This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal’s head.
Det er allment anerkjent at bruk av generell anestesi kan undergrave relevansen av elektrofysiologiske eller mikroskopiske data innhentet fra et levende dyr hjerne. Videre er den omstendelige gjenvinning fra anestesi begrenser frekvensen av gjentatte opptak / avbildnings episoder i langsgående studier. Derfor er nye metoder som ville tillate stabile opptak fra ikke-bedøvede oppfører mus ventes å avansere innen cellulære og kognitive nevrovitenskap. Eksisterende løsninger spenner fra ren fysisk tvang til mer avanserte metoder, som lineære og sfæriske tredemøller brukes i kombinasjon med datagenerert virtuell virkelighet. Her er en ny metode som er beskrevet der en head-fast mus kan flytte rundt en luft-løftet mobile homecage og utforske omgivelsene under stress-frie forhold. Denne metoden gjør det mulig for forskere å utføre atferdstester (f.eks, læring, tilvenning eller roman objekt gjenkjenning) samtidig medto-foton mikroskopisk avbildning og / eller patch-clamp opptak, alle kombinert i et enkelt eksperiment. Dette video-artikkelen beskriver bruken av våkne dyr hode fikseringsanordning (mobil homecage), demonstrerer fremgangsmåten av animalsk tilvenning, og eksemplifiserer en rekke mulige anvendelser av fremgangsmåten.
En spennende ny trend i Neurosciences er å utvikle eksperimentelle tilnærminger for molekylær-og celle sondering av nevrale nettverk i hjernen av våken, oppfører gnagere. Slike tilnærminger holder lover å kaste nytt lys over nevrofysiologiske prosesser som ligger til grunn motorisk funksjon, sensorisk integrasjon, persepsjon, læring, hukommelse, samt skade progresjon, nevrodegenerasjon og genetiske sykdommer. Videre opptak fra våken dyrets hjerne holder løftet i utvikling av nye terapeutiske midler og behandlinger.
Det er en økende bevissthet om at anestesi, noe som har vært vanlig i nevrofysiologiske eksperimenter, kan påvirke de grunnleggende mekanismene for hjernens funksjon, som potensielt kan føre til feilaktige tolkning av eksperimentelle funn. Dermed blir mye brukt bedøvelse ketamin øker hurtig dannelse av nye dendrittutløperne og forbedrer synaptiske funksjon 1; En annen mye brukt anesthetic isofluran ved kirurgisk anestesi nivåer helt undertrykker spontan kortikal aktivitet i nyfødte rotter og blokkerer spindel-burst svingninger i voksne dyr 2. I dag er bare et begrenset antall tilnærminger aktiver eksperimenter i ikke-bedøvet mus ved hjelp av to-foton mikroskopisk bildebehandling eller patch-clamp opptak. Disse metodene kan deles inn i fritt bevegelige og hode-faste preparater.
Den unike attraktivitet av et fritt bevegelige dyr preparatet er at det tillater vurdering av naturlig atferd, inkludert hele kroppsbevegelser under navigasjon. En måte å bilde inne i hjernen til en fritt glidende gnager er å feste en miniatyrisert hodemontert mikroskop eller fiberskop 3-5. Men miniatyriserte enheter har en tendens til å ha begrenset optisk ytelse i forhold til objektiv-basert to-foton mikroskopi, og kan ikke være lett kombineres med hele cellen patch-clamp opptak seks.
Den exibrodd løsninger for hode-feste en våken gnager har stolt primært enten på fysisk tvang 7,8 eller på trening dyret til å stille frivillig hodestøttene ni. En annen populær metode er å la dyrets lemmer å flytte ved å plassere den på, for eksempel, en sfærisk tredemølle 10; denne tilnærmingen er ofte kombinert med datagenerert virtuell virkelighet. Elektrofysiologiske eksperimenter på hode-fast mus har for det meste brukt ekstracellulære innspillinger og ble brukt til å studere sentrale regulering av kardiovaskulær funksjon 11, effektene av anestesi på nerveaktiviten 12, hørsels respons i hjernestammen 13 og informasjonsbehandling 14. Den banebrytende intracellulære / hel-celle opptak i våken oppfører dyr ble utført på 2000-tallet, og har fokusert på nevral aktivitet knyttet til persepsjon og bevegelse 15-20. Rundt samme tid ble de første mikroskopiske imaging studier på våken mus pubblert, hvor to-fotonmikroskopi ble anvendt i det sensoriske cortex i rotter fysisk i setet 7, og på mus som kjører på en sfærisk tredemølle 21..
