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Genes,Brain and Behavior(2015)14:534–542doi:10.1111/gbb.12232 NPY-Y1coexpressed with NPY-Y5receptors modulate anxiety but not mild social stress response in mice
A.Longo?,?,A.Oberto?,?,§,P.Mele?,?,
L.Mattiello?,M.G.Pisu??,P.Palanza??,
M.Serra??,??and C.Eva?,?,§,?
?Neuroscience Institute of the Cavalieri-Ottolenghi Foundation Orbassano(Turin),?Department of Neuroscience,University of Turin,§Neuroscience Institute of Turin,?Department of Clinical and Biological Sciences,University of Turin,Turin,
??Neuroscience Institute,National Research Council of Italy (CNR),Cagliari,??Department of Neuroscience,University of Parma,Parma,and??Department of Life and Environmental Sciences and Center of Excellence for Neurobiology of Dependence,University of Cagliari,Cagliari,Italy
*Corresponding author:C.Eva,PhD,Neuroscience Institute of the Cavalieri-Ottolenghi Foundation(NICO),Regione Gonzole 10,University of Turin,10043Orbassano(Turin),Italy.E-mail: carola.eva@unito.it
The Y1and Y5receptors for neuropeptide Y have overlapping functions in regulating anxiety.We pre-viously demonstrated that conditional removal of the Y1receptor in the Y5receptor expressing neurons in juvenile Npy1r Y5R?/?mice leads to higher anxiety but no changes in hypothalamus-pituitary-adrenocortical axis activity,under basal conditions or after acute restraint stress.In the present study,we used the same conditional system to analyze the speci?c con-tribution of limbic neurons coexpressing Y1and Y5receptors on the emotional and neuroendocrine responses to social chronic stress,using different housing conditions(isolation vs.group-housing)as a model.We demonstrated that control Npy1r2lox male mice housed in groups show increased anxiety and hypothalamus-pituitary-adrenocortical axis activ-ity compared with Npy1r2lox mice isolated for six weeks immediately after weaning.Conversely,Npy1r Y5R?/?con-ditional mutants display an anxious-like behavior but no changes in hypothalamus-pituitary-adrenocortical axis activity as compared with their control littermates, independently of housing conditions.These results sug-gest that group housing constitutes a mild social stress for our B6129S mouse strain and they con?rm that the conditional inactivation of Y1receptors speci?cally in Y5receptor containing neurons increases stress-related anxiety without affecting endocrine stress responses. Keywords:3α,5α-TH PROG,anxiety,conditional knockout mice,group housing,hypothalamus-pituitary-adrenocortical axis,isolation,mild social stress,neuropeptide Y,Y1receptor, Y5receptor Received2March2015,revised26May2015and2July2015, accepted for publication5July2015
Neuropeptide Y(NPY)is a widely distributed peptide in the CNS,where it regulates anxiety and stress responses (Eva et al.2006).Pharmacologic and genetic studies sug-gest that NPY induces anxiolytic effects(Dumont et al. 2009;Eva et al.2006;Kormos&Gaszner2013)and counter-acts corticotropin releasing hormone(CRH)-mediated stress responses(Russo et al.2012;Sajdyk et al.2006)via the acti-vation of Y1receptor(Y1R)in amygdala,hippocampus and locus coeruleus(Bertocchi et al.2011;Broqua et al.1995; Heilig et al.1993;Lin et al.2010;Sajdyk et al.2008).In the rodent brain,the Y1R colocalizes with Y5receptor(Y5R) in the hippocampus and basolateral amygdala(BLA;Oberto et al.2007;Wolak et al.2003)where they have overlap-ping functions in regulating anxiety(Sorensen et al.2004). We previously showed that conditional inactivation of Npy1r restricted to Y5R-containing neurons in adult Npy1r Y5R?/?mice increased anxiety without affecting basal or acute stress-activated hypothalamus-pituitary-adrenocortical(HPA) axis activity(Longo et al.2014).These data suggest that NPY, acting on Y1R,might activate distinct neuronal circuits to reg-ulate anxiety-related behaviors and stress responses. Isolation rearing in rats constitutes a stress in terms of social deprivation and adversely impacts cognitive,emotional and social functions(Lyons et al.1999;Ruis et al.1999; Sapolsky et al.1997;Serra et al.2008).Social isolation in rats induces alterations in the structure and function of GABA A receptors and decreases the cerebrocortical concentrations of3α,5α-TH PROG(Serra et al.2000),a neuroactive steroid derived from progesterone that binds,with high af?nity, GABA A receptors(Lambert et al.1995;Majewska et al.1986; Paul&Purdy1992),thereby eliciting anxiolytic,anticonvulsant and hypnotic–anesthetic effects in vivo(Belelli et al.1989; Bitran et al.1993;Concas et al.1996;Kokate et al.1999). Isolation in adult male mice may not be markedly stressful because of their territoriality(Brain1975;Palanza et al.2001). In fact,isolation rearing in male mice increases locomotor activity in response to novelty and decreases anxiety-like behavior(Abramov et al.2004;Bartolomucci et al.2003;Guo et al.2004;Hilakivi et al.1989;Voikar et al.2005),suggesting an improved ability to cope with novel situations.
To further explore the speci?c contribution to stress responses of Y1R coexpressed with Y5R in limbic neurons, we investigated the effect of chronic stressful social con-text,induced by different housing conditions(isolation vs. group-housing with siblings),on the emotional and neuroen-docrine functions of Npy1r Y5R?/?conditional mutants and
534?2015John Wiley&Sons Ltd and International Behavioural and Neural Genetics Society
Housing conditions and Y1R/Y5R containing neurons
Npy1r2lox control mice.We demonstrate that group-housed Npy1r2lox and Npy1r Y5R?/?mice showed increased anxiety and HPA-axis activity when compared with Npy1r2lox and Npy1r Y5R?/?mice isolated for6weeks immediately after weaning,respectively.Conversely,Npy1r Y5R?/?mice display an anxious-like behavior but no changes in HPA-axis activity as compared with their control littermates,independently of housing conditions.This suggests that,in this B6129S mouse strain,group housing is a chronic stressful social context and the anxiolytic Y1R speci?cally expressed in Y5R containing neurons is not involved in stress responses. Materials and methods
Animals and housing
Mice were housed in a temperature(22±1°C)and humidity-controlled(50±10%)room on a12-h light/dark cycle (0800h to2000h)and had ad libitum access to food and water. All experiments were conducted in accordance with the European Community Council Directive of24November198686/609/EEC and 6106/10/EU and approved by the University of Turin Ethical Commit-tee for animal research and by the Italian Ministry of Health(License No.180/2006-B).The generation of Npy1r2lox and Npy1r Y5R?/?B6129S(129/SvJ,C57BL/6N derived strain)mice was achieved as previously described(Longo et al.2014).
Brie?y,a targeting vector for homologous recombination in embryonic stem(ES)cells was designed to introduce loxP sites around exons2–3,which code for the entire region of Npy1r, and a frt-neo-frt cassette≈450bp excised from p146(kind gift of William Wisden)upstream the ATG of Npy1r exon2.The lin-earized targeting vector was electroporated into5×107R1ES cells (129Sv×129sv-CP)(Nagy et al.1993)to create a conditional Npy1r allele(Npy1r+/loxP-neo),containing two loxP sites and a frt?anked neomycin selection cassette.R1-ES cells of a correct targeted clone for the recombination(Npy1r+/loxP-neo)were then expanded for injection into C57BL/6N blastocysts that were implanted into pseudopregnant ICR female recipients.We obtained11chimeric founders and three chimeric males transmitted the R1-ES cells and produced offspring with agouti coat color.PCR and Southern blot analysis of tail DNA revealed that50%of the pups con-tained the gene targeted Npy1r allele.Subsequently,the neo gene was removed by?p-mediated recombination in vivo,by crossing Npy1r+/loxP-neo offsprings with?p expressing C57BL/6N FLP deleter mice(Dymecki1996).To obtain mice with Npy1r gene inactivation restricted to Y5R expressing neurons(Npy1r Y5R?/?)three different mouse lines[Npy1r2lox,Tg Y5RitTA/Y1RVenus mice(Oberto et al.2007), and Tg LC1(Schonig et al.2002)]underwent speci?c breeding pro-tocol:we generated(1)a Npy1r2lox/TgY5RitTA/Y1RVenus mouse line, expressing the tTA in a cell type-speci?c manner under the control of the Npy5r promoter and(2)a Npy1r2lox/TgLC1line,encoding the tTA-responsive Cre transgene Tg LC1.Region-speci?c inactivation of Npy1r was achieved by crossing Npy1r2lox/TgY5RitTA/Y1RVenus with Npy1r2lox/Tg LC1mice.Doxycycline treatment of mothers(50mg/l in drinking water,1%sucrose)from conception to birth keeps the Cre transgene inactive in embryos and synchronizes the postna-tal Cre activation.In Npy1r2lox/Y5RitTA/Y1RVenus/LC1mice(Npy1r Y5R?/?mice)Cre expression was fully activated at approximately P40. Npy1r2lox(Npy1r2lox/TgY5RitTA/Y1RVenus,Npy1r2lox,Npy1r2lox/Tg LC1)and Npy1r2lox/TgY5RitTA/Y1RVenus/LC1(Npy1r Y5R?/?)were maintained in a randomly segregating population derived from more than300 different breeding pairs and each litter provided the same num-ber of experimental animals to both Npy1r2lox and Npy1r Y5R?/?groups.Npy1r2lox control mice(Npy1r2lox/TgY5RitTA/Y1RVenus,Npy1r2lox, Npy1r2lox/Tg LC1)did not show any recombination of the?oxed Npy1r allele and were used as control littermates in all experiments(Crusio et al.2009).
