Name: methods for intravenous self administration in a mouse model
Uploaded: 2012-12-08T14:00:00
Duration: 12 min 9 s
Description: Watch this Scientific Journal Video about Methods for Intravenous Self Administration in a Mouse Model at JoVE.com

Forsøg på dyr blev udført i overensstemmelse med de retningslinjer og regler, der er fastsat af den canadiske Råd Animal Care og McGill University Animal Omsorgsudvalget. Denne forskning blev støttet af midler ydet til KG fra den canadiske Institutes of Health Research (CIHR). Ingen interessekonflikter erklæret.

1. Stalde og akklimatisering Procedures <ol><li> Mandlige og kvindelige CD-1 mus er opstaldet med samme køn søskende, op til 5 dyr pr bur, i standard plastbure indeholder beta chip sengetøj og nestlet pads, med trådgitteret låg til at rumme vandflasker og fødevarer. Standard musefoder og vand er tilgængeligt ad libitum i bure i hele eksperimentet. </li><li> Mus holdes på en omvendt lys-mørke-skema (lys på fra 22:00 til 10:00). </li><li> Mus får lov til at akklimatisere til IVSA test rum i en uge forud for starten af ​​eksperimentelle procedurer. </li></ol> 2. Operant træning <ol><li> Alle adfærdsmæssige procedurer, herunder sødet mælk træning og stiknarkomaners selvadministration sessioner udføres ved hjælp operantkamre måler 15,9 x 14 x 12,7 cm, der er udstyret med 2 ultra-følsomme mus løftestænger, dipper kopper, stimulus lys og mikroliter sprøjtepumper (Med Associatesates Inc., St. Albans, VT, USA). Chambers er forbundet med en computer ved hjælp Med Associates Smart CR-interface og Med-PC-software til at programmere tidsplanen for forstærkning og indsamler data. </li><li> Naive mus er vant til de operantkamre og trænet til at løftestang presse under anvendelse af en sødet mælk belønning (0,1 ml), der præsenteres i en dipper kop. Operant mælk træning gennemføres i 1-hr sessioner i 5-7 dage. Afslutning af et fast forhold (FR) tidsplan på den aktive arm resulterede i præsentationen af ​​dipper kop og belysningen af ​​en stimulus lys. Det sødede mælkeopløsning bestod af sucrose (10 mg / ml, ACS Reagent, Sigma-Aldrich Inc. St-Louis MO, USA) tilsat til sødmælk (3,25% fedt). Operant træning med en sødet mælk opløsning eller en flydende føde forstærker er en udbredt teknik til at lette operant reagere på mus 1,2. </li></ol> 3. Fremstilling af udstyr til brug under kirurgi RequIRED udstyr - 20, 23 og 26 gauge nåle, grinder, 1 cc sprøjter, Tygon slanger, loddekolbe, heparin, antibiotika og smertestillende. <ol><li> Udarbejde en nål til at styre indføringen af ​​kateteret i halsvenen ved barbering ned en 20 gauge nål. Akslen af ​​nålen er barberet ned med en slibemaskine til dannelse af en kanal inde i nålen til at lede kateterrøret ind i venen. Nålen kanal skal omhyggeligt kontrolleres for enhver metalaffald, som kan være blevet deponeret. Eventuelle forhindringer til kanalen skal skrabes ved hjælp af fine spids tang. </li><li> Tilpasse to 1-cc sprøjter, der skal benyttes til skylning og kontrol af kateteret. Forbered to sprøjter ved at tage 12 cm stykker af Tygon-røret og fastgøre dem ved den ene ende til 26 gauge nåle fastgjort til 1 cc sprøjter, og strækker den anden ende af røret over 23 gauge nåle. En sprøjte skal fyldes med 0,9% sterilt saltvand og den anden med en hepariniseret ticarcillin opløsning (33 mg ticarcillinog 0,3 mg heparin per 10 ml sterilt saltvand). </li><li> Forbered kateterkanylen caps. Tygon-røret strækkes over en 23 gauge nål, og skåret 1 cm fra facet. Smelt den åbne ende af røret for at skabe en tyk tætning. Slangen skal smeltes, således at hele kanylen hætte er lang nok til at passe over kateterkanylen, med den lukkede ende slutter helt over enden af ​​kanylen. Bemærk, at hvis hætten er for lang, er der risiko for, at den vil blive bøjet og efterfølgende gennemboret under processen med fastgørelse af skrue på kateteret dækker. Således omhyggelige forberedelse af katetret hætter anbefales, for at tilvejebringe en god pasform til kanylen. </li><li> Forbered den nødvendige løsninger. Alle reagenser købt hos Sigma-Aldrich Inc. (St-Louis MO, USA). <ol class="lalpha"><li> Hepariniseret ticarcillin opløsning kateter skylning - opløses 0,33 g ticarcillin (dinatriumsalt) og 0,003 g heparin i 10 ml sterilt saltvand. 