalian M6a,M6b,and PLP/DM20 (Schweitzer et al.,2006).Proteolipids appeared earlier in evolution than myelin,so their involvement in myelina-tion was acquired later.In agreement,DM20(or its ortholog DM a)emerged approximately when myelin evolved,and the PLP-speci?c domain,which apparently increases the ef?ciency of proteolipid targeting to myelin (Trapp et al.,1997),was acquired later(Schweitzer et al.,2006).The high amino acid identity between DM20and the neuronal proteins M6a and M6b can explain the functional homology in inducing?lopodium formation.

Development of hippocampal neurons in vitro requires the sprouting of neurites from the cell body, and these processes develop later into dendrites and axons(Dotti et al.,1988).Although the exact mecha-nism underlying neurite development is not completely understood,recent studies have demonstrated that?lo-podium formation is a crucial step for the initiation

Fig.6.Induction of?lopodium formation in N2a and COS7cells.a:

N2a cells were transfected with GFP,GFP::M6a,GFP::M6b TMD x,

GFP::M6b TMD/,GFP::M6b a TMD x,GFP::M6b a TMD/,or

GFP::DM20.One day after transfection,cells were?xed,and F-actin

was stained with phalloidin.Percentage of cells bearing?lopodia as

manifested by phalloidin staining was assesed.As a control,nontrans-

fected cells from the same coverslips were used.Results are expressed

as mean value of the group(6SEM)of at least six coverslips coming

from a minimum of two independent experiments.Signi?cant differ-

ences were determined using Student’s t-test,two tailed.*P<0.05,

**P<0.01between transfected and nontransfected cells for each

group.#P<0.01against increase in M6a(ratio between transfected

and nontransfected cells).Results are shown in b for N2a cells and in

c for COS7cells.Scale bar510l m.

PLP Family,Stress,and Filopodium Formation1305 Journal of Neuroscience Research

neuritogenesis of cortical neurons both in vivo and in vitro(Dent et al.,2007;Kwiatkowski et al.,2007).The spatial and temporal pattern of expression of M6a and M6b(Yan et al.,1996)suggest a possible role for these proteins in brain development.According to our studies (Alfonso et al.,2005,and present work),they might be involved in the?rst steps of neurite outgrowth and neu-ronal maturation.In agreement,recent studies have shown a role for M6a in neurite extension in retinal cells (Zhao et al.,2008).M6a was also found to be involved in the differentiation of neurons derived from mouse and human embryonic stem cells(Michibata et al., 2008a,b).

In oligodendrocytes,the capacity of M6b and DM20to induce process outgrowth could be useful at the initial stages of myelination,in which extension of processes has been observed(Trapp et al.,1997).A dis-tinct function for DM20other than a structural compo-nent of myelin or an involvement in oligodendrocyte differentiation and survival has long been conceived (Knapp,1996;Nadon et al.,1997),especially as it is expressed in diverse nonmyelinating cells as myocardio-cytes,embryonic CNS cells,and neuroblastomas (Campagnoni et al.,1992;Ikenaka et al.,1992;Timsit et al.,1992).In accordance,in the present work,we describe a novel function for DM20in?lopodium for-mation that is not restricted to neuronal cells as observed by overexpression in COS7cells.

Possible Mechanisms Underlying the Induction of Filopodium Formation by Proteolipids

PLP family members have been studied for over50 years(Folch et al.,1951).They were?rst associated with process extension15years ago(Lagenaur et al., 1992)and have only been implicated in?lopodium for-mation recently(present work,and Alfonso et al.,2005). The mechanisms through which these proteins induce ?lopodium formation remain unknown,but several pos-sibilities exist in light of different?ndings.All of the PLPs involved in?lopodium formation share a common putative structure that includes two extracellular loops. These extracellular domains could interact with external ligands,which is supported by the fact that incubation of live cultured neurons with antibodies against M6a inter-feres with neurite extension(Lagenaur et al.,1992). PLPs might also interact with other membrane proteins, as was found for PLP and DM20,which were shown to form a complex with integrins in oligodendrocytes (Gudz et al.,2002).M6a was found to interact with l-opiod receptor and has been suggested to act as a scaf-folding molecule in the regulation of G protein-coupled receptors endocytosis and intracellular traf?cking(Wu et al.,2007).Similarly,M6b was found to interact with the human serotonin transporter,and it is speculated that it may play a role in its traf?cking regulation(Fjorback et al.,2009).

PLPs and their molecular interactions could be associated with lipid-enriched membrane microdomains,or lipid rafts,which have been associated with intracellu-lar membrane transport and cell signalling(Simons and Ikonen,1997;Simons and Toomre,2000).In accord-ance,DM20was found to participate in intracellular molecular transport(Nadon and West,1998),and PLP and DM20were found to interact with cholesterol (Simons et al.,2000).Moreover,aberrant expression of PLP perturbs the cellular distribution of cholesterol (Simons et al.,2002),and this has been associated with impaired protein traf?cking(Butchbach et al.,2004).