Påfølgende in vivo mikroskopi og elektrofysiologiske studier viste at et hode fiksering forberedelse kan med hell kombineres med atferds paradigmer basert på forbena bevegelser, lukt anerkjennelse, visping, og slikker 8,22-25. Mus plassert på sfærisk tredemølle kan trenes til å navigere i virtuelle visuelle miljøet generert av en datamaskin 10,26. Intracellulære / ekstracellulære opptakene viste at i en head-fast dyr navigerer slikt virtuelt miljø, kan aktivering av hippocampus sted celler oppdages 27. I en virtuell visuelle miljøet, mus viser normal bevegelse relaterte theta rytme i det lokale feltet potensial og theta-fase presesjon under aktiv bevegelse 27. Nylig, den romlige og tidsmessige activity mønstre av nevronale populasjoner ble registrert optisk mus i løpet av arbeidsminnet beslutning oppgaver i et virtuelt miljø 28.
Til tross for å ha slått banebrytende forskning, har den sfæriske tredemølle utformingen flere iboende begrensninger. Først er det dyret som kreves for å flytte på et ubegrenset overflaten av en roterende luft løftet ballen, noe som utgjør ingen konkrete hindringer som vegger eller hindringer. Denne begrensningen er bare delvis kompensert av datagenererte "virtuell virkelighet", for visuell inngang er kanskje mindre effektive på mus og rotter i forhold til det taktile sanseinntrykk (som whisker-trykks-eller slikke), hvor disse arter naturligvis avhengige på. For det andre kan en betydelig krumning av kulens overflate være ubehagelig for laboratoriemus som brukes til å vandre på et plant gulv i burene. Til slutt gjengir selve diameteren på ballen (minst 200 mm for mus og 300 mm for rotter) den vertikale størrelsen på sfærisktredemølle enhet relativt stor. Dette gjør det vanskelig å kombinere sfærisk tredemølle med de fleste kommersielt tilgjengelige mikros oppsett, og krever ofte å bygge et nytt oppsett rundt tredemølle ved hjelp av skreddersydde mikroskop rammer.
Her er en ny metode som er beskrevet der en head-fast mus kan flytte rundt en luft-løftet mobile homecage som har flatt gulv og konkrete vegger, og utforske det fysiske miljøet under stress-frie forhold. Denne artikkelen viser prosedyrene for musetrening og hodet fiksering, og gir representative eksempler der to-foton mikroskopi, iboende optisk bildebehandling og patch-clamp opptak er utført i hjernen av våken oppfører mus.
To better understand brain physiology and pathology, research must be performed on a variety of preparation complexity levels, utilizing the most appropriate techniques for each preparation. At present, a wide range of neuroscience methodologies (from full-body fMRI to sub-organelle STED microscopy) are readily applied to anaesthetized animals, while experiments on awake and behaving animals have represented a significant methodological challenge.
Here, a novel approach is described where a laboratory animal, despite being firmly head-fixed, can move around an air-lifted mobile homecage and explore its tangible environment under stress-free conditions. The head-fixed behaving animal preparation presented here provides a number of crucial advantages. First, electrophysiological or imaging data obtained with this method are uncompromised neither by anesthesia nor by constrain-induced stress. Positioning of the mouse into the mobile homecage is quick and does not require anesthetizing the animal even transiently. Second, the air-lifted homecage ensures the mechanical stability that is needed to quantify changes in fine neuronal morphology and to record single-cell electrophysiological activity in awake animals. Finally, the mobile homecage’s design is more compact in comparison to the spherical treadmill, thus allowing positioning the mobile homecage under a standard upright microscope for two-photon imaging or patch-clamp recording in awake mouse’s brain.