0,03 ml af opløsningen skylles thrgående kateteret dagligt. Den antibiotiske opløsning administreres til at forebygge blodpropper og infektioner i at udvikle sig. </li><li> Amikicin (antibiotika) til subkutan injektion - En enkelt subkutan injektion gives efter kirurgi i en dosis på 10 mg / kg for at forhindre postoperative infektioner. </li><li> Ketoprofen (analgetikum) til subkutan injektion - En enkelt subkutan injektion gives efter kirurgi i en dosis på 5 mg / kg til at håndtere enhver postoperativ smerte. </li></ol></li></ol> 4. Kateter implantering Nødvendigt udstyr og reagenser: Isofluran, sterilt saltvand, alkohol (70%), 1 og 3 cc sprøjter fyldt med sterilt saltvand, 1-cc tilpassede sprøjter, antibiotika og smertestillende opløsninger fremstillet i afsnit 3 ovenfor, mus katetre (CamCaths, Cambridgeshire, UK ), kateter covers (krystal hætter fra HRS Scientific, Montreal, Quebec), øje smøremiddel, 4 cm plast bar at ophøje blodåre (dettekan være fremstillet af en plast vatpind), polysporin, sterile vatpinde og gaze, buede og lige pincet, arterie klemmer, fine sakse. <img src="/files/ftp_upload/3739/3739step1.jpg" alt="Trin 1" fo:content-width="3in" fo:src="/files/ftp_upload/3739/3739step1highres.jpg"/><ol><li> Hjælp af aseptiske standardteknikker den kirurgiske bænk, de kirurgiske instrumenter, og katetrene er steriliseret forud for operationen. Korrekte steriliseringsteknikker indbefatter damp autoklavering i de kirurgiske instrumenter og katetere købt fra CamCaths. Glasperle sterilisering kan også anvendes på spidsen af ​​de kirurgiske instrumenter. Ethylenoxidsterilisering kan anvendes på mere sarte katetre eller materialer, som risiko smeltning. En mere detaljeret beskrivelse af gnaver kirurgiske aseptiske teknikker kan findes i vedlagte referencer 1,2. Opsætning af bænken, er instrumenter og næse kegle til vedligeholdelse af anæstesi illustreret i billedet. </li><li> Mus er bedøvet med isofluran gas og holdt under anæstesi ved anvendelse af en vejrtrækning rør under et udsugningssystem. Eye smøremiddel (Tears Naturale PM) påføres på begge øjne for at forhindre dem i at tørre ud under proceduren. Et passende kirurgiske margin er barberet på dyrets ryg og omkring halsen. Før nogen indsnit er lavet, er de kirurgiske områder begrænses vha. sterile forhæng. </li><li> For at fremstille katetret til indføring i det højre atrium af hjertet, det overskydende kateterrøret afskåret 1,2 cm fra pæren af ​​katetret. Dette er den optimale længde fastsat for voksne CD-1-mus, 8 uger gamle, 20-25 g legemsvægt. Længden af ​​kateterrør kan være nødvendigt at justere en smule (ved forsøg), ud fra stammen, størrelse og alder af musene. Før indsættelse, er sprøjte indeholdende steril saltopløsning (afsnit 3.2) fastgjort til kateterkanylen, og kateteret skylles og kontrolleres for lækager. Holddenne sprøjte er fastgjort til kateteret under hele den kirurgiske procedure. Det vil blive brugt til at skylle kateteret, og trække tilbage blod i trin 4,7. </li><li> Efter sterilisation i 70% alkohol, en 2 cm lang midscapular snit begynder midt på ryggen og slutter lige under halsen for at rumme bunden af ​​kateteret. Bindevæv skal tvinges fra hinanden med en pincet for at gøre plads for kateteret bund under huden. </li><li> At anbringe dyret på ryggen, en anden flad 1-2 cm diagonal incision i højre nøgleben går opad til dyr kæbe, efter at området er blevet penslet med 70% alkohol. Halsvenen vil blive fundet overfladisk under huden af ​​halsen. Som forberedelse til indføring af kateteret, er røret fra bunden af ​​kateteret trækkes gennem indsnit på ryggen og bringes tæt på halsvenen ved at lede slangen under huden lidt over den højre skulder. Enden af ​​kateterrøret erderefter fastgjort til en arterie klemme og placeret i dyrets side for at holde den på plads. </li><li> Den højre halsvene er placeret ved forsigtigt at bevæge sig væk overfladisk binde-og fedtvæv fra snit omkring dyrets hals. Bindevævet omkring venen er brudt fra hinanden ved hjælp af buede pincet og venen derpå hævet under anvendelse af en steril plastik bar. Løse åbne sutur knob er lavet rundt om hver ende af venen og kateterrøret er ført gennem den øverste knude og