M6a has also been proposed to act as a calcium channel(Mukobata et al.,2002).Accordingly,alterations in intracellular calcium concentration have been shown to be involved in the regulation of neurite extension and ?lopodium/spine activity(Lau et al.,1999;Nikonenko et al.,2002).Alternative molecular pathways have been described;however,more research is needed to elucidate the mechanisms underlying?lopodium formation induced by proteolipid proteins

CONCLUSIONS

In the present work,we described a conserved cel-lular function and stress-mediated regulation among members of the PLP family,namely,M6a,M6b,and DM20.Our results suggest that proteolipids may be involved in stress-related disorders such as depression.In fact,one member of this family,M6a,has been associ-ated with a subgroup of patients with depressive symp-toms among individuals diagnosed with schizophrenia (Boks et al.,2007).Taken together,this work and recent ?ndings suggest that PLPs should be considered in mood disorders such as depression.

ACKNOWLEDGMENTS

We thank C.Scorticati and all members of our laboratory for critical reading of the manuscript and extensive discussion and A.Genaro and L.Frick for mRNA samples of stressed and control mice.

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选择twoball-2k.bin，点击打开。步骤3：选择端口首先把实验板通过USB延长线连接到电脑上，然后右击“我的电脑”，选择“管理”，单击设备管理器，点击端口前的加号将其展开，当发现这个时，说明驱动的安装和实验板的下载电路应该是没什么问题的，这里的可以看出端口是COM14。其次是选择好端口，如下图所示：步骤4：下载程序到单片机（注意的是STC的单片机需要重新给系统上电才能下载到单片机）点击下图所示的Download/下载按钮当出现下图所示的提示时，如果实验板是在通电的情况下，则按一下实验板的开关稍等两秒左右，再按一下开关重新给实验板上电，稍等片刻就下载成功。如果实验板是在不通电的情况下，则按一下实验板的开关重新给实验板上电，稍等片刻就下载成功下载成功的提示如下图：下载过程中如果端口选择对的情况下，出现如下图所示：原因在于连电脑USB插口松动。解决办法：1、重新把延长线从实验板上拔掉，然后再插上。

金龙STM32F207开发板用户手册

1.概述金龙STM32开发板用户手册芯片描述 -ARM32-bit Cortex-M3CPU -120MHz maximum frequency,150DMIPS/1.25DMIPS/MHz -Memory protection unit Memories -Up to1Mbyte of Flash memory -Up to128+4Kbytes of SRAM -Flexible static memory controller (supports Compact Flash,SRAM,PSRAM,NOR,NAND memories) -LCD parallel interface,8080/6800modes Clock,reset and supply management -1.8to3.6V application supply and I/Os -POR,PDR,PVD and BOR -4to25MHz crystal oscillator -Internal16MHz factory-trimmed RC -32kHz oscillator for RTC with calibration -Internal32kHz RC with calibration Low power -Sleep,Stop and Standby modes -VBAT supply for RTC, C32bit backup registers 20 optional4KB backup SRAM C12-bit,0.5us A/D converters 3 -up to24channels -up to6MSPS in triple interleaved mode C12-bit D/A converters 2 General-purpose DMA -16-stream DMA controller centralized FIFOs and burst support Up to17timers -Up to twelve16-bit and two32-bit timers Debug mode -Serial wire debug(SWD)&JTAG interfaces -Cortex-M3Embedded Trace Macrocell Up to140fast I/O ports with interrupt capability -51/82/114/140I/Os,all5V-tolerant Up to15communication interfaces C I2C interfaces(SMBus/PMBus) -Up to3 -Up to6USARTs(7.5Mbit/s,ISO7816interface,LIN,IrDA,modem control)

KR-51开发板使用说明

KR-51/AVR开发板使用说明声明：本指导教程和配套程序仅在开发和学习中参考，不得用于商业用途，如需转载或引用，请保留版权声明和出处。请不要在带电时拔插芯片以及相关器件。自行扩展搭接导致不良故障，本公司不负任何责任。产品不定时升级，所有更改不另行通知，本公司有最终解释权。一、开发板硬件资源介绍 1 .开发板支持USB 程序下载（宏晶科技STC系列单片机） 2. 开发板支持AT89S51 ，AT89S52 单片机下载（需要配合本店另外下载器下载） 3. 开发板支持ATmega16，ATmega32 AVR 单片机下载（需要配合本店另外转接板和下载器使用） 4. 开发板供电模式为：电脑USB 供电（USB 接口）和外部5V 电源供电（DC5V接口） 5. 开发板复位方式：上电复位和51按键复位 6. 外扩电源：通过排针外扩5路5V 电源，3路3.3V电源方便连接外部实验使用 7. 所有IO 引脚全部外扩，方便连接外部实验使用 8. 开发板集成防反接电路，防止接反，保护开发板二、开发板功能模块介绍（1 ）8 位高亮度贴片led 跑马灯；（2） 4 位共阳数码管显示；（3）LCD1602 和LCD12864液晶屏接口；（4） 1 路无源蜂鸣器；（5） 1 路ds18b20 温度测量电路（与DHT11 温湿度接口共用）；（6） 1 路红外接口电路（7） 4 路独立按键（8） 1 路CH340 USB转串口通讯电路（全面支持XP/WIN7/WIN8系统）；（9）1路蓝牙模块接口（可做蓝牙测试板，USB转蓝牙）；（10）1路2.4G模块接口；（11）1路WiFi模块接口（可做WiFi测试板，USB转WiFi）三开发板跳线选择本开发板接线简单，适合初学者使用，开发板各模块的跳线使用注意事项：烧写程序时，拔掉蓝牙模块，WiFi模块，J10处用跳线帽短接1,3和2,4。蓝牙模块和WiFi模共用串口，不能同时使用。使用1602、12864液晶接口时请拔下数码管J4 跳线帽。以下是几个主要跳线的使用说明；

