Denne metode skaber en konkret, velkendt miljø for musen til at navigere og udforske under mikroskopiske billeddannelse eller encellede elektrofysiologiske optagelser, som kræver fast fiksering af dyrets hoved.
Det er almindeligt anerkendt, at brugen af generel anæstesi kan underminere relevansen af elektrofysiologiske eller mikroskopiske data fra et levende dyr hjerne. Desuden langvarige opsving fra anæstesi begrænser hyppigheden af gentagne optagelse / imaging episoder i longitudinelle studier. Derfor er nye metoder, som ville give stabile optagelser fra ikke-bedøvede opfører mus forventes at fremme områderne cellulære og kognitive neurovidenskab. Eksisterende løsninger spænder fra blotte fysiske tilbageholdenhed til mere sofistikerede metoder, såsom lineære og sfæriske løbebånd anvendes i kombination med computer-genereret virtuel virkelighed. Her er en ny metode, der er beskrevet, hvor en hoved-fikseret mus kan flytte rundt en luft-løftet mobil homecage og udforske sine omgivelser under stress-fri betingelser. Denne metode giver forskerne at udføre adfærdsmæssige test (f.eks læring tilvænnings eller roman objekt anerkendelse) samtidig medto-foton mikroskopisk billedbehandling og / eller patch-clamp optagelser, alt sammen kombineret i et enkelt eksperiment. Denne video-artiklen beskriver anvendelsen af den vågne dyrehoved fikseringsindretning (mobil homecage), viser de procedurer, animalske tilvænning, og et eksempel på en række mulige anvendelser af fremgangsmåden.
En spændende nyere tendens i neurovidenskab er at udvikle eksperimentelle tilgange til molekylær og cellulær sondering af neuronale netværk i hjernen på vågen, opfører gnavere. Sådanne tilgange holder lover at kaste nyt lys over neurofysiologiske processer, der ligger til grund for motorik, sansemotorisk integration, perception, indlæring, hukommelse, samt skade progression, neurodegeneration og genetiske sygdomme. Desuden optagelse fra vågen dyrets hjerne lover i udvikling af nye terapeutiske midler og behandlinger.
Der er en voksende bevidsthed om, at anæstesi, som har været almindeligt anvendt i neurofysiologiske forsøg, kan påvirke de grundlæggende mekanismer af hjernens funktion, der kan føre til fejlagtig fortolkning af eksperimentelle resultater. Således udbredte bedøvelsesmiddel ketamin hurtigt øger dannelsen af nye dendritiske Torner og forbedrer synaptisk funktion 1; en anden almindeligt anvendt anesthetic isofluran ved kirurgisk anæstesi niveauer helt undertrykker spontan kortikale aktivitet hos nyfødte rotter og blokerer spindel-burst svingninger i voksne dyr 2. På nuværende tidspunkt er kun et begrænset antal metoder gør det muligt forsøg i ikke-bedøvede mus ved hjælp af to-foton mikroskopisk billedbehandling eller patch-clamp optagelser. Disse fremgangsmåder kan opdeles i frit bevægelige og hoved-faste præparater.
Den unikke tiltrækningskraft et frit bevægelige forberedelse dyr er at det giver vurdering af naturlig adfærd, herunder hele kroppen bevægelser under navigation. En måde at billedet i hjernen på en frit bevægelig gnaver er at fastgøre en miniaturiseret hovedmonteret mikroskop eller fiberscope 3-5. Men miniaturiserede enheder tendens til at have begrænset optisk ydeevne i forhold til objektiv-baserede to-foton mikroskopi, og ikke let kan kombineres med hele celle patch-clamp optagelser 6.
Den existing løsninger til head-fastsættelse af en vågen gnaver har primært påberåbt enten fysisk tilbageholdenhed 7,8 eller på at træne dyr til at udstille frivillig nakkestøtte 9. En anden populær metode er at tillade dyrets lemmer til at bevæge sig ved at placere den på, fx en kugleformet løbebånd 10; denne tilgang er ofte kombineret med computer-genereret virtuel virkelighed. Elektrofysiologiske eksperimenter på head-faste mus har for det meste brugt ekstracellulære optagelser og blev brugt til at studere central regulering af hjerte-kar-funktion 11, effekter af anæstesi på neuronal aktivitet 12, det auditive reaktion i hjernestammen 13 og informationsbehandling 14. De banebrydende intracellulære / hel-celle optagelser i vågen opfører dyr blev udført i 2000'erne og har fokuseret på neurale aktivitet relateret til perception og bevægelse 15-20. Omkring samme tid blev de første mikroskopiske billeddiagnostiske undersøgelser på vågne mus publeret, hvor to-foton mikroskopi blev brugt i den sensoriske cortex fysisk fastholdes rotter 7 og på mus, der kører på en kugleformet løbebånd 21.
Efterfølgende in vivo mikroskopi og elektrofysiologi undersøgelser viste, at et hoved fiksering præparat med held kan kombineres med adfærdsmæssige paradigmer baseret på forben bevægelser, lugt anerkendelse, piske og slikke 8,22-25. Mus placeret på den sfæriske løbebånd kan trænes til at navigere den virtuelle visuelle miljø genereret af en computer 10,26. Intracellulære / ekstracellulære optagelser viste i et head-fast dyr navigere sådan virtuelt miljø, kan aktivering af hippocampus sted celler påvises 27. I et virtuelt visuelt miljø, mus demonstrere normal bevægelse-relaterede theta rytme i det lokale potentiale felt og theta-fase præcession under aktiv bevægelse 27. For nylig, den rumlige og tidsmæssige Aktivitety mønstre af neuronale populationer blev registreret optisk mus under arbejdet beslutningstagere hukommelse opgaver i et virtuelt miljø 28.
Trods have aktiveret banebrydende forskning, den sfæriske løbebånd design har flere iboende begrænsninger. Først dyret kræves for at flytte på et ubegrænset overflade af en roterende luft-løftet kugle, der udgør ingen håndgribelige forhindringer såsom vægge eller barrierer. Denne begrænsning er kun delvist kompenseret af computer-genereret "virtual reality", fordi det visuelle er nok mindre effektive på mus og rotter sammenlignet med den taktile sanseindtryk (f.eks whisker-touch eller slikke), som disse arter naturligvis afhængige på. For det andet kan stor krumning af kugleoverfladen være ubehageligt for laboratoriemus anvendes til at gå på et fladt gulv i deres bure. Endelig, det store diameter af bolden (mindst 200 mm for mus og 300 mm for rotter) gør den lodrette størrelse af den sfæriskeløbebånd enhed relativt stor. Dette gør det vanskeligt at kombinere sfærisk løbebånd med størstedelen af kommercielt tilgængelige mikroskopi opsætninger, og kræver ofte at bygge en ny setup omkring løbebåndet ved hjælp af skræddersyede mikroskop rammer.
Her er en ny metode, der er beskrevet, hvor en hoved-fikseret mus kan flytte rundt en luft-løftet mobil homecage der har et fladt gulv og materielle vægge, og udforske det fysiske miljø under stress-fri betingelser. Denne artikel viser procedurerne i mus uddannelse og hoved fiksering og giver repræsentative eksempler, hvor to-foton mikroskopi, iboende optisk billedbehandling og patch-clamp optagelser udføres i hjernen af vågen opfører 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 |