sløjfes over suturtråd at hvile løsnet over den højre skulder. </li><li> Før indføring, våde både 20 gauge indføringsnålen og vene med sterilt saltvand for at reducere friktion. Nålen holdes parallelt med venen, og indsættes forsigtigt nær bunden af den forhøjede vene (Bemærk: cirka 0,5 cm af nålespidsen skal indtaste den vene). Ved hjælp af en pincet, kateterrøret glide ned skaftet af kanylen i venen. Resistens tyder på, at røret is inden for bindevæv og ikke i venen. Skub 0,03 cc af saltvand gennem venen at sikre, at der ikke er utætheder. Utætheder ville indikere, at venen kan gennemhulles eller at kateterrøret placering skal justeres. Bemærk: For at kontrollere, om slangen er i venen, forsøge at udarbejde nogle blod på vedlagte saltvand sprøjte. Hvis blodet ikke umiddelbart kan opstilles, kan venen eller hjerte væg være okkludere kateterspidsen, eller venen er ikke blevet gennemboret, justere røret og prøve igen. Needle reinsertion kan være nødvendig, hvis blod stadig ikke kan udarbejdes. </li><li> For at sikre kateteret på plads, skubbes kateteret pæren til indsætningspunktet og fjern nålen. Bind den nederste knude og derefter trække kateteret flugter baren før binde den anden knude lige over pæren. Test igen for at se, om blodet kan trækkes op og løsne knuderne lidt, hvis nødvendigt. Tuck kateterrøret under huden og suturventrale snit omkring dyrets hals. Anvende Polysporin helbrede hurtigt med en steril vatpind eller en anden antibiotisk salve fortrinsvis indeholdende noget analgetikum til den lukkede incision. </li><li> Med dyret på dets underliv, placere kateteret base under huden på ryggen i den forberedte indsnit. Sørg for, at overskydende slange minimalt er ført og godt skjult under kateteret basis for at minimere risikoen for at dyret tygger og piercing det. Sy snit på begge sider af kateteret base, og anvende Polysporin helbrede hurtigt under anvendelse af en steril vatpind. </li><li> Skyl kateteret med 0,03 cc af den hepariniserede ticarcillin løsning ved hjælp af sprøjten med slangen fastgjort til det (afsnit 3,2). Cap kanylen med plastikkanylen og skru på den hvide kateter dæksel. I nogle mus, som er frisk fra kirurgi kan blod løbe ud af kateteret før kanylen er udjævnet. Det er vigtigt at re-skylle dyret og rapidly udskifte kanylen hætte før blod har mulighed for at strømme ud. Dyrene skal skylles på daglig basis for at opretholde kateter åbenhed. </li><li> Efter aftørring ned injektionen området mellem dyrene bagben med 70% ethanol, injiceres subkutant den analgetiske ketoprofen i en dosis på 5 mg / kg på den ene side, og det antibiotiske amikicin i en dosis på 10 mg / kg på den anden side. </li><li> Efter anæstesi afbrydes, har dyrene lov at komme sig i et rent bur med let adgang til foder og vand i 5 til 7 dage. Mus anbringes i et opvarmet skab natten over for at forhindre post-operative hypotermi. </li></ol><img src="/files/ftp_upload/3739/3739step2.jpg" alt="Trin 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/3739/3739step2highres.jpg"/> 5. Behavioural test - Intravenøs selvadministration <ol><li> Forud for adfærdsmæssige forsøg katetre skyllet med 0,9% sterilt saltvand. Mus derefter placered i operantkamre og forbundet med infusionen linjer og infusionspumper. Aktive håndtag presser resultere i en 3,2 sek 18 pi lægemiddelinfusion kombineret med belysningen af ​​en stimulus lys. Hver løftestang presse er efterfulgt af en 8 sek timeout periode, hvor stimulus lyser. </li><li> Efter den operant session, er mus katetre skylles med den hepariniserede ticarcillin løsning, før det returneres til deres hjem bur. </li><li> Mus får lov til selv at administrere i 3 på hinanden følgende 2-timers sessioner på hver dosis. Doser blev præsenteret i tilfældig rækkefølge for hver mus, som vist i det følgende afsnit. </li><li> Kateter åbenhed vurderes dagligt ved at sikre, at både saltvand og antibiotikaopløsning kan skylles gennem katetret. Desuden kan en ketamin / midazolam test udføres som beskrevet i vedlagte reference 5. Kort fortalt, tegn på anæstesi såsom immobilitet inden for 5 sek af en infusion af fra 0,02 til 0,03 ml ketamin (15 mg / ml)eller midazolam (0,75 mg / ml) midazolam er bevis på et patent kateter 5. </li></ol>