路虎开发板用户手册

路虎NXP LPC1768开发板用户手册

1、概述路虎开发板采用 NXP公司 LPC1768 ARM是一款基于第二代 ARM Cortex-M3内核的微控制器，是为嵌入式系统应用而设计的高性能、低功耗的 32位微处理器，适用于仪器仪表、工业通讯、电机控制、灯光控制、报警系统等领域。路虎开发板板载 USB仿真器，支持 USB2.0 Device，具有双 CAN接口、RS-485接口等功能。路虎开发板配套丰富的例程和详尽的资料，方便用户快速进行项目开发。功能特点：强大的 MCU内核：Cortex-M3 ●处理速率高达 100MHz，并包含一个支持 8个区的存储器保护单元（MPU） ●内置嵌套向量中断控制器（NVIC） ● 512KB片上 Flash程序存储器，支持在系统编程（ISP）和在应用编程（IAP） ● 64KB SRAM可供高性能 CPU通过指令总线、系统总线、数据总线访问 ● AHB多层矩阵上具有 8通道的通用 DMA控制器（GPDMA） ●支持SSP、UART、AD/DA、定时器、GPIO等，并可用于存储器到存储器的传输 ●标准 JTAG测试/调试接口以及串行线调试和串行线跟踪端口选项 ●仿真跟踪模块支持实时跟踪 ● 4个低功率模式：睡眠、深度睡眠、掉电、深度掉电

●单个 3.3V电源（2.4V – 3.6V） ●工作温度：-40 °C - 85°C ●不可屏蔽中断（NMI）输入 ●片内集成上电复位电路 ●内置系统节拍定时器（SysTick），方便操作系统移植。丰富的板载资源： 1、2路 RS232串行接口（使用直通串口线、其中一路串口支持 ISP下载程序） 2、2路 CAN总线通信接口（CAN收发器：SN65VHD230） 3、RS485通信接口（485收发器：SP3485） 4、RJ45-10/100M Ethernet网络接口（以太网 PHY：DP83848） 5、DA输出接口(可做 USB声卡实验、板载扬声器和扬声器输出驱动) 6、AD输入接口（可调电位器输入） 7、彩色液晶显示接口(可接 2.8寸或 3.2寸 TFT 320X240彩屏) 8、USB2.0接口,USB host及 USB Device接口。 9、SD/MMC卡(SPI)接口(提供带 FAT12、FAT16、FAT32文件系统)

RK3188开发板使用手册v1.0

RK3188开发板使用手册v1.0 一.安装RockUsb驱动 (2) 二.查看串口输出信息 (5) 三.烧写/下载固件 (8) 四.Kernel开发 (11) 五.Android开发 (12) 六.制作固件升级包update.img (13) 七.Recovery系统 (14) 八.Android系统USB操作 (17)

一.安装RockUsb驱动 Rockusb驱动放在RK3188\tools\RockusbDriver文件夹中当你第一次使用RK3188SDK开发板时，接好USB线，按住“VOL+(RECOVERY)”按键上电，会要求安装驱动，按下面的图示步骤进行安装：图1 选择“否，暂时不(T)”，点击“下一步”进入图2所示界面

图2 选择“从列表或指定位置安装（高级）”，点击下一步，进入图3界面图3 选择你的驱动所存放的目录，点击“下一步”开始安装驱动，如图4所示

图4 完成以后可以在设备管理器看到设备已经安装成功图5

二.查看串口输出信息 RK3188SDK开发板没有使用普通的串口，而是使用USB口来输出串口信息，你可以用一根特殊的USB调试线将开发板上的USB口连接到你的电脑中来查看串口信息。 1、在连接USB口之前，请先安装PL-2303USB转串口驱动 2、驱动安装完成后，再使用USB线将开发板上名为“UART0”的USB口连接到PC 中，然后你应该可以在设备管理器中看到一个新设备，如下所示： 3、使用串口工具查看开发板的输出信息。在这边我以Windows自带的超级终端为例说明串口的配置： a、点击开始->所有程序->附件->通讯->超级终端点击确定 b、选择正确的COM口：