For the maintenance of the gene targeted and the transgenic lines animals were backcrossed to C57BL/6N mice.The coat color of the investigated animals was still mixed‘mainly black’indicating that the genetic background of the investigated popu-lation is very heterogeneous,which suggests that the observed phenotype is independent from the genetic background(Silva et al.1997).
Immediately after weaning(4weeks),male mice were assigned into four different groups:Npy1r2lox and Npy1r Y5R?/?single-housed and Npy1r2lox and Npy1r Y5R?/?group-housed with siblings(3–4 Npy1r2lox and Npy1r Y5R?/?brothers per cage).After6weeks,mice were tested for anxiety-like behavior(69mice)and,the day after, weighed and sacri?ced for in situ hybridization or perfused for immunohistochemical analysis.For3α,5α-TH PROG quanti?cation, 27mice were immediately sacri?ced after6weeks of different housing conditions.None of the animals were excluded from the analysis.
In situ hybridization for Npy1r mRNA
In situ hybridization was performed on coronal brain sections(14μm thick).Four different40-/45-mer oligonucleotide S35-labeled probes were simultaneously used to increase the signal and the reaction was carried out as previously described(Longo et al.2014),accord-ing to the protocol reported by Wisden and Morris(2002).The area of interest of clearly distinguishable nuclei was de?ned following the boundaries of the labeled regions[CA1pyramidal cell layer(CA1), dentate gyrus granule cell layer(DG)],while three to four spots were used for each slice in poorly contrasted regions[BLA and CA3 pyramidal cell layer(CA3)].Optical densities were measured and averaged using a Rodbard calibration to convert gray levels in opti-cal density(OD)unit,following the instruction in I MAGEJ(Rasband, W.S.,I MAGEJ,U.S.National Institutes of Health,Bethesda,Mary-land,USA,https://www.doczj.com/doc/7215409957.html,/ij/,1997–2014).Background was measured by averaging three to?ve spots(OD unit)in the sur-rounding blank region of the autoradiogram then subtracted from the correspondent nucleus value.Brain nuclei coordinates relative to bregma line were:BLA about?1.46to?1.70mm;DG,granu-lar layer?1.46to?1.70mm;CA1and CA3(pyramidal layer)?1.58 to?1.94mm.
Immunohistochemistry
Mice were given an anesthetic overdose of Zoletil(25mg/kg,i.p.) and transcardially perfused with4%paraformaldehyde in phos-phate buffered saline(PBS).Immunostainings of CRH and glucocor-ticoid receptor(GR)were carried out on free-?oating brain coronal sections(25μm),using the ABC Vectastain kit(Vector Laboratories, Burlingame,CA,USA).For CRH,after endogenous peroxidase block-ing in2%H2O2in0.1M PBS for30min,sections were blocked with 10%normal goat serum(NGS)and incubated for48h with primary rabbit CRH antibody(1:4000;Peninsula Laboratories,Bachem,UK). After washing in PBS0.3%Triton sections were incubated for1h with a secondary biotinylated goat anti-rabbit antibody(1:300;Vector Lab-oratories)followed by the ABC reagent.Peroxidase enzyme activity was visualized by0.05%diaminobenzidine tetrahydrochloride(DAB; SigmaAldrich,Milan,Italy).For each animal three to?ve sections of central amygdala(CeA)(?1.46mm relative to bregma)and three to four sections of medial parvocellular division of the paraventricular nuclei(PVNmp)(?0.82mm relative to bregma)were analyzed.For GR,after endogenous peroxidase blocking in0.5%H2O2in0.1M PBS for15min,sections were blocked with10%NGS in DayI buffer (PBS containing0.3%Triton X-100and1%bovine serum albumin) for1h and incubated overnight at room temperature with primary GR antibody(Santa Cruz Biotechnology,Heidelberg,Germany)at1:1000 dilution in DayI solution.After washing in DayII buffer(1:3dilution of DayI in0.1M PBS),sections were incubated for1h with a secondary biotinylated goat anti-rabbit antibody(1:300;Vector Laboratories)fol-lowed by the ABC reagent.Peroxidase was reacted with0.05% DAB and0.003%H2O2,mounted on slides and air-dried overnight. For each animal,?ve to seven sections of CA1were analyzed.
I MAGEJ1.48q software(NIH)was used to measure CRH and GR immunoreactivities through a threshold method and the number of positive pixels and the extension of area of interest were used to determine the fractional area covered by the speci?c signal.
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Longo et al.
Immuno?uorescence
Immunostaining of Y1R was carried on free-?oating vibratome-cut brain coronal sections(50μm)as previously described(Longo et al. 2014).Primary Y1R antibody was kindly provided by Janice Urban and was used at1:2000dilution.Brie?y,after the incubation with the biotinylated secondary antibody,?oating sections were subjected to tyramide ampli?cation(TSA Fluorescence System,Perkin Elmer, Milan,Italy).Sections were then mounted and coverslipped with Mowiol and pictures of brain regions were taken with a confocal microscope(Nikon D-Eclipse C1,Nikon,Chiyoda,Tokyo,Japan). Behavioral studies
At the end of the6weeks of isolation or group-housing,elevated plus maze(EPM)and open?eld(OF)were performed close to shifting phase timings(from0800h to1000h for the OF or1700h to1900h for EPM),when mice are more active.In the groups of mice that were analyzed by both of the tests,EPM and OF were performed the same day.On test day,mice were transported to the testing room and left undisturbed for1h before testing.The experiments were performed under dim white light conditions(2lux). For the EPM,mouse was placed in the center of the plus maze [comprising two open arms(30×5×0.20cm3,light intensity5.3lux) and two closed arms(30×5×15cm3walls,light intensity3.5lux)], facing an open arm and it was allowed to explore the maze for 5min.The cumulative time spent in and the number of entries into open and closed arms was recorded.For the OF,animal was placed in the corner of the arena(50×50×50cm3)with a light intensity of1.4lux,for5min.Total distance traveled,time spent and number of entries in the central area(25×25)were recorded.At the end of each trial,the apparatus was accurately cleaned up with ethanol2%and water.Data were analyzed using Ethovision XT9 video track system(Noldus Information Technology,Wageningen, The Netherlands).
Serum collection and analysis
A group of mice that were analyzed by both the EPM and the OF were given an anesthetic overdose of Zoletil(25mg/kg,i.p.)24-h after the beginning of behavioral tests.Blood samples were collected from the atrium,centrifuged and serum was frozen at?20°C until analyzed. Serum was assayed for corticosterone levels by using commercially available kits(MP Biomedicals,Santa Ana,CA,USA;RIA).
3α,5αTH-PROG extraction and assay
Steroids present in cerebral cortical homogenates were extracted three times with ethyl acetate,and the combined organic phases were dried under vacuum.The resulting residue was dissolved in 5ml of n-hexane and applied to a SepPak silica cartridge(Waters, Milford,MA,USA),and components were eluted with n-hexane and2-propanol(7:3,v/v).Steroids were separated and further puri-?ed by high-performance liquid chromatography(HPLC)on a5-μm Lichrosorb-diol column(250by4mm;Phenomenex,Torrance,CA, USA)with a discontinuous gradient of2-propanol(0–30%,v/v)in n-hexane.The recovery(70–80%)of3α,5αTH-PROG through the extraction and puri?cation procedures was monitored by adding trace amounts of tritiated3α,5αTH-PROG(6000–8000cpm; 20–80Ci/mmol)to the cortical homogenate.3α,5αTH-PROG was quanti?ed by radioimmunoassay as previously described (Serra et al.2000)with a speci?c antibody to3α,5αTH-PROG (Purdy et al.1990).Protein concentration was measured by the method of Lowry et al.(1951)with bovine serum albumin as standard.