<ol>
	<li>Rocha, B. A., Ator, R., Emmett-Oglesby, M. W., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intravenous Cocaine Self-Administration in Mice Lacking 5-HT1B Receptors. Pharmacology Biochemistry and Behavior. 57 (3), 407-412 (1997).</li><li>Zapata, A., Chefer, V. I., Ator, R., Shippenberg, T. S., Rocha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+sensitization+and+enhanced+dopamine+response+in+the+nucleus+accumbens+after+intravenous+cocaine+self-administration+in+mice.">Behavioural sensitization and enhanced dopamine response in the nucleus accumbens after intravenous cocaine self-administration in mice.</a> European Journal of Neuroscience. 17 (3), 590-596 (2003).</li><li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Rodent+Surgery+for+the+New+Surgeon.">Principles of Rodent Surgery for the New Surgeon.</a> J. Vis. Exp. (47), e2586 (2011).</li><li>Hoogstraten-Miller, S. L., Brown, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in Aseptic Rodent Surgery Current Protocols in Immunology. , 1.12-1.14 (2001).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Intravenous+Drug+Self-Administration+in+Rats+and+Mice.">Chronic Intravenous Drug Self-Administration in Rats and Mice.</a> Current Protocols in Neuroscience. . 9, 20.1-20.40 (2005).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+quantitative+trait+loci+for+cocaine+induced+activation+in+the+AcB/BcA+series+of+recombinant+congenic+strains.">Confirmation of quantitative trait loci for cocaine induced activation in the AcB/BcA series of recombinant congenic strains.</a> Pharmacogenetics. 13, 329-338 (2003).</li><li>Gill, K. J., Boyle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+drug-induced+psychomotor+activation+in+mice.">Genetic influences on drug-induced psychomotor activation in mice.</a> Genes, Brain &#38; Behavior. 7, 859-868 (2008).</li><li>Boyle, A. E., Gill, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+AXB/BXA+recombinant+inbred+lines+of+mice+to+the+locomotor+activating+effects+of+cocaine:+A+QTL+analysis.">Sensitivity of AXB/BXA recombinant inbred lines of mice to the locomotor activating effects of cocaine: A QTL analysis.</a> Pharmacogenetics. 11 (3), 255-264 (2001).</li><li>Boyle, A. E., Gill, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+verification+of+previously+identified+QTLs+for+cocaine-induced+activation+using+a+panel+of+B6.A+chromosome+substitution+strains+(CSS)+and+A/JxC57Bl/6J+F2+mice.">A verification of previously identified QTLs for cocaine-induced activation using a panel of B6.A chromosome substitution strains (CSS) and A/JxC57Bl/6J F2 mice.</a> Psychopharmacology. 207 (2), 325-3234 (2009).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychomotor+stimulant+effects+of+cocaine+in+rats+and+15+mouse+strains.">Psychomotor stimulant effects of cocaine in rats and 15 mouse strains.</a> Experimental and Clinical Psychopharmacology. 19 (5), 321-341 (2011).</li><li>Thomsen, M., Caine, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+drug+self-administration+in+mice:+practical+considerations.">Intravenous drug self-administration in mice: practical considerations.</a> Behavior genetics. 37 (1), 101-118 (2007).</li></ol>