智慧校园宿舍管理系统安装使用说明书v1.2

PM-03-005-04 成都易科士信息产业有限公司研发中心高校宿舍管理系统软件安装使用说明书本文档是成都易科士信息产业有限公司文档。任何使用、复制、公开此文档的行为都必须经过成都易科士信息产业有限公司的书面允许。

前言概述本文档介绍大中专院校宿舍管理系统的软件的安装和使用。读者对象本文档（本指南）适用于所有使用该系统对的用户。符号约定在本文中可能出现下列标志，它们所代表的含义如下。修改记录修改记录累积了每次文档更新的说明。最新版本的文档包含以前所有文档版本的更新内容。

目录前言 (ii) 1 产品说明 (6) 1.1 概述 (6) 1.2 目标 (6) 2 技术规格说明 (7) 2.1 软件版本 (7) 3 系统安装手册 (7) 3.1 数据库初始化 (7) 3.2 服务端安装 (9) 3.3 客户端安装 (9) 4 服务端操作使用说明 (10) 4.1 服务端配置概述 (10) 4.1.1 如何配置数据库？ (10) 4.1.2 备份计划 (10) 4.1.3 如何注册信息? (11) 5 客户端操作使用说明 (12) 5.1 系统主要功能概述 (12) 5.1.1 基础信息管理 (12) 5.1.2 公寓信息管理 (12) 5.1.3新生入住管理 (12) 5.2 系统详细功能一览 (12) 5.3登录 (13) 5.4系统相关配置 (14) 5.4.1 系统相关配置 (14) 5.4.2 登录设置 (16) 5.4.3 密码修改 (17) 5.5 基础信息配置 (17) 5.5.1 物品种类管理 (17) 5.5.2 学年学期管理 (22) 5.5.3代码字典 (23) 5.5.4校区管理 (23) 5.5.5院系管理 (23) 5.5.6专业管理 (24) 5.5.7班级管理 (25) 5.5.8辅导员管理 (26)

51单片机开发板使用手册

STU_MAIN单片机开发板使用手册第一章STU_MAIN 单片机开发板简介 (2) 1.1 单片机开发板概述 (2) 1.2 单片机开发板载资源介绍 (2) 1.3 STU_MAIN 单片机开发板接口说明 (4) 1.4 如何开始学习单片机 (5) 第二章软件使用方法 ......................... . (6) 2.1 KEIL 软件的使用方法 (6) 2.2 STC-ISP 软件的安装与使用 (13) 2.3 使用USB 口下载程序时设置步骤 (18) 第三章STU_MAIN 开发板例程详细介绍 (21) 3.1 准备工作 (21) 3.2 安装STC-ISP下载程序 (21) 3.3 闪烁灯 (22) 3.4 流水灯 (23) 3.5 单键识别 (25) 3.6 利用定时器和蜂鸣器唱歌 (28) 3.7 DS18B20 温度测量显示实验 (31) 3.8 LCD1602 字符液晶显示 (36) 3.9 串口通讯实验 (39) 3.10 基于DS1302的多功能数字钟实验 (41) 3.11 EEPROM X5045 实验 (47)

第一章STU_MAIN 单片机开发板简介 1.1 单片机开发板概述 STU_MAIN 单片机开发板是经过精心设计开发出的多功能MCS-51 单片机开发平台。该开发板集常用的单片机外围资源、串口调试下载接口于一身，可以让您在最短的时间内，全面的掌握单片机编程技术。该开发板特别适合单片机初学者、电子及通信等专业的课程设计以及电子爱好者自学使用。 STU_MAIN 单片机开发板可作为单片机课程的配套设备，课程从最基本的预备知识开始讲起，非常详细的讲解KEIL 编译器的使用，包括软件仿真、测定时间、单步运行、全速运行、设置断点、调试、硬件仿真调试、变量观察等，整个过程全部用单片机的C 语言讲解，从C 语言的第一个主函数MAIN 讲起，一步步一条条讲解每一个语法、每条指令的意思，即使对单片机一巧不通，对C 语言一无所知，通过本课程的学习也可以让你轻松掌握MCS-51 单片机的C 语言编程。全新的讲课风格，跳过复杂的单片机内部结构知识，首先从单片机的应用讲起，一步步深入到内部结构，让学生彻底掌握其实际应用方法，把MCS-51单片机的所有应用、每个部分都讲解的非常清晰明了，授课教师在教室前面用电脑一条一条写程序，旁边用STU_MAIN 单片机开发板逐个实验的演示，给学生解释每条指令的意思及原理，通过一学期的学习让学生完全掌握单片机的C 语言编程及单片机外围电路设计的思想。以实践为主、学生现场写程序、直接下载到开发板观察现象。 1.2 单片机开发板载资源介绍一. STU_MAIN单片机开发板(串口直接下载程序) 本开发板以STC 公司生产的STC90C54RD+ 单片机做核心控制芯片,它是一款性价比非常高的单片机，它完全兼容ATMEL 公司的51/52系列单片机，除此之外它自身还有很多特点，如：无法解密、低功耗、高速、高可靠、强抗静电、强抗干扰等。其次STC 公司的单片机内部资源比起ATMEL 公司的单片机来要丰富的多，它内部有1280 字节的SRAM、8-64K 字节的内部程序存储器、2-8K 字节的ISP 引导码、除P0-P3 口外还多P4 口(PLCC封装)、片内自带8路8位AD(AD 系列)、片内自带EEPROM、片内自带看门狗、双数据指针等。目前STC 公司的单片机在国内市场上的占有率与日俱增,有关STC 单片机更详细资料请查阅相关网站。 STU_MAIN单片机开发板可完全作为各种MCS-51单片机的开发板，用汇编语言或C 语言对其进行编程。当用STC 公司的单片机时，直接用后面介绍的串口线将开发板与计算机串口相连，按照STC 单片机下载操作教程便可下载程序，