Statistical analysis
All the quantitative results were analyzed by two-way analysis of variance,followed by Newman-Keuls test,for multiple comparisons. Data were analyzed using the STATISTICA software(StatSoft,Inc). All data are expressed as mean±SEM,and the level of statistical signi?cance was set at P<0.05.Results
Effect of housing conditions on Npy1r expression
The effect of different housing conditions and genotype on
Npy1r gene expression was?rst analyzed by semiquanti-
tative in situ hybridization.No differences of Npy1r mRNA
expression were observed in the cerebral cortex,in the hip-
pocampal CA1and CA3pyramidal cell layers,in the DG gran-
ule cell layer,and in the BLA of group-housed compared to
isolated Npy1r2lox control mice.The conditional inactivation
of Npy1r led to a signi?cant reduction of Npy1r mRNA in
the hippocampus and BLA of Npy1r Y5R?/?mice,compared
with Npy1r2lox mice,as previously demonstrated(Longo
et al.2014)(Fig.1a,b).Two-way analysis of variance(ANOVA)
for genotype and housing conditions indicated a signi?cant
effect of genotype in the CA1(F1,9=24.29;P=0.0008)and CA3(F1,9=17.26;P=0.0025)pyramidal cell layers,in the DG granule cell layer(F1,9=43.92;P=0.0001),and in the BLA(F1,9=6.73;P=0.0289).Accordingly,the immuno?uo-rescence staining of coronal brain sections using an anti-Y1R
antibody con?rmed that housing conditions did not in?uence
the expression of Y1R in the hippocampus and the amyg-
dala of isolated and group-housed Npy1r2lox mice,whereas
conditional inactivation of Npy1r led to a reduction of Y1R
expression in the CA1and CA3,in the DG,and in the BLA of
Npy1r Y5R?/?mice,compared with Npy1r2lox mice(Fig.1c). Effect of housing conditions on anxiety behavior
of Npy1r2lox and Npy1r Y5R?/?mice
Group-housed Npy1r2lox control mice showed increased
anxiety-like behavior in the EPM(Fig.2a)and in the OF
(Fig.2b)compared with isolated Npy1r2lox control mice.
Npy1r Y5R?/?mice show increased anxiety-related behav-
ior in both of the tests compared with Npy1r2lox siblings,
independently of the housing conditions(Fig.2a,b).In the
EPM test,two-way ANOVA for housing conditions and geno-
type revealed a signi?cant effect of housing conditions and
genotype for percent of entries in open arms(F1,65=33.32, P=0.0001and F1,65=7.57,P=0.0077,respectively),and of housing conditions,genotype,and housing conditions
and genotype interaction for percent of time in open
arms(F1,65=24.88,P=0.0000;F1,65=9.61,P=0.0029 and F1,65=7.16,P=0.0094,respectively).Consistently, group-housed mice showed a signi?cant decrease of total
entries in the arms,compared with isolated mice(iso-
lated Npy1r2lox:36±3.1;isolated Npy1r Y5R?/?:38±2.7;
group-housed Npy1r2lox:31±2.3;group-housed Npy1r Y5R?/?:
31±2.3)suggestive of a lower exploratory behavior(two-way ANOVA for housing conditions:F1,65=4.691,P=0.034).The signi?cant effect of housing conditions and genotype inter-
action for percent of time in open arms seems to be
mainly due to the large differences in the absolute val-
ues between isolated and group-housed mice that are
more anxious than isolated mice.In OF test,there was
a signi?cant effect of housing conditions and genotype
for distance traveled in the center(F1,28=4.33,P=0.047 and F1,28=5.56,P=0.026,respectively).A similar effect, although not signi?cant,of housing conditions and genotype was observed in the number of total entrance in the center
536Genes,Brain and Behavior(2015)14:534–542
Housing conditions and Y1R/Y5R containing
neurons
Figure 1:Expression of Npy1r messenger RNA (mRNA)and Y1receptor (Y1R)peptide in the brain of isolated or group-housed Npy1r 2lox control and Npy1r Y5R ?/?conditional mutants.(a)Representative autoradiograms of in situ hybridization of Npy1r mRNA on brain coronal sections from isolated and group-housed Npy1r 2lox and Npy1r Y5R ?/?mice.(b)Quantitative signal intensity (OD)analysis of in situ hybridization revealed a signi?cant decrease of Npy1r mRNA expression in the DG granule cell layer,CA1and CA3pyramidal cell layers of hippocampus and in the BLA of Npy1r Y5R ?/?mice compared with control Npy1r 2lox littermates independently of the housing conditions.Data are expressed as relative OD and are the mean ±SEM from two independent experiments;n =3(group-housed Npy1r Y5R ?/?and Npy1r 2lox )and four (isolated Npy1r Y5R ?/?)mice.*P <0.05and **P <0.01vs.isolated or grouped-housed Npy1r 2lox mice,by Newman–Keuls (Scale bar =1.5mm).(c)The Y1R immuno?uorescence con?rmed the reduced Y1R expression in DG,CA1and CA3of hippocampus and in the BLA of grouped housed and isolated Npy1r Y5R ?/?mice compared with Npy1r 2lox siblings (Scale bar =50μm).BLA,basolateral amygdala;CA1,CA1stratum pyramidale;CA3,CA3stratum pyramidale;gr,granular cell layer;mol,molecular layer;po,polymorphic-layer;pyr,pyramidal cell layer;rad,stratum radiatum;slu,stratum lucidum.
of the OF (isolated Npy1r 2lox :16.8±4.1;isolated Npy1r Y5R ?/?:10.8±2.7;group-housed Npy1r 2lox :12.8±2.6;group-housed Npy1r Y5R ?/?:8.8±2.9)and in the %of time spent in the center of the OF (isolated Npy1r 2lox :7.7±0.7;isolated
Npy1r
Y5R ?/?: 5.6±1.8;group-housed Npy1r 2lox : 6.5±0.9;group-housed Npy1r Y5R ?/?: 3.9±1.1).Housing conditions and genotype failed to affect locomotor activity of Npy1r 2lox and Npy1r Y5R ?/?mice,as evidenced by total distance moved in the OF (Fig.2b).
Effect of housing conditions on physiological functions of Npy1r 2lox and Npy1r Y5R ?/?mice
Independently of genotype,group-housed mice displayed lower density of CRH immunoreactive ?bers in the CeA and of GR immunoreactive neurons in the CA1,and higher density of CRH cell bodies in the medial parvocellular divi-sion of the paraventricular nuclei (PVNmp)and of corticos-terone serum levels,compared with isolated mice,which suggests increased central drive of the HPA-axis activity (Bertocchi et al.2011;Dimitrov et al.2007;Noguchi et al.2010)(Figs.3,4).Mice raised in group also showed higher cerebrocortical concentrations of 3α,5αTH-PROG than iso-lated mice (Fig.3).
Two way ANOVA revealed a signi?cant effect of hous-ing conditions for CRH immunoreactivity (IR)in the CeA (F 1,21=6.364,P =0.020),CRH-IR in the PVNmp (F 1,27=21.2,P <0.001),GR-IR (F 1,15=13.815,P =0.0021),corticosterone (F 1,19=8.51,P =0.0089)and 3α,5αTH-PROG (F 1,23=26.25,P =0.0000).No differences in CRH-IR and GR-IR,corti-costerone and 3α,5αTH-PROG,were observed between
Npy1r 2lox and Npy1r Y5R ?/?mice,independently of hous-ing conditions.No signi?cant differences in body weight were observed among experimental groups [(g),isolated Npy1r 2lox =32.4±1.3;isolated Npy1r Y5R ?/?=29.9±0.8;
group-housed Npy1r 2lox =29.4±1.5;
group-housed Npy1r
Y5R ?/?=28.5±0.9].Discussion
Animal models that involve a social context,such as hous-ing conditions,are more appropriate than conventional ani-mal models of stress,including restraint,to mimic a stress-ful situation that an animal can meet in its real life events and for which behavioral and neuroendocrine responses had been shaped by evolutionary processes (Palanza et al.2001).A number of studies have clearly demonstrated that group-housed male mice may show behavioral (e.g.Barnard et al.1991;Kaliste-Korhonen &Eskola 2000),endocrine (e.g.Brain &Benton 1983)and immune (e.g.Bohus &Koolhaas 1991)indices of stress as a consequence of aggressive inter-actions between cage-mates.