Ingen interessekonflikter erklæret.

Dyremodeller af stofmisbrug er særligt nyttige til forståelse af genetiske grundlag for narkotikarelaterede adfærdsmønstre. For eksempel viser mus med forskellige genetiske profiler arvelige forskelle i deres følsomhed over for kokain 6-8 og hjælpe med at identificere potentielle gen kandidater mediere den fænotypiske variation 9. De intravenøse kateterisation, der er beskrevet i dette dokument er blevet anvendt med stor succes til at undersøge lægemiddel IVSA i forskellige stammer af mus og mus af forskellige genetiske baggrunde 10. De procedurer, der er vist i denne video fremhæve vigtige faktorer til at fokusere på under og efter kateterisering kirurgi med henblik på at få pålidelige intravenøse selvadministration data. For det første anbringelse af kateterrøret i højre atrium er af afgørende betydning, for at forhindre katetret svigt på blot-blodpropper. Under operation er det vigtigt at sikre, at kateteretende er uhindret, og ikke okkluderet af enten hjertet eller venen væv. For det andet er daglig kateter rødmen kræves både før og efter operant møder for at undgå tilstopninger. Endelig skal kateterkanylen være dækket konstant med både kanylen hætter og krystal dækker, når dyrene er i deres bure for at forhindre indtrængen af ​​snavs. Mindre blokering af kateterkanylen kan fjernes med en fin 26 gauge nål, dog daglig skylning med den hepariniserede antibiotika løsning er påkrævet, især på dage, hvor dyrene er ikke testet for IVSA. For at udføre dyr overlevelse kirurgi, et godt kendskab til aseptiske teknikker, analgesi og anæstesi er nødvendig. Mens denne video ikke erstatter egentlig kirurgisk uddannelse, kan den bruges som en rettesnor for forskere, der ønsker at tilegne sig de teknikker, der er nødvendige for dette paradigme.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Ticarcillin dinatriumsalt </td><td> Sigma-Aldrich </td><td> T5639-1G </td><td> 1 gram flaske </td></tr><tr><td> Tears Naturale PM (Eye smøremiddel) </td><td> Alcon </td><td> N / A </td><td> 3,5 gram rør Dette øje smøremiddel kan findes i de fleste apoteker. Enhver ophthalmisk eye smøremiddel kan anvendes. </td></tr><tr><td> Kokain </td><td> Medisca Pharmaceutique </td><td> 0723-06 </td><td> 1 gram flaske </td></tr><tr><td> Ketamin </td><td> Medisca Pharmaceutique </td><td> 1754-04 </td><td> 25 gram flaske </td></tr><tr><td> Midazolam </td><td> Medisca Pharmaceutique </td><td> 2519-06 </td><td> 1 gram flaske </td></tr><tr><td> Heparin natriumsalt </td><td> Sigma-Aldrich </td><td> H4784-250 mg </td><td> 250 gram flaske </td></tr><tr><td> Ketoprofen </td><td> Sigma-Aldrich </td><td> K1751-1G </td><td> 1 gram flaske </td></tr><tr><td> Amikicin sulfatsalt </td><td> Sigma-Aldrich </td><td> A2324-5G </td><td> 5 gram flaske </td></tr><tr><td> Saccharose ACS Reagent </td><td> Sigma-Aldrich </td><td> S5016-500G </td><td> 500 gram flaske </td></tr><tr><td> Standard mus katetre med blødt mesh </td><td> CamCaths </td><td> MIVSA/20/26/BC-1S </td><td> Tubing specifikationer: 26SWG; 0,451; 0,254 </td></tr><tr><td> Crystal cap applikator </td><td> HRS Videnskabelige </td><td> 313CAC </td><td> Dette er den hvide kateter hætte. </td></tr><tr><td> Tygon Micro Bore Tubing </td><td> Thomas Scientific </td><td> 9561S41 </td><td> Tubing specifikationer (boring x OD x vægtykkelse i inches): 0,01 x 0,03 x 0,01 </td></tr>
 </tbody>
</table>