STM32F429开发板用户手册

STM32F429开发板用户手册介绍 STM32F429(32F429IDISCOVERY)开发板可以帮助你去学习高性能STM32F4系列，并去开发你自己的应用。它包含了一个STM32F429ZIT6和一个嵌入ST-LINK/V2调试接口，2.4吋TFTLCD，64MbitsSDRAM，ST微机电陀螺仪，按键和USB OTG接口。

1约定下表提供了一些约定惯例，目前的文档可能会用到。

2快速入门 STM32F429开发板是一种廉价且易于上手的开发套件，可以让使用者快速评估和开始STM32F4的开发工作。在安装和使用产品以前，请接收评估产品许可协议。 2.1启动跟随以下顺序来设置STM32F429开发板并开始开发应用： 1、确认跳线JP3和CN4被设置为“on”（开发模式） 2、连接STM32F429Discovery开发板CN1到PC，使用USB电缆（type A/mini-B）,开发板上电。 3、屏幕上以下应用可用：时钟日历和游戏视频播放器和图片浏览器（播放浏览USB大容量存储器上的视频和图片）性能显示器（观察CPU负载和图形测试）系统信息 4、演示软件，也像其他软件例程，运行你用来开发STM32F4。 5、从例程开始开发你自己的应用吧。 2.2系统要求 ?Windows PC(XP,Vista,7) ?USB type A to mini-B cable 2.3支持STM32F429开发板的开发工具 ?Altium:TASKING?VX-Toolset ?Atollic:TrueSTUDIO ?IAR:EWARM ?Keil?:MDK-ARM 2.4订购码要订购STM32F429Discovery kit，请使用STM32F429I-DISCO订购码。 3特性 STM32F429Discovery开发板提供一下特性： ?S TM32F429ZIT6具有2MB闪存，256KB的RAM，LQFP144封装。 ?板载ST-LINK/V2，带有选择模式跳线，可以作为独立的ST-LINK/V2使用。 ?板电源：通过USB总线或外部3V或5V电源。 ?L3GD20：ST微机电动作传感器，3轴数字输出陀螺仪 ?TFT LCD，2.4寸，262K色RGB，240*230分辨率 ?SDRAM64Mbits(1Mbit x16-bit x4-bank)，包含自动刷新模式和节能模式 ?六个LED： LD1（红绿）：USB通信 LD2（红）：3.3V电源两个用户LED LD3（绿），LD4红两个USBOTG LED：LD5(绿)VBUS和LD6OC（过流） ?两个按键（user and reset）

智慧校园视频监控安防方案解析【最新版】

智慧校园视频监控安防方案解析本方案将以物联网技术为基础围绕校园进行全方位、全天候的全面安全防范，最大限度的降低各种安全隐患，建成一套以学校为中心的智能化视频监控系统解决方案。一、需求分析 1、高清智能监控需求校园安全越来越受全社会瞩目，夜间幽暗的校园环境隐藏着诸多危机，传统监控系统在夜间必须启动红外灯补光，因此视频被迫转为黑白画面，一旦危机发生，黑白视频在画面上会丢失许多色彩上的细节，给事后查证工作造成一定困难。被动式监控系统并不能制止安全事件的发生，因此需要建设一套高清晰、智能化的监控系统，提前发现危险，及时制止，将危险降到最低，营造一个安全、温馨的校园环境。 2、人员管理需求学生安全问题一直以来都是校方和学生家长最重视的问题，保护学生在上学期间安全有保障、不受到来自外界的安全威胁是校方的职