In accordance with previous studies (Wersinger et al.2002),we observed a high level of aggression of Npy1r 2lox mice,compared to typical levels observed in the C57BL/6N strain,possibly due their mixed genetic background (P .Palanza,personal communication).
Here we showed that group-housing of our mouse strain increases anxiety-like behavior in the EPM and in the OF and activates HPA axis,as shown by the increase of
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al.
Figure 2:Anxiety-like behavior of isolated and group-housed Npy1r 2lox and Npy1r Y5R ?/?mice.Two way ANOVA revealed that group housing conditions (group-housed mice vs.isolated mice)and conditional inactivation of Npy1r in Y5R containing neurons signi?cantly increases anxiety levels in the EPM and OF tests (see Results ).Group-housed Npy1r 2lox and Npy1r Y5R ?/?mice showed signi?cantly lower frequency of entries and percent of time spent in the open arms of the EPM (a),and lower percent of distance traveled in the center of the OF (b,left panel)compared with isolated Npy1r 2lox mice and Npy1r Y5R ?/?mice,respectively.In the same behavioral tests,Npy1r Y5R ?/?mice showed an anxious phenotype compared with Npy1r 2lox siblings,independently of housing conditions.Genotype and housing conditions failed to affect total distance traveled by Npy1r 2lox and Npy1r Y5R ?/?mice in the OF (b,right panel).EPM:data are the mean ±SEM;n =12(isolated Npy1r 2lox ),15(isolated Npy1r Y5R ?/?)and 21(group-housed Npy1r 2lox and Npy1r Y5R ?/?)mice from 7to 11litters.**P <0.01vs.isolated Npy1r 2lox mice.OF:data are the mean ±SEM;n =6(isolated Npy1r 2lox ),5(isolated Npy1r Y5R ?/?),10(group-housed Npy1r 2lox )and 11(group-housed Npy1r Y5R ?/?)mice from 4to 5litters.
corticosterone serum levels and CRH-IR in PVNmp and the decrease of CRH-IR in CeA and GR-IR in the CA1(Noguchi et al.2010).These ?ndings are consistent with previous stud-ies showing that individually housed male mice of different strains showed decreased anxiety-like behavior in different experimental paradigms (Abramov et al.2004;Bartolomucci et al.2003;Guo et al.2004;Hilakivi et al.1989;Palanza et al.2001;Voikar et al.2005),and lower corticosterone plasma levels compared with their group-housed counterpart males (Mucignat-Caretta et al.2014).However,contrasting reports on the possible stressfulness of isolation in mice exist,and other studies found opposite or no effects of group housing on emotional behavior and endocrine system (Arndt et al.2009;Koike et al.2009;Liu et al.2013;Zhang et al.2012).Differences among studies in the species,the laboratory strain,the duration and timing of isolation and the procedural variables in testing anxious behavior may explain these discrepancies.Thus,we cannot rule out that the lack of backcrossing of our B6129S mouse strain onto C57BL/6N strain of mice could impact on the results (Crusio et al.2009).Several lines of evidence suggest that,in rodents,NPY participates in the regulation of anxious responses (Dumont &Quirion 2014;Dumont et al.2009;Eva et al.2006;Kormos &Gaszner 2013)and has marked anti-stress effects (Russo et al.2012;Sajdyk et al.2006)via the activation of Y1R in amygdala,hippocampus and locus coeruleus (Bertocchi et al.2011;Broqua et al.1995;Heilig et al.1993;Lin et al.2010;Sajdyk et al.2008).Genetic and pharmacologic experiments
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Housing conditions and Y1R/Y5R containing
neurons
Figure3:Physiological functions in isolated and group-housed Npy1r2lox and Npy1r Y5R?/?mice.Two way ANOVA revealed that group housing conditions(group-housed mice vs.isolated mice)signi?cantly decreases glucocorticoid receptor immunoreactivity (GR-IR)in the CA1and corticotropin releasing hormone immunoreactive(CRH-IR)?bers in the CeA,and signi?cantly increases CRH-IR cell bodies in the medial parvocellular division of the paraventricular nucleus(PVNmp)and corticosterone serum levels compared with isolation,which suggests increased central drive of the HPA-axis activity(see Results).In addition,group housing signi?cantly increases cerebrocortical concentrations of3α,5α-TH PROG compared with isolated mice,independently of the genotype.Data are expressed as the mean±SEM.Immunoreactivity,fractional area×02:GR-IR:n=5(group-housed Npy1r2lox and Npy1r Y5R?/?and isolated Npy1r2lox) and4(isolated Npy1r Y5R?/?)mice from2to4litters;CeA CRH-IR:n=7(isolated Npy1r2lox and Npy1r Y5R?/?),5(group-housed Npy1r2lox) and6(group-housed Npy1r Y5R?/?)mice from2to4litters;PVNmp CRH-IR:n=7(isolated Npy1r2lox),8(isolated Npy1r Y5R?/?and group-housed Npy1r2lox)and9(group-housed Npy1r Y5R?/?)mice from2to4litters;corticosterone serum levels(ng/ml):n=6(isolated and group-housed Npy1r2lox),n=5(isolated Npy1r Y5R?/?)and n=7(group-housed Npy1r Y5R?/?)mice from2to5litters.3α,5α-TH PROG (ng/g of tissue)n=6(isolated and group-housed Npy1r2lox),n=8(isolated Npy1r Y5R?/?),n=7(group-housed Npy1r Y5R?/?)from2to4 litters.##P<0.01vs.isolated Npy1r2lox;#P<0.05vs.isolated Npy1r2lox;**P<0.01vs.isolated Npy1r Y5R?/?mice;*P<0.05vs.isolated Npy1r Y5R?/?mice by Newman–Keuls.
suggested that in addition to the Y1R,the Y5R subtype in the BLA plays a signi?cant role in anxiety-related behaviors (Domschke et al.2008;Roseboom et al.2014;Sajdyk et al. 2002;Sorensen et al.2004).
We have previously shown that the conditional inactivation of Npy1r selectively in neurons coexpressing Y5R increases anxiety but is not associated with the activation of HPA-axis activity(Longo et al.2014).In the present study,we further demonstrated that Npy1r Y5R?/?conditional mutants display a strong anxious phenotype in the EPM and OF,indepen-dently of housing conditions,but show identical levels of CRH in the CeA and PVNmp,of GR in the CA1and of corticos-terone serum levels,compared to their control littermates. These data con?rm that Y1Rs expressed in Y5R containing neurons in the limbic system play a major role in the control of anxious behavior but fails to affect stress-related markers. This suggest that NPY,acting on Y1R,might activate dis-tinct neuronal circuits to regulate anxiety-and stress-related responses.On the other hand,given that Y1R and Y5R colo-calization may result in receptor heterodimerization and in enhanced or altered function of several physiologic systems (Mashiko et al.2009),the anxious phenotype of Npy1r Y5R?/?mice might be due to both the conditional inactivation of Npy1r and the Y5R altered expression/function. Experimental evidence suggested a functional interaction between GABA and NPY that may be important in the reg-ulation of anxiety-like behavior(Dimitrov et al.2007;Sajdyk et al.2006).
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Longo et
al.
Figure 4:Representative pictures of corticotropin releasing hormone and nuclear glucocorticoid receptor immunoreactive staining of brain coronal sections from isolated and group-housed Npy1r 2lox and Npy1r Y5R ?/?mice.Group-housed mice showed a decrease of the density of nuclear glucocorticoid immunoreactive (GR-IR)staining in the CA1pyramidal cell layer (a)and of corticotropin releasing hormone immunoreactive (CRH-IR)?bers in the central amygdala (b)and an increase of CRH-IR cell bodies in the medial parvocellular division of the paraventricular nuclei (PVNmp)(c)compared to isolated mice,independently of the genotype.(Scale bar =100μm).