methods for intravenous self administration in a mouse model

Dyremodeller er blevet udviklet for at studere de selvforstærkende virkninger af narkotika, herunder den intravenøse self-administration (IVSA) paradigme. Fordelene ved at anvende en IVSA paradigme for at studere de forstærkende egenskaber af misbrugsstoffer, såsom kokain nævnes, at lægemidlet er selvadministreres i stedet for eksperimentator-administreres, kan tidsplanen for forstærkning ændres, og nøjagtige målinger af mængden af lægemiddel indtages samt timingen og mønster af IV injektioner kan opnås. Endvidere den intravenøse administration undgår potentielle confounds relateret til first-pass metabolisme eller smag, og producerer hurtige stigninger i blod og hjerne lægemiddelniveauer. Som skitseret i denne video, kan intravenøs selvadministration opnås uden forudgående mad restriktioner eller forudgående lægemiddel træning efter en omhyggelig kateteranbringelse under kirurgi og omhyggelig daglig kateter rødmen og vedligeholdelse. Eksperimentelle procedurer er beskrevet i dennepapir omfatter en beskrivelse af stalde og akklimatisering metoder, operant træning med sødet mælk løsninger, og kateter implantering.

<img src="/files/ftp_upload/3739/3739fig1.jpg" alt="Figur 1" /> Figur 1. Mønstret for at reagere for intravenøs medicin selvadministration vil variere efter narkotika, dosisinterval ansat og musestamme. Den fremlagte Figuren viser kokain selvadministration data på grundlag af vellykkede kateterisering operationer ved hjælp af proceduren beskrevet i videoen. Figuren viser de gennemsnitlige (± SEM) kokain infusioner tjent og gennemsnitlig (± SEM) kokainforbrug (mg / kg legemsvægt) over et område på 4 kokain doser præsenteres i en vilkårlig rækkefølge på en FR1 tidsplan for forstærkning. Abscisse: dosis af selvadministrerede lægemiddel pr infusion. Venstre ordinat: samlet antal infusioner optjent i løbet af 2-hr operant session. Højre ordinat: total kokain indtagelse i mg / kg i løbet af 2-hr test session. Alle 13 katetre forblev patent for hele undersøgelsen (fire uger). En envejs ANOVA udført på dosis afslørede, at mice blev administrering kokain i en dosisafhængig måde [F (1,12) = 42,8, p &lt;0,05]. Der er en stigning i kokainforbrug over dosisresponskurven [F (3,36) = 29,6, p &lt;0,05] på trods af et fald i arm presning ved de højere doser. Hvert datapunkt repræsenterer gennemsnittet af 3 forsøg sessioner på hver kokain dosis (± SEM) opsamlet i CD-1-mus (n = 13 / dosis, mænd og kvinder kombineret). Sammenligninger af aktiv (lægemiddel-forstærket) vs inaktiv arm reagerer over dosisresponskurven blev fremstillet under anvendelse af tovejs-ANOVA for at sikre, at musene skelner mellem de to arme. For de CD1-mus, viser analysen præference for den aktive løftestang [F (1,12) = 10,255, p &lt;0,05], over hele dosis-responskurve (data ikke vist her).

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Methods for Intravenous Self Administration in a Mouse Model

Den intravenøse selvadministration (IVSA) paradigme anses for at være den gyldne standard i behandlingen de forstærkende egenskaber af misbrugsstoffer hos gnavere. Dette håndskrift beskriver de eksperimentelle procedurer og kirurgiske teknikker er nødvendige for at få pålidelige IVSA data. Især er omhyggelig kateter implantation og vedligeholdelse fremhævet.

Fremgangsmåder til intravenøs selvindgivelse i en musemodel

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

methods-for-intravenous-self-administration-in-a-mouse-model

Research

JoVE Journal

Neuroscience

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Mikrodialyse af ethanol under operant ethanol selvforvaltning og ethanol bestemmelse ved gaschromatografi

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Metoder til undersøgelse af virkningsmekanismer for antipsykotika i<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

Hornhindens Micropocket assay: En model af angiogenese i mus Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunhistokemiske og Calcium Imaging Methods in whole- mount rotteretina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Metoder til Cell-attached kapacitansmålinger i Mus Adrenal chromaffinceller Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Neurobehavioral vurdering i en musemodel af Neonatal hypoksiske iskæmisk hjerneskade

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Metoder og Tips til intravenøs indgift af Adeno-associeret Virus at rotter og evaluering af centralnervesystemet transduktion

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Vurdering af blod - hjerne barrieren permeabilitet af intravenøs Infusion af FITC-mærket Albumin i en musemodel af neurodegenerativ sygdom

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...