All animal experiments were performed in accordance with guidelines approved by the IACUC at the University of Nebraska Medical Center.
1. Before surgery
2. Start of surgery
3. Craniotomy
4. Viral injections
5. Implantation of cranial window
6. Post operation
7. Animal habituation for imaging
8. Multiphoton imaging
The quality of the cranial window can be assessed by how crisp the neuronal structures appear. In a good window, dendritic spines are clearly visible (Figure 1). With the structural and positional data stored, the same animal can be imaged repeatedly for days, weeks, or months to examine the same cells (Figure 1). The images in Figure 1 were obtained from the forelimb region of the primary motor cortex (in a 5 mm window). A variety of parameters can be measured, including density and dynamics of dendritic spines and axonal boutons to study structural synaptic plasticity. Astrocytic activity can be studied by analyzing the spatiotemporal dynamics of calcium signaling (Figure 2). Depending on the microscope capabilities (i.e., resonant scanners, piezo motor controlling the objective) and the experimental question, time-lapse imaging can be performed in a single focal plane or in a volume or multiregion to monitor calcium activity at the desired acquisition frequency.
Figure 1: Repeated imaging of dendrites and astrocytes over days. A dendrite expressing tdTomato (magenta) allows the identification and repeated imaging of GCaMP6f-expressing astrocytes (white) in close proximity. Ca2+ activity within astrocytes is shown at different time points on different days. Synaptic structural plasticity can be concurrently imaged. Two new spines that appeared on Day 2 are marked with arrows. While one spine persisted and was visible on Day 5 (blue arrow), the other spine was eliminated (green arrow). Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 2: Imaging of astrocytic calcium signaling. Images showing Ca2+ activity from an astrocyte expressing GCaMP6f. Panels show Ca2+ activity recorded from a 4 µm volume at different time points during the acquisition. Calcium signaling in processes and microdomains can be observed during the resting periods. A global event encompassing the entire cell can be observed during a locomotion bout at 188 s. Scale bar = 10 µm. Please click here to view a larger version of this figure.
15o Pointed Blade | Surgistar | 6500 | Surgery Tools |
19 G Needles | BD | 305186 | Surgery Supply |
AAV1-CAG-FLEX-tdTomato | Addgene | 28306-AAV1 | Viral Vector |
AAV1-CaMKII-0-4-Cre | Addgene | 105558-AAV1 | Viral Vector |
Acteone | Fisher Scientific | A16P4 | Reagent |
Alcohol Prep Pads | Fisher Scientific | Covidien 5750 | Surgery Supply |
Beveler | Narishige | Equipment | |
Borosilicate Glass | World Precision Instruments | TW100F-4 | Surgery Supply |
Carbide Burs | SS White Dental | 14717 | Surgery Tools |
Carprofen (Rimadyl), 50 mg/mL | Zoetis Mylan Institutional, LLC. | Drug | |
Compressed Air | Fisher Scientific | 23-022-523 | Surgery Supply |
Cotton Tip Applicators | Puritan | 836-WC NO BINDER | Surgery Supply |
Cover Glass, No. 1 thickness, 3 mm/5 mm | Warner Instruments | 64-0720, 64-0700 | Surgery Supply |
Dental Drill | Aseptico | Equipment | |
Dexamethasone, 4 mg/mL | Mylan Institutional, LLC. | Drug | |
Dissecting Microscope | Nikon | Equipment | |
Duralay Liquid (dental cement liquid) | Patterson Dental | 602-8518 | Reagent |
Duralay Powder (dental cement powder) | Patterson Dental | 602-7932 | Reagent |
Enrofloxacin, 2.27% | Bayer | Drug | |
Eye Ointment | Dechra | 17033-211-38 | Surgery Supply |
Fiber Lite High Intensity Illuminator | Dolan-Jenner Industries | Equipment | |
Forceps (Large) | World Precision Instruments | 14099 | Surgery Tools |
Forceps (Small) | World Precision Instruments | 501764 | Surgery Tools |
GCaMP6f B6; 129S-Gt(ROSA)26Sortm95.1(CAGGCaMP6f)Hze/J | The Jackson Laboratory | Stock No: 024105 | Mouse line |
Germinator | Fisher Scientific | Equipment | |
GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J | The Jackson Laboratory | Stock No: 012586 | Mouse Line |
Headplate | Neurotar | Model 1, Model 3 | Surgery Supply |
Hemostatic forceps | World Precision Instruments | 501705 | Surgery Tools |
Holder for 15o Pointed Blade | World Precision Instruments | 501247 | Surgery Tools |
Holder for Scalpel Blades | World Precision Instruments | 500236 | Surgery Tools |
Iodine Prep Pads | Avantor | 15648-926 | Surgery Supply |
Isoflurane | Piramal | Surgery Supply | |
Isoflurane table top system with Induction Box | Harvard Apparatus | Equipment | |
Isoflurane Vaporizer | SurgiVet | Equipment | |
Krazy Glue | Office Depot | KG517 | Reagent |
Loctite 401 | Henkel | 40140 | fast-curing instant adhesive