The BLA is an important component of neuronal circuitry controlling anxiety-related behaviors where both the Y1R and the Y5R appear to be required for the anxiolytic response to NPY .Previous studies suggested that,in the rodent limbic system,the Y1R and Y5R are colocalized in the BLA (Oberto et al.2007;Wolak et al.2003)and are coexpressed in 50%of GABAergic interneurons (Longo et al.2014),which have strong synaptic control over principal neurons –the pyrami-dal cells (Muller et al.2006;Rainnie et al.2006).Therefore,the inactivation of Npy1r in neurons coexpressing Y5R could have profound effects on the synchronous activity of the BLA and may explain the phenotype of Npy1r Y5R ?/?mice.The amygdala was identi?ed as a key structure for mediating the anxiolytic effects of 3α,5α-TH PROG,as its direct infu-sion into the amygdala was followed by a signi?cant increase in the number of entries and the time spent in the open arms of the EPM (Akwa et al.1999)and its anxiolytic-like effects vary as a function of intracerebral microinfusion site (Engin &Treit 2007).We have previously shown that a sus-tained increase in the brain concentrations of neuroactive steroids,induced by pharmacological treatment or by physio-logical conditions,results in the upregulation of Npy1r gene expression in the amygdaloid complex,suggesting that these compounds might play an important functional role in regulat-ing the modulatory effect of NPY on emotional behavior (Eva et al.2008;Ferrara et al.2001;Oberto et al.2002).
Conversely,adaptation of GABAergic transmission to chronic stress,apparently,does not involve Y1R-mediated transmission in the amygdala,since exposure to repeated restraint stress,which induced repetitive and transient increases in the concentrations of 3α,5α-TH PROG in the cerebral cortex,failed to affect Npy1r gene expression in this region (Mele et al.2004).
Accordingly,here we showed that group housing increases the concentrations of 3α,5α-TH PROG in the cerebral cortex
of both Npy1r Y5R ?/?and Npy1r 2lox mice,compared with their isolated counterparts.
The ?nding that both chronic restraint and mild social stress increase cerebrocortical concentrations of 3α,5α-TH PROG may be surprising since,consistent with its ability to facilitate GABAergic neurotransmission,this neuroactive steroid exhibits potent anxiolytic-like effects in a variety of animal models (Gasior et al.1999).The increase in neu-roactive steroid concentrations induced by chronic stress might represent a homeostatic mechanism for the restora-tion of GABA A receptor function and be physiologically rel-evant in protection against stress-induced anxiety.On the other hand,cerebrocortical levels might not re?ect dynamic release patterns into different compartments of the brain.Further investigations focused on different brain areas are needed to ascertain putative differences between socially isolated and group-housed mice.In conclusion,the current study expands on our ?nding on Npy1r Y5R ?/?mice phenotype by showing that the inactivation of Npy1r in neurons coex-pressing Y5R increases anxiety but is not involved in promot-ing resilience to a mild social stress,such as group housing seems to be in this B6129S mouse strain,since no changes in stress-related markers were observed between conditional mutants and their control littermates.This ?nding provides additional information about the NPY-related neuronal path-ways involved and the speci?c role of Y1R coexpressed with Y5R in limbic neurons in decreasing anxious behavior and stress susceptibility,suggesting a potential strategy of novel therapeutics for a variety of anxiety-and stress-related disorders.
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Acknowledgments
We thank Dr J.Urban for polyclonal antibody and protocols for Y1R immunohistochemistry.Grants:PRIN2005057519_002, and2008PLKP3E_003from MIUR,Fondazione CRT,Torino to C.E.;‘Neuroscience program’,Compagnia di San Paolo,Torino to C.E.and P.P.;PRIN2005057519_001from MIUR to M.S.A.L. fellowships from the Health Ministry of Regione Piemonte and Fondazione CRT.
542Genes,Brain and Behavior(2015)14:534–542
英语介词用法大全 TTA standardization office【TTA 5AB- TTAK 08- TTA 2C】
介词（The Preposition）又叫做前置词，通常置于名词之前。它是一种虚词，不需要重读，在句中不单独作任何句子成分，只表示其后的名词或相当于名词的词语与其他句子成分的关系。中国学生在使用英语进行书面或口头表达时，往往会出现遗漏介词或误用介词的错误，因此各类考试语法的结构部分均有这方面的测试内容。 1. 介词的种类 英语中最常用的介词，按照不同的分类标准可分为以下几类： (1). 简单介词、复合介词和短语介词 ①.简单介词是指单一介词。如： at , in ,of ,by , about , for, from , except , since, near, with 等。②. 复合介词是指由两个简单介词组成的介词。如： Inside, outside , onto, into , throughout, without , as to as for , unpon, except for 等。 ③. 短语介词是指由短语构成的介词。如： In front of , by means o f, on behalf of, in spite of , by way of , in favor of , in regard to 等。 (2). 按词义分类 {1} 表地点（包括动向）的介词。如： About ,above, across, after, along , among, around , at, before, behind, below, beneath, beside, between , beyond ,by, down, from, in, into , near, off, on, over, through, throught, to, towards,, under, up, unpon, with, within , without 等。 {2} 表时间的介词。如： About, after, around , as , at, before , behind , between , by, during, for, from, in, into, of, on, over, past, since, through, throughout, till(until) , to, towards , within 等。 {3} 表除去的介词。如： beside , but, except等。 {4} 表比较的介词。如： As, like, above, over等。 {5} 表反对的介词。如： againt ,with 等。 {6} 表原因、目的的介词。如： for, with, from 等。 {7} 表结果的介词。如： to, with , without 等。 {8} 表手段、方式的介词。如： by, in ,with 等。 {9} 表所属的介词。如： of , with 等。 {10} 表条件的介词。如：
介词用法知多少 介词是英语中最活跃的词类之一。同一个汉语词汇在英语中可译成不同的英语介词。例如汉语中的“用”可译成：（1）用英语（in English）；（2）用小刀（with a knife）；（3）用手工（by hand）；（4）用墨水（in ink）等。所以，千万不要以为记住介词的一两种意思就掌握了这个介词的用法，其实介词的用法非常广泛，搭配能力很强，越是常用的介词，其含义越多。下面就简单介绍几组近义介词的用法及其搭配方法。 一. in, to, on和off在方位名词前的区别 1. in表示A地在B地范围之内。如： Taiwan is in the southeast of China. 2. to表示A地在B地范围之外，即二者之间有距离间隔。如： Japan lies to the east of China. 3. on表示A地与B地接壤、毗邻。如： North Korea is on the east of China. 4. off表示“离……一些距离或离……不远的海上”。如： They arrived at a house off the main road. New Zealand lies off the eastern coast of Australia. 二. at, in, on, by和through在表示时间上的区别 1. at指时间表示： （1）时间的一点、时刻等。如： They came home at sunrise （at noon, at midnight, at ten o’clock, at daybreak, at dawn）. （2）较短暂的一段时间。可指某个节日或被认为是一年中标志大事的日子。如： He went home at Christmas （at New Year, at the Spring Festival, at night）. 2. in指时间表示： （1）在某个较长的时间（如世纪、朝代、年、月、季节以及泛指的上午、下午或傍晚等）内。如： in 2004, in March, in spring, in the morning, in the evening, etc （2）在一段时间之后。一般情况下，用于将来时，谓语动词为瞬间动词，意为“在……以后”。如： He will arrive in two hours. 谓语动词为延续性动词时，in意为“在……以内”。如： These products will be produced in a month. 注意：after用于将来时间也指一段时间之后，但其后的时间是“一点”，而不是“一段”。如： He will arrive after two o’clock. 3. on指时间表示： （1）具体的时日和一个特定的时间，如某日、某节日、星期几等。如： On Christmas Day（On May 4th）, there will be a celebration. （2）在某个特定的早晨、下午或晚上。如： He arrived at 10 o’clock on the night of the 5th. （3）准时，按时。如： If the train should be on time, I should reach home before dark. 4. by指时间表示： （1）不迟于，在（某时）前。如：
With的用法全解 with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、 with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词； 2. with或without-名词/代词+副词； 3. with或without-名词/代词+介词短语； 4. with或without-名词/代词 +动词不定式； 5. with或without-名词/代词 +分词。 下面分别举例： 1、 She came into the room，with her nose red because of cold.（with+名词+形容词，作伴随状语）
2、 With the meal over ， we all went home.（with+名词+副词，作时间状语） 3、The master was walking up and down with the ruler under his arm。（with+名词+介词短语，作伴随状语。） The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.（with+名词+不定式，作伴随状语）He could not finish it without me to help him.（without+代词 +不定式，作条件状语） 5、She fell asleep with the light burning.（with+名词+现在分词，作伴随状语） Without anything left in the with结构是许多英 语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 二、with结构的用法 with是介词，其意义颇多，一时难掌握。为帮助大家理清头绪，以教材中的句子为例，进行分类，并配以简单的解释。在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件（详见上述例句）。 1.带着，牵着…… (表动作特征)。如： Run with the kite like this.