责所在，也是学生家长们选择孩子入学场所最关心的条件之一。为了保障学生在上学时安全到校，放学时安全离校，在校期间不受到来自外界不良分子的安全威胁，需要对进出学校的外来人员进行有效管理，对校门外潜在风险降到最低。 3、车辆管理需求为维护校内师生良好的工作和生活秩序，保障师生人身安全，减少各类事故发生，营建良好的校园环境，需要对进出学校的车辆做智能化管理，通过人为的手段无法做到24小时有效的管理，需要借助技术手段，实现校园车辆的有效管理，针对教职工车辆进行自动识别放行，对外来车辆进行识别并提醒登记。 4、应急处理需求校园安保工作的主要任务是打造一个和谐、舒适的平安校园，但即使校园安防工作做得非常完善，还是有可能存在很多突发事件，给校园安保工作带来很大的压力。过去校园视频监控系统主要用于事后的追溯，缺乏提供事前预警、事中处理的机制。因此，如何进行全局资源的调度整合，快速处理此类突发事件，争取更多的主动性，尽可能的降低事件的影响，也成为校园安防需要解决的问题。

百问网精智JZ2440开发板使用手册 S3C2440

百问网·精智JZ2440使用手册提示：除了QT外，可以不看本手册，参考《嵌入式Linux应用开发完全手册》及视频即可

第1章嵌入式Linux开发环境构建 (4) 1.1 安装Ubuntu 9.10 (4) 1.1.1 安装VMware (4) 1.1.2 安装Ubuntu 9.10 (13) 1.2 安装Ubuntu下的开发工具 (20) 1.3 安装Windows下的开发工具 (22) 第2章精智JZ2440开发板烧写程序方法 (23) 2.1 使用JTAG工具烧写开发板 (23) 2.1.1 Windows下并口JTAG驱动安装 (23) 2.1.2 Windows下OpenJTAG驱动安装 (29) 2.1.3 Ubuntu下驱动程序的安装 (29) 2.1.4 JTAG烧写软件oflash的用法 (29) 2.2 通过u-boot烧写整个系统 (29) 2.2.1 在Windows下使用dnw和u-boot烧写系统 (30) 2.2.2 在Linux下使用dnw和u-boot烧写系统 (31) 第3章板上Linux系统搭建 (33) 3.1 修改、编译、使用u-boot (33) 3.1.1 使用补丁修改、编译u-boot (33) 3.1.2 u-boot使用方法 (33) 3.2 修改、编译、使用Linux内核 (36) 3.2.1 使用补丁修改、编译内核 (36) 3.2.2 使用uImage (36) 3.3 修改、编译QT (36) 3.3.1 编译依赖的软件 (36) 3.3.2 使用补丁修改、编译QT (39) 3.4 构造根文件系统 (39) 3.4.1 基于最小根文件系统制作QT文件系统 (39) 3.4.2 制作YAFFS2、JFFS2文件系统映象文件 (42)

STM32F0-DISCOVERY用户手册

1/30 文档ID 022910第1版2012年3 月 STM32F0DISCOVERY STM32F0探索套件 UM1525 前言 STM32F0DISCOVERY 是意法半导体STM32F0系列微控制器的探索套件，用于帮助你探索STM32F0 Cortex-M0微控制器的功能，轻松开发应用设计。STM32F0探索套件基于1颗STM32F051R8T6微控制器，组件包括ST-LINK/V2嵌入式调试工具、LED 指示灯、按键和1个原型板。图1： STM32F0 探索套件用户手册

2/30UM1525 文档ID 022910第1版目录目录 1. 约定....................................................................................................................................52. 快速入门 (6) 2.1 开始使用........................................................................................................ 62.2 系统要求..........................................................................................................62.3 支持STM32F0探索套件的开发工具链 .......................................................62.4 订货代码. (6) 3. 特性....................................................................................................................................74. 硬件与原理图.. (8) 4.1 STM32F051R8T6 微控制器 ..........................................................................114.2 嵌入式ST-LINK/V2编程器/调试器 . (13) 4.2.1 使用ST-LINK/V2向板载STM32F0烧录和调试代码 ............................14 4.2.2 使用ST-LINK/V2向外部STM32应用板烧录和调试代码. (15) 4.3电源和电源选择............................................ 164.4 LED 指示灯 ...................................................................................................164.5 按键................................................................................................................164.6 JP2（Idd ）﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍ 16 4.7 OSC 时钟 -----------------------------------------------------------------------------174.7.1 OSC 时钟电源 .............................................................................................174.7.2 OSC 32kHz 时钟电源 17 4.8 焊桥.................................................................................................................184.9 扩展连接器.. (19) 5. 尺寸图..............................................................................................................................266. 原理图..............................................................................................................................277. 修改历史记录 (30)

单片机开发板操作手册.