Firm head fixation in the mobile homecage requires implantation of a specially designed four-winged metal holder, with a round opening in the center for optical or electrical access to the underlying brain region. These metal holders are attached to the skull by means of a combination of glue, dental cement and a small bolt screwed into the skull bone. This surgical procedure was developed based on a large number of previously published procedures, and was found to result in a stable and reproducible cranial window preparation. For in vivo electrophysiological experiments, a moon-shaped window34, a small size craniotomy (less than 0.5 mm)32, and a drilled glass-covered preparation35 have been utilized. Here, the “inverted” cranial window was implanted with either a large (3.5 mm diameter) or small (less than 0.5 mm diameter) craniotomy. Minimizing brain movement is critical for stable single cell recordings, which is why it is advisable to perform small size craniotomies for electrophysiological experiments. Upon implantation of the cranial window for optical imaging experiments, the animals are allowed to recover for at least 2 or 3 weeks, during which period the window first transiently loses its transparency and then regains it (with a 50-70% yield, depending on the genetic background of the mouse strain). Transparency of the cranial window and stability of the dental cement “cap” attached to the skull can be verified by means of a regular binocular microscope and physical inspection during animal handling. At the end of the 2-3 week recovery period, those animals that exhibit any signs of residual post-operational inflammation or mechanical defects in the dental cement should be excluded from the experiments and terminated.
The optimal age for starting training the mice is 2-4 months (corresponding to the body weight of 20-40 g). In younger animals, anchoring of the dental cement “cap” to the skull can be unreliable, which may decrease its resilience to the mechanical stress that is imposed by locomotion of the head-fixed mouse in the mobile homecage. Although male and female mice appear equally willing to navigate in mobile homecage, there is a tendency to achieve better percentage of cranial windows regaining their transparency in female mice (data not shown). Hence, in order to ensure a balanced mix of genders in the cohort of animals selected for imaging, implanting cranial windows in approximately 30% more male mice is recommended. Social interactions are known to improve the animals’ well-being and reduce stress, therefore it is advisable that littermates are operated and trained in parallel and kept together in group-housing cages.
In contrast to the procedures published for the spherical treadmill preparation13, the method utilizing the mobile homecage does not require anesthetizing the mouse at the moment of head fixation. This difference is important because it allows to rule out any residual effects that even a brief and “light” anesthesia episode is likely to have on the physiological measurements obtained shortly after. Indeed, even though in the studies where head fixation was done under anesthesia and the actual experiments were started after a brief waiting period13, one cannot exclude possible long-lasting effects of the brief anesthesia episode on the experimental data. Other studies have relied on water deprivation for systematic habituation of the animals to head fixation and used water reward as the means of motivating the animal to remain immobile36. However, the reward-based head fixation method limits the choice of applicable behavioral tests and, importantly, occupies one of the well-established stimulus–reward associations. In contrast, the method of mouse habituation to head fixation in mobile homecage does not require water deprivation and subsequent reward.
Supplementing the mobile homecage with a water delivery system is recommended for long-lasting experiments. The animal training sessions and experiments presented here were done during daytime (between 8 a.m. and 6 p.m.), which corresponds to the physiologically passive period for those mice that are kept under the standard 12-hr light schedule (lights on at 6 a.m. and off at 6 p.m.). Since the water intake is directly associated with the mouse’s activity, during the passive period mice do not require water delivery if the duration of a training/imaging/recording session does not exceed 2 hr. In addition to the timing and duration of the training sessions, one needs to address the issue of the optimal number of sessions required for habituating the animals to mobile homecage. To this end, two criteria were used to evaluate stress induced by head fixation procedures: i) weight loss, and ii) locomotor activity level. As shown in Figure 6, weight loss reaches the average level of 6% on training day 2, and is completely reversed by training day 4 (Figure 6A). Consistently with the weigh dynamics, the locomotor activity level of head-fixed animals is suppressed on the first day of training but stabilizes by training day 4 (Figure 6D). Based on these measurements, we suggest that the minimal duration of the mouse training period on mobile homecage is 4 days, as described in the protocol hereby.
Use of the air-lifted, flat-floored mobile homecage allows adding complex tasks (sensorimotor, perceptional, and cognitive) to the training paradigms for head-fixed mice. In the present study two protocols of behavioral tests are presented. Both protocols utilize odor cues and can be combined with longitudinal imaging/recordings in the mouse cortex. Although the mobile homecage is manufactured from nonabsorbent materials, one still needs to take into account possible interferences between the smell of the device and test odor(s). Another factor that may interfere with visual/tactile cues of a behavioral experiment is the junction between the wall and the insert, which is not seamless and may, therefore, be perceived by the animal as a landmark. It is worth noticing here that, in order to minimize animal’s distress during such interventions as placement of an odor-presenting cotton to the mobile homecage wall, the experimentalist should practice to perform such interventions as quickly as possible and avoid prolonged handling of the carbon cage. Alternative strategies for novel smell/object presentation are conceivable, e.g., placing hydrogel-based solution drops or objects (such as food chips) onto small shelves attached to the inner surface of the carbon cage wall at the height compatible with the animal’s head positioning.