精神分裂症的病因及发病机理 精神分裂症病因：尚未明，近百年来的研究结果也仅发现一些可能的致病因素。（一）生物学因素1．遗传遗传因素是精神分裂症最可能的一种素质因素。国内家系调查资料表明：精神分裂症患者亲属中的患病率比一般居民高6.2倍，血缘关系愈近，患病率也愈高。双生子研究表明：遗传信息几乎相同的单卵双生子的同病率远较遗传信息不完全相同 的双卵双生子为高，综合近年来11项研究资料：单卵双生子同病率（56.7%），是双卵双生子同病率（12.7%）的4.5倍，是一般人口患难与共病率的35-60倍。说明遗传因素在本病发生中具有重要作用，寄养子研究也证明遗传因素是本症发病的主要因素，而环境因素的重要性较小。以往的研究证明疾病并不按类型进行遗传，目前认为多基因遗传方式的可能性最大，也有人认为是常染色体单基因遗传或多源性遗传。Shields发现病情愈轻，病因愈复杂，愈属多源性遗传。高发家系的前瞻性研究与分子遗传的研究相结合，可能阐明一些问题。国内有报道用人类原癌基因Ha-ras-1为探针，对精神病患者基因组进行限止性片段长度多态性的分析，结果提示11号染色体上可能存在着精神分裂症与双相情感性精神病有关的DNA序列。2．性格特征：约40%患者的病前性格具有孤僻、冷淡、敏感、多疑、富于幻想等特征，即内向
型性格。3．其它：精神分裂症发病与年龄有一定关系，多发生于青壮年，约1/2患者于20～30岁发病。发病年龄与临床类型有关，偏执型发病较晚，有资料提示偏执型平均发病年龄为35岁，其它型为23岁。80年代国内12地区调查资料：女性总患病率（7.07%。）与时点患病率（5.91%。）明显高于男性（4.33%。与3.68%。）。Kretschmer在描述性格与精神分裂症关系时指出：61%患者为瘦长型和运动家型，12.8%为肥胖型，11.3%发育不良型。在躯体疾病或分娩之后发生精神分裂症是很常见的现象，可能是心理性生理性应激的非特异性影响。部分患者在脑外伤后或感染性疾病后发病；有报告在精神分裂症患者的脑脊液中发现病毒性物质；月经期内病情加重等躯体因素都可能是诱发因素，但在精神分裂症发病机理中的价值有待进一步证实。（二）心理社会因素1．环境因素①家庭中父母的性格，言行、举止和教育方式（如放纵、溺爱、过严）等都会影响子女的心身健康或导致个性偏离常态。②家庭成员间的关系及其精神交流的紊乱。③生活不安定、居住拥挤、职业不固定、人际关系不良、噪音干扰、环境污染等均对发病有一定作用。农村精神分裂症发病率明显低于城市。2．心理因素一般认为生活事件可发诱发精神分裂症。诸如失学、失恋、学习紧张、家庭纠纷、夫妻不和、意处事故等均对发病有一定影响，但这些事件的性质均无特殊性。因此，心理因素也仅属诱发因
with的用法大全----四级专项训练with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、 with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词; 2. with或without-名词/代词+副词; 3. with或without-名词/代词+介词短语; 4. with或without-名词/代词+动词不定式; 5. with或without-名词/代词+分词。 下面分别举例：
1、 She came into the room，with her nose red because of cold.(with+名词+形容词，作伴随状语) 2、 With the meal over ， we all went home.(with+名词+副词，作时间状语) 3、The master was walking up and down with the ruler under his arm。(with+名词+介词短语，作伴随状语。) The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.(with+名词+不定式，作伴随状语) He could not finish it without me to help him.(without+代词 +不定式，作条件状语) 5、She fell asleep with the light burning.(with+名词+现在分词，作伴随状语) 6、Without anything left in the cupboard， she went out to get something to eat.(without+代词+过去分词，作为原因状语) 二、with结构的用法 在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件(详见上述例句)。
1.I don`t like a ___(rain) day. 2.Please tell Tom ___(call)me back. 3.____ is the weather like? It`s snowy. How. What. Where. Why. 4.____ ia it going? It`s great. How. What. Where. When. 5.Is he____? No, he’s ___ in the river. A. swims,fishing B. swimming, running C. swim,walking D. running, swimming 6.Hello!is that Linda speaking? Yes.______ Who’s that? Who’s it? Who are you? I’m Linda. 7.He often ____(watch)TV in the evening. But now he _____(read)a book. 8.My uncle is a ___(cook). 9.It’s a nice ____. A weather photo salad rice 10.After supper Mary often ___ along the river. Take a walk takes walk taking a walk takes a walk 11.How’s it going? _____. The weather is so cold. Preety good. Great. Terrible. Not bad. 12.Beijing ___ (have)a long history. 13.I am having a great time ____(visit)my grandmother. 14.We feel very ___ after the ___ (relax) vacation. 15.We are ___ vacation in Dalian. A in B on C at Dof 16.What ____ when it’s sunny? Aare you doing B do you do C does you D is he do 17.Tom like to go ___(skate) 18.Kate is very tired, so she needs ____(relax) 19.Would you like to ____ a photofor us? Sure. Agive B make C take Dget 20.Could you help me put up the maps on the wall?_____ A.No problem B. I hope so C. That’s all right D.That’s a good idea. 21.China plan to let Jim ____the Xisha Islands> Visit visits visiting visited 22.Where is Grace? She ____ IN THE yard. Reads read is reading was reading 23.Britain is ___ European country and Singapore is ___ Asian country A,a,a a, an the , an a ,the 1.I don`t like a ___(rain) day. 2.Please tell Tom ___(call)me back. 3.____ is the weather like? It`s snowy. How. What. Where. Why. 4.____ ia it going? It`s great. How. What. Where. When. 5.Is he____? No, he’s ___ in the river. A. swims,fishing B. swimming, running C. swim,walking D. running, swimming 6.Hello!is that Linda speaking? Yes.______ Who’s that? Who’s it? Who are you? I’m Linda. 7.He often ____(watch)TV in the evening. But now he _____(read)a book. 8.My uncle is a ___(cook). 9.It’s a nice ____. A weather photo salad rice 10.After supper Mary often ___ along the river. Take a walk takes walk taking a walk takes a walk 11.How’s it going? _____. The weather is so cold. Preety good. Great. Terrible. Not bad. 12.Beijing ___ (have)a long history. 13.I am having a great time ____(visit)my grandmother. 14.We feel very ___ after the ___ (relax) vacation. 15.We are ___ vacation in Dalian. A in B on C at Dof 16.What ____ when it’s sunny? Aare you doing B do you do C does you D is he do 17.Tom like to go ___(skate) 18.Kate is very tired, so she needs ____(relax) 19.Would you like to ____ a photofor us? Sure. Agive B make C take Dget 20.Could you help me put up the maps on the wall?_____ A.No problem B. I hope so C. That’s all right D.That’s a good idea. 21.China plan to let Jim ____the Xisha Islands> Visit visits visiting visited 22.Where is Grace? She ____ IN THE yard. Reads read is reading was reading 23.Britain is ___ European country and Singapore is ___ Asian country A,a,a a, an the , an a ,the
with用法归纳 （1）“用……”表示使用工具，手段等。例如： ①We can walk with our legs and feet. 我们用腿脚行走。 ②He writes with a pencil. 他用铅笔写。 （2）“和……在一起”，表示伴随。例如： ①Can you go to a movie with me? 你能和我一起去看电影'>电影吗？ ②He often goes to the library with Jenny. 他常和詹妮一起去图书馆。 （3）“与……”。例如： I’d like to have a talk with you. 我很想和你说句话。 （4）“关于，对于”，表示一种关系或适应范围。例如： What’s wrong with your watch? 你的手表怎么了？ （5）“带有，具有”。例如： ①He’s a tall kid with short hair. 他是个长着一头短发的高个子小孩。 ②They have no money with them. 他们没带钱。 （6）“在……方面”。例如： Kate helps me with my English. 凯特帮我学英语。 （7）“随着，与……同时”。例如： With these words, he left the room. 说完这些话，他离开了房间。 [解题过程] with结构也称为with复合结构。是由with+复合宾语组成。常在句中做状语，表示谓语动作发生的伴随情况、时间、原因、方式等。其构成有下列几种情形： 1.with+名词(或代词)+现在分词 此时，现在分词和前面的名词或代词是逻辑上的主谓关系。 例如:1)With prices going up so fast, we can't afford luxuries. 由于物价上涨很快，我们买不起高档商品。（原因状语） 2)With the crowds cheering, they drove to the palace. 在人群的欢呼声中，他们驱车来到皇宫。（伴随情况） 2.with+名词(或代词)+过去分词 此时，过去分词和前面的名词或代词是逻辑上的动宾关系。
in，on ，at，by ，of ，with 介词区别与用法 in用在年月季节前，还有上午、下午等固定习语里 at用于传统的节日前，如at Christmas等；还有固定词组：at noon, at night;在点时间前用at 如at 7.15 on 用于具体的日期前，星期几，几号，包括那天的上午下午晚上等，如on Friday afternoon with: 一、with表拥有某物 Mary married a man with a lot of money . 马莉嫁给了一个有着很多钱的男人。 I often dream of a big house with a nice garden . 我经常梦想有一个带花园的大房子。 The old man lived with a little dog on the lonely island . 这个老人和一条小狗住在荒岛上。 二、with表用某种工具或手段 I cut the apple with a sharp knife . 我用一把锋利的刀削平果。 Tom drew the picture with a pencil . 汤母用铅笔画画。 三、with表人与人之间的协同关系 make friends with sb talk with sb quarrel with sb struggle with sb fight with sb play with sb work with sb cooperate with sb I have been friends with Tom for ten years since we worked with each other , and I have never quarreled with him . 自从我们一起工作以来，我和汤母已经是十年的朋友了，但我们从没有吵过架。 四、with 表原因或理由 John was in bed with high fever . 约翰因发烧卧床。 He jumped up with joy . 他因高兴跳起来。 Father is often excited with wine . 父亲常因白酒变的兴奋。 五、with 表“带来”，或“带有,具有”，在…身上，在…身边之意 The girl with golden hair looks beautiful . 那个金头发的女孩看起来漂亮。 The famous director will come to the meeting with the leading actor and actress .