单片机开发板操作手册一、概述 1，多功能单片机开发板，板载资源非常丰富，仅是包括的功能（芯片）有：步进电机驱动芯片ULN2003、八路并行AD转换芯片ADC0804、八路并行DA转换芯片DAC0832、光电耦合（转换）芯片MOC3063、八路锁存器芯片74HC573、实时时钟芯片DS1302及备用电池、 IIC总线芯片A T24C02、串行下载芯片MAX232CPE，双向可控硅BTA06-600B、 4*4矩阵键盘、 4位独立按键、 DC5V SONGLE继电器、 5V蜂鸣器、八位八段共阴数码管 5V稳压集成块78M05 八路发光二极管显示另还有功能接口（标准配置没有芯片但留有接口，可直接连接使用）：单总线温度传感器DS18B2接口、红外线遥控接收头SM003接口8、蓝屏超亮字符型液晶1602接口、蓝屏超亮点阵图形带中文字库液晶12864接口、 2（4）相五线制小功率步进电机接口、外接交流（7V-15V）电源接口 USB直接取电接口镀金MCU晶振座 40DIP锁紧座外接电源和5V稳压电源的外接扩展接口及MCU所有IO口扩展2，可以完成的单片机实验： 1、LED显示实验（点亮某一个指示灯、流水灯）， 2、八位八段数码管显示实验（你可以任意显示段字符和数字以及开发板所有功能芯片的显示）， 3、液晶显示（1602液晶显示、12864点阵中文图形液晶显示、可以显示出开发板所有功能芯片的操作）， 4、继电器的操作 5、蜂鸣器的操作（你可以编写程序让它发出美妙动听的歌声） 6、可控硅的操作（胆大的朋友就利用这一独有的功能吧，你见过实验室温度实验箱没有，它的驱动就是这样的；聪明的朋友就可以自己写个程序把把加热温度温度恒定在（X±0.5）度的范围内了 7、步进电机的操作（这个是迈向自动化控制的第一步，现在的数控机床、机器人呀

AX301用户手册REV1.0

FPGA黑金开发平台用户手册 AX301 REV 1.0 芯驿电子科技（上海）有限公司黑金动力社区 https://www.wendangku.net/doc/cc16834356.html,

目录一、简介 (3) 二、电源 (6) 三、FPGA (7) 1)JTAG接口 (8) 2)配置芯片：M25P16 (8) 3)FPGA供电引脚 (9) 4)FPGA时钟输入引脚 (10) 四、50M有源晶振 (11) 五、SDRAM (11) 六、EEPROM 24LC04 (14) 七、PS/2接口 (15) 八、实时时钟DS1302 (16) 九、数码管 (17) 十、USB转串口 (19) 十一、VGA接口 (20) 十二、蜂鸣器 (23) 十三、SD卡槽 (24) 十四、扩展口 (25) 十五、LED (27) 十六、按键 (28) 十七、摄像头接口 (29) 十八、7寸TFT接口 (30)

FPGA黑金开发平台学生版2014款正式发布了，此款开发平台是在前两款学生版的基础上升级而来，型号为：AX301。虽然是一款入门级的FPGA开发平台，但是我们不管是在PCB设计上，还是在整个构造上都花费了很多的心思，真可谓“简约而不简单”。一、简介在这里，对这款FPGA开发平台进行简单的功能介绍。此款开发板使用的是ALERA公司的CYCLONE IV系列FPGA，型号为EP4CE6F17C8，256个引脚的FBGA封装。根据ALTERA官方的数据，CYCLONE IV相对CYCLONE III来说，功耗减少25%，如下图所示此款FPGA的资源如下图所示：

其中，主要的参数，逻辑单元LE：6272；乘法器LAB：392； RAM：276480bit； IO数量：179个；内核电压：1.15V-1.25V(推荐1.2V); 工作温度：0-85℃ 图为整个系统的结构示意图：

EXCD1开发板使用手册

EXCD‐1开发板参考手册北京中教仪装备技术有限公司 2010年3月

目录 1 概述 (1) 2 板上资源 (2) 3 使用说明 (3) 3.1使用前准备 (3) 3.2输入时钟 (4) 3.3LED，拨码开关和按键 (5) 3.47段数码管 (9) 3.5VGA接口 (10) 3.6RS232串口 (12) 3.7PS/2鼠标键盘接口 (13) 3.8I/O扩展接口 (14) 3.9SRAM和F LASH存储器 (16) 联系我们地址：北京市西城区德外大街4号C座邮编：100120 客户服务热线：4006061700 传真 : 010- 58582440 销售Email : ECsales@https://www.wendangku.net/doc/cc16834356.html, 技术支持Email：Ecservice@https://www.wendangku.net/doc/cc16834356.html, 网址：https://www.wendangku.net/doc/cc16834356.html,