Mobile homecage allows head-fixed animals to perform a wide range of two-dimensional movements including horizontal locomotion, situp, grooming, whisking, licking, nose-poking, skilled front paw movements, and wall touching with forelimbs, as illustrated in the present study. Using mobile homecage and the protocols presented here, researchers can study the sensorimotor neuronal system with a high level of control over both the stimulation conditions and the behavioral read-outs. Furthermore, studies of cognitive abilities in awake mice can be performed during conditioning, spatial navigation and decision-making tasks.
There are several practical limitations of this method. First, a significant amount of pressurized air is needed to achieve the homecage-lifting power and to perform long-lasting experiments. Second, the mobile homecage in its present implementation is only 18 cm in diameter, and therefore provides a relatively small and simple space in comparison to virtual reality, where a complex experimental environment can be designed without any spatial restrictions. Third, during whisker stimulation and reward-based experiments presented here, a device was used that limits the possibility the wall-contact for the mouse. Addition of an external visual or sensory stimulation channel (such as an eye-directed light projector) would require designing a more ergonomic and compact device in comparison to the multiple-screen or dome-projection solutions that have been used in the spherical treadmill experiments.
In summary, the use of the head-fixed mice moving in the air-lifted mobile homecage greatly facilitates the studies that combine cellular, molecular and behavioral levels of observation and manipulation within a single experiment. Specific applications illustrated here include two-photon microscopic imaging, intrinsic optical signal imaging and patch-clamp recordings in non-anesthetized behaving mice. It is expected that this approach will open new horizons in experimentation on awake, behaving mouse and serve as a useful tool for both drug development and basic research of brain function.
The authors have nothing to disclose.
The authors thank Prof. Eero Castren for his valuable comments on the manuscript. The work is supported by grants from The Academy of Finland, Centre for International Mobility of Finland, and Finnish Graduate School of Neuroscience (Brain and Mind Doctoral Program).
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Tweezers Stainless Steel, 115mm | XYtronic | XY-2A-SA | |
Animal trimmer, shaving machine | Aesculap | Isis GT420 | |
Binocular Microscope | Zeiss | Stemi 2000 | |
Biological Temperature Controller with stainless steel heating pad | Supertech | TMP-5b | |
Blunt microsurgical blade | BD | REF 374769 | |
Borosilicate tube with filament | Sutter Instruments | BF120-69-10 | For patch pipette production |
Camera | Foscam | FI8903W | Night visibility |
Carprofen | Pfizer | Rimadyl vet | |
Dental cement | DrguDent, Dentsply | REF 640 200 271 | |
Dexamethasone | FaunaPharma | Rapidexon vet | |
Disposable drills | Meisinger | HP 310104001001008 | |
Dulbeco’s PBS 10X | Sigma | D1408 | |
Dumont #5 forceps, 110 mm | FST | 91150-20 | |
Eyes-lubricant | Novartis | Viscotears | For eyes protection during operation and as viscose solution for immersion |
Foredom drill control | Foredom | FM3545 | |
Foredom micro motor handpiece | Foredom | MH-145 | |
Four-winged metal holder | Neurotar | ||
Head Holder for Mice | Narishige | SG-4N | Assembled on stereotaxic instrument |
Hemostasis Collagen Sponge | Avitene, Ultrafoam BARD | Ref 1050050 | |
Imaris | Bitplane | ||
Ketamine | Intervet | Ketaminol vet | |
Kwik-Sil | WPI | ||
Mai Tai DeepSee laser | Spectra-Physics | ||
Micro dressing forceps, 105 mm | Aesculap | BD302R | |
Microelectrode puller | Narishige | PC-10H | Vertical puller for glass pipette production |
Micromanipulator | Sensapex | ||
Mini bolt | Centrostyle | Ref. 00343 s/steel M1.0x4.5 | |
Mobile Homecage | Neurotar | ||
Multiphoton Laser Scanning Microscope | Olympus | FV1000MPE | |
Nonwoven swabs 5×5 | Molnlycke Health Care | Mesoft | Surgical tampons |
Polyacrylic glue | Henkel | Loctite 401 | |
Round glass coverslip | Electron Microscopy Sciences | ||
1.5 thickness | |||
Small animal stereotaxic instrument | David Kopf Instruments | 900 | |
Student iris scissors, straight 11.5 cm | FST | 91460-11 | |
Xylazine | Bayer Health Care | Rompun vet |