独立主格篇 独立主格，首先它是一个“格”，而不是一个“句子”。在英语中任何一个句子都要有主谓结构，而在这个结构中，没有真正的主语和谓语动词，但又在逻辑上构成主谓或主表关系。独立主格结构主要用于描绘性文字中，其作用相当于一个状语从句，常用来表示时间、原因、条件、行为方式或伴随情况等。除名词/代词+名词、形容词、副词、非谓语动词及介词短语外，另有with或without短语可做独立主格，其中with可省略而without不可以。*注：独立主格结构一般放在句首，表示原因时还可放在句末；表伴随状况或补充说明时，相当于一个并列句，通常放于句末。 一、独立主格结构： 1. 名词/代词+形容词 He sat in the front row, his mouth half open. Close to the bank I saw deep pools, the water blue like the sky. 靠近岸时，我看见几汪深池塘，池水碧似蓝天。 2. 名词/代词+现在分词 Winter coming, it gets colder and colder. The rain having stopped, he went out for a walk.
The question having been settled, we wound up the meeting. 也可以The question settled, we wound up the meeting. 但含义稍有差异。前者强调了动作的先后。 We redoubled our efforts, each man working like two. 我们加倍努力，一个人干两个人的活。 3. 名词/代词+过去分词 The job finished, we went home. More time given, we should have done the job much better. *当表人体部位的词做逻辑主语时，不及物动词用现在分词，及物动词用过去分词。 He lay there, his teeth set, his hands clenched, his eyes looking straight up. 他躺在那儿，牙关紧闭，双拳紧握，两眼直视上方。 4. 名词/代词+不定式 We shall assemble at ten forty-five, the procession to start moving at precisely eleven. We divided the work, he to clean the windows and I to sweep the floor.
精神分裂症的发病原因是什么 精神分裂症是一种精神病，对于我们的影响是很大的，如果不幸患上就要及时做好治疗，不然后果会很严重，无法进行正常的工作和生活，是一件很尴尬的事情。因此为了避免患上这样的疾病，我们就要做好预防，今天我们就请广州协佳的专家张可斌来介绍一下精神分裂症的发病原因。 精神分裂症是严重影响人们身体健康的一种疾病，这种疾病会让我们整体看起来不正常，会出现胡言乱语的情况，甚至还会出现幻想幻听，可见精神分裂症这种病的危害程度。 (1)精神刺激：人的心理与社会因素密切相关，个人与社会环境不相适应，就产生了精神刺激，精神刺激导致大脑功能紊乱，出现精神障碍。不管是令人愉快的良性刺激，还是使人痛苦的恶性刺激，超过一定的限度都会对人的心理造成影响。 (2)遗传因素：精神病中如精神分裂症、情感性精神障碍，家族中精神病的患病率明显高于一般普通人群，而且血缘关系愈近，发病机会愈高。此外，精神发育迟滞、癫痫性精神障碍的遗传性在发病因素中也占相当的比重。这也是精神病的病因之一。 (3)自身：在同样的环境中，承受同样的精神刺激，那些心理素质差、对精神刺激耐受力低的人易发病。通常情况下，性格内向、心胸狭窄、过分自尊的人，不与人交往、孤僻懒散的人受挫折后容易出现精神异常。 (4)躯体因素：感染、中毒、颅脑外伤、肿瘤、内分泌、代谢及营养障碍等均可导致精神障碍，。但应注意，精神障碍伴有的躯体因素，并不完全与精神症状直接相关，有些是由躯体因素直接引起的，有些则是以躯体因素只作为一种诱因而存在。 孕期感染。如果在怀孕期间，孕妇感染了某种病毒，病毒也传染给了胎儿的话，那么，胎儿出生长大后患上精神分裂症的可能性是极其的大。所以怀孕中的女性朋友要注意卫生，尽量不要接触病毒源。 上述就是关于精神分裂症的发病原因，想必大家都已经知道了吧。患上精神分裂症之后，大家也不必过于伤心，现在我国的医疗水平是足以让大家快速恢复过来的，所以说一定要保持良好的情绪。
介词with的用法大全 With是个介词，基本的意思是“用”，但它也可以协助构成一个极为多采多姿的句型，在句子中起两种作用；副词与形容词。 with在下列结构中起副词作用： 1.“with+宾语+现在分词或短语”，如： (1) This article deals with common social ills, with particular attention being paid to vandalism. 2.“with+宾语+过去分词或短语”，如： (2) With different techniques used, different results can be obtained. (3) The TV mechanic entered the factory with tools carried in both hands. 3.“with+宾语+形容词或短语”，如： (4) With so much water vapour present in the room, some iron-made utensils have become rusty easily. (5) Every night, Helen sleeps with all the windows open. 4.“with+宾语＋介词短语”，如： (6) With the school badge on his shirt, he looks all the more serious. (7) With the security guard near the gate no bad character could do any thing illegal. 5.“with+宾语＋副词虚词”，如： (8) You cannot leave the machine there with electric power on. (9) How can you lock the door with your guests in? 上面五种“with”结构的副词功能，相当普遍，尤其是在科技英语中。 接着谈“with”结构的形容词功能，有下列五种： 一、“with+宾语+现在分词或短语”，如： (10) The body with a constant force acting on it. moves at constant pace. (11) Can you see the huge box with a long handle attaching to it ? 二、“with+宾语+过去分词或短语” (12) Throw away the container with its cover sealed. (13) Atoms with the outer layer filled with electrons do not form compounds. 三、“with+宾语+形容词或短语”，如： (14) Put the documents in the filing container with all the drawers open.
with用法小结 一、with表拥有某物 Mary married a man with a lot of money . 马莉嫁给了一个有着很多钱的男人。 I often dream of a big house with a nice garden . 我经常梦想有一个带花园的大房子。 The old man lived with a little dog on the lonely island . 这个老人和一条小狗住在荒岛上。 二、with表用某种工具或手段 I cut the apple with a sharp knife . 我用一把锋利的刀削平果。 Tom drew the picture with a pencil . 汤母用铅笔画画。 三、with表人与人之间的协同关系 make friends with sb talk with sb quarrel with sb struggle with sb fight with sb play with sb work with sb cooperate with sb I have been friends with Tom for ten years since we worked with each other, and I have never quarreled with him . 自从我们一起工作以来，我和汤姆已经是十年的朋友了，我们从没有吵过架。 四、with 表原因或理由 John was in bed with high fever . 约翰因发烧卧床。 He jumped up with joy . 他因高兴跳起来。 Father is often excited with wine . 父亲常因白酒变的兴奋。 五、with 表“带来”，或“带有,具有”，在…身上，在…身边之意
精神分裂症的病因是什么 精神分裂症是一种精神方面的疾病，青壮年发生的概率高，一般 在16～40岁间，没有正常器官的疾病出现，为一种功能性精神病。 精神分裂症大部分的患者是由于在日常的生活和工作当中受到的压力 过大，而患者没有一个良好的疏导的方式所导致。患者在出现该情况 不仅影响本人的正常社会生活，且对家庭和社会也造成很严重的影响。 精神分裂症常见的致病因素： 1、环境因素：工作环境比如经济水平低低收入人群、无职业的人群中，精神分裂症的患病率明显高于经济水平高的职业人群的患病率。还有实际的生活环境生活中的不如意不开心也会诱发该病。 2、心理因素：生活工作中的不开心不满意，导致情绪上的失控，心里长期受到压抑没有办法和没有正确的途径去发泄，如恋爱失败， 婚姻破裂，学习、工作中不愉快都会成为本病的原因。 3、遗传因素：家族中长辈或者亲属中曾经有过这样的病人，后代会出现精神分裂症的机会比正常人要高。 4、精神影响：人的心里与社会要各个方面都有着不可缺少的联系，对社会环境不适应，自己无法融入到社会中去，自己与社会环境不相
适应，精神和心情就会受到一定的影响，大脑控制着人的精神世界， 有可能促发精神分裂症。 5、身体方面：细菌感染、出现中毒情况、大脑外伤、肿瘤、身体的代谢及营养不良等均可能导致使精神分裂症，身体受到外界环境的 影响受到一定程度的伤害，心里受到打击，无法承受伤害造成的痛苦，可能会出现精神的问题。 对于精神分裂症一定要配合治疗，接受全面正确的治疗，最好的 疗法就是中医疗法加心理疗法。早发现并及时治疗并且科学合理的治疗，不要相信迷信，要去正规的医院接受合理的治疗，接受正确的治 疗按照医生的要求对症下药，配合医生和家人，给病人创造一个良好 的治疗环境，对于该病的康复和痊愈会起到意想不到的效果。
介词“with”的用法 1、同, 与, 和, 跟 talk with a friend 与朋友谈话 learn farming with an old peasant 跟老农学习种田 fight [quarrel, argue] with sb. 跟某人打架 [争吵, 辩论] [说明表示动作的词, 表示伴随]随着, 和...同时 change with the temperature 随着温度而变化 increase with years 逐年增加 be up with the dawn 黎明即起 W-these words he left the room. 他说完这些话便离开了房间。2 2、表示使用的工具, 手段 defend the motherland with one s life 用生命保卫祖国 dig with a pick 用镐挖掘 cut meat with a knife 用刀割肉3
3、说明名词, 表示事物的附属部分或所具有的性质]具有; 带有; 加上; 包括...在内 tea with sugar 加糖的茶水 a country with a long history 历史悠久的国家4 4、表示一致]在...一边, 与...一致; 拥护, 有利于 vote with sb. 投票赞成某人 with的复合结构作独立主格,表示伴随情况时，既可用分词的独立结构，也可用with的复合结构： with +名词(代词)+现在分词/过去分词/形容词/副词/不定式/介词短语。例如： He stood there, his hand raised. = He stood there, with his hand raise.他举手着站在那儿。 典型例题 The murderer was brought in, with his hands ___ behind his back A. being tied B. having tied C. to be tied D. tied 答案D. with +名词(代词)+分词+介词短语结构。当分词表示伴随状况时，其主语常常用
with[wIT] prep.1.与…(在)一起，带着：Come with me. 跟我一起来吧。/ I went on holiday with my friend. 我跟我朋友一起去度假。/ Do you want to walk home with me? 你愿意和我一道走回家吗 2.(表带有或拥有)有…的，持有，随身带着：I have no money with me. 我没有带钱。/ He is a man with a hot temper. 他是一个脾气暴躁的人。/ We bought a house with a garden. 我们买了一座带花园的房子。/ China is a very large country with a long history. 中国是一个具有历史悠久的大国。3.(表方式、手段或工具)以，用：He caught the ball with his left hand. 他用左手接球。/ She wrote the letter with a pencil. 她用铅笔写那封信。4.(表材料或内容)以，用：Fill the glass with wine. 把杯子装满酒。/ The road is paved with stones. 这条路用石头铺砌。5.(表状态)在…的情况下，…地：He can read French with ease. 他能轻易地读法文。/ I finished my homework though with difficulty. 虽然有困难，我还是做完了功课。6.(表让步)尽管，虽然：With all his money, he is unhappy. 尽管他有钱，他并不快乐。/ With all his efforts, he lost the match. 虽然尽了全力，他还是输了那场比赛。7.(表条件)若是，如果：With your permission, I’ll go. 如蒙你同意我就去。8.(表原因或理由)因为，由于：He is tired with work. 他工作做累了。/ At the news we all jumped with joy. 听到这消息我们都高兴得跳了起来。9.(表时间)当…的时候，在…之后：With that remark, he left. 他说了那话就离开了。/ With daylight I hurried there to see what had happened. 天一亮我就去那儿看发生了什么事。10. (表同时或随同)与…一起，随着：The girl seemed to be growing prettier with each day. 那女孩好像长得一天比一天漂亮。11.(表伴随或附带情况)同时：I slept with the window open. 我开着窗户睡觉。/ Don’t speak with your mouth full. 不要满嘴巴食物说话。12.赞成，同意：I am with you there. 在那点上我同你意见一致。13.由…照看，交…管理，把…放在某处：I left a message for you with your secretary. 我给你留了个信儿交给你的秘书了。/ The keys are with reception. 钥匙放在接待处。14 (表连同或包含)连用，包含：The meal with wine came to ￡8 each. 那顿饭连酒每人8英镑。/ With preparation and marking a teacher works 12 hours a day. 一位老师连备课带批改作业每天工作12小时。15. (表对象或关系)对，关于，就…而言，对…来说：He is pleased with his new house. 他对他的新房子很满意。/ The teacher was very angry with him. 老师对他很生气。/ It’s the same with us students. 我们学生也是这样。16.(表对立或敌对)跟，以…为对手：The dog was fighting with the cat. 狗在同猫打架。/ He’s always arguing with his brother. 他老是跟他弟弟争论。17.(在祈使句中与副词连用)：Away with him! 带他走！/ Off with your clothes! 脱掉衣服！/ Down with your money! 交出钱来！ 【用法】1.表示方式、手段或工具等时(=以，用)，注意不要受汉语意思的影响而用错搭配，如“用英语”习惯上用in English，而不是with English。2.与某些抽象名词连用时，其作用相当于一个副词：with care=carefully 认真地/ with kindness=kindly 亲切地/ with joy=joyfully 高兴地/ with anger=angrily 生气地/ with sorrow=sorrowfully 悲伤地/ with ease=easily 容易地/ with delight=delightedly 高兴地/ with great fluency =very fluently 很流利地3.表示条件时，根据情况可与虚拟语气连用：With more money I would be able to buy it. 要是钱多一点，我就买得起了。/ With better equipment, we could have finished the job even sooner. 要是设备好些，我们完成这项工作还要快些。4.比较with 和as：两者均可表示“随着”，但前者是介词，后者是连词：He will improve as he grows older. 随着年龄的增长，他会进步的。/ People’s ideas change with the change of the times. 时代变了，人们的观念也会变化。5.介词with和to 均可表示“对”，但各自的搭配不同，注意不要受汉语意思的影响而用错，如在kind, polite, rude, good, married等形容词后通常不接介词with而接to。6.复合结构“with+宾语+宾语补足语”是一个很有用的结构，它在句中主要用作状语，表示伴随、原因、时间、条件、方式等；其中的宾语补足语可以是名词、形容词、副词、现在分词、过去分词、不定式、介词短语等：I went out with the windows open. 我外出时没有关窗户。/ He stood before his teacher with his head down. 他低着头站在老师面前。/ He was lying on the bed with all his clothes on. 他和衣躺在床上。/ He died with his daughter yet a schoolgirl. 他去世时，女儿还是个小学生。/ The old man sat there with a basket beside her. 老人坐在那儿，身边放着一个篮子。/ He fell asleep with the lamp burning. 他没熄灯就睡着了。/ He sat there with his eyes closed. 他闭目坐在那儿。/ I can’t go out with all these clothes to wash. 要洗这些衣服，我无法出去了。这类结构也常用于名词后作定语：The boy with nothing on is her son. 没穿衣服的这个男孩子是她儿子。 （摘自《英语常用词多用途词典》金盾出版社） - 1 -