1 概述 EXCD-1是一款易于使用的开发板，它能够实现大量基于FPGA 的数字系统。开发板采用Xilinx Spartan 3E FPGA 器件，板上资源丰富，有2Mbytes 的Flash 和1Mbytes 的快速SRAM ，以及各种输入输出设备，可以方便的设计具有各种功能的数字系统，也可设计含有Xilinx MicroBlaze 软核的嵌入式处理器系统。EXCD-1开发板有5个扩展接口，可灵活的扩展各种功能模块，如数模转换模块，模数转换模块，LCD 显示模块等。 VGA 接口RS232PRom PS2LEDs 晶振图1-1 EXCD-1开发板

2板上资源 displays 4 buttons port2 图2-1 EXCD-1板上资源框图 1.Xilinx Spartan 3E XC3S500E PQ208 FPGA器件 z10,476 逻辑单元 z1,164 CLBs z73Kbits分布式RAM z360Kbits块RAMs z20个专用乘法器 z4个DCMs z158个用户I/O管脚 z PQ208管脚封装 2.时钟：50MHZ晶振输入 3.高速异步SRAM z512K × 16bits 4.Flash存储器 z1M ×16bits 5.配置Flash： XCF04S

Xilinx 开发板用户手册

SP605 Hardware User Guide UG526 (v1.6) July 18, 2011

? Copyright 2009–2011 Xilinx, Inc. Xilinx, the Xilinx logo, Artix, ISE, Kintex, Spartan, Virtex, Zynq, and other designated brands included herein are trademarks of Xilinx in the United States and other countries. All other trademarks are the property of their respective owners. DISCLAIMER The information disclosed to you hereunder (the “Materials”) is provided solely for the selection and use of Xilinx products. To the maximum extent permitted by applicable law: (1) Materials are made available "AS IS" and with all faults, Xilinx hereby DISCLAIMS ALL WARRANTIES AND CONDITIONS, EXPRESS, IMPLIED, OR ST ATUTORY, INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY, NON-INFRINGEMENT, OR FITNESS FOR ANY PARTICULAR PURPOSE; and (2) Xilinx shall not be liable (whether in contract or tort, including negligence, or under any other theory of liability) for any loss or damage of any kind or nature related to, arising under, or in connection with, the Materials (including your use of the Materials), including for any direct, indirect, special, incidental, or consequential loss or damage (including loss of data, profits, goodwill, or any type of loss or damage suffered as a result of any action brought by a third party) even if such damage or loss was reasonably foreseeable or Xilinx had been advised of the possibility of the same. Xilinx assumes no obligation to correct any errors contained in the Materials, or to advise you of any corrections or update. Y ou may not reproduce, modify, distribute, or publicly display the Materials without prior written consent. Certain products are subject to the terms and conditions of the Limited Warranties which can be viewed at https://www.wendangku.net/doc/cc16834356.html,/warranty.htm; IP cores may be subject to warranty and support terms contained in a license issued to you by Xilinx. Xilinx products are not designed or intended to be fail-safe or for use in any application requiring fail-safe performance; you assume sole risk and liability for use of Xilinx products in Critical Applications: https://www.wendangku.net/doc/cc16834356.html,/warranty.htm#critapps. Revision History The following table shows the revision history for this document. Date Version Revision 10/07/09 1.0Initial Xilinx release. 11/09/09 1.1?Updated Figure1-17 and Figure1-23. ?Changed speed grade from -2 to -3. ?Miscellaneous typographical edits. 02/01/10 1.1.1Minor typographical edits to Table1-24 and Table1-25. 05/18/10 1.2Updated Figure1-2. Added Note 6 to Table1-11. Updated board connections for SFP_TX_DISABLE in Table1-12. Added note about FMC LPC J63 connector in 18. VITA 57.1 FMC LPC Connector. Updated U1 FPGA Pin column for FMC_LA00_CC_P/N in Table1-28. Updated description of PMBus Pod and TI Fusion Digital Power Software GUI in Onboard Power Regulation. Updated Appendix B, VITA 57.1 FMC LPC Connector Pinout, and Appendix C, SP605 Master UCF. 06/16/10 1.3Updated 2. 128 MB DDR3 Component Memory. Added note 1 to Table1-30. 09/24/10 1.4Updated description of Fusion Digital Power Software in Onboard Power Regulation. 02/16/11 1.5Revised oscillator manufacturer information from Epson to SiTime in Table1-1. Revised oscillator manufacturer information from Epson to SiTime on page page23. Deleted note on page 44 referring to J55: “Note: This header is not installed on the SP605 as built.” Revised values for R50 and R216 in Figure1-12. Revised oscillator manufacturer information from Epson to SiTime on page page69. 07/18/11 1.6Corrected “jitter” to “stability” in section Oscillator (Differential), page23. Revised the feature and notes descriptions for reference numbers 6 and 12 in Table1-1, page10. Revised FPGA pin numbers for ZIO and RZQ in Table1-4, page14. Added Table1-29, page52, Table1-31, page55, and table notes in Table1-30. SP605 Hardware User Guide https://www.wendangku.net/doc/cc16834356.html, UG526 (v1.6) July 18, 2011

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智慧校园视频监控安防方案解析【最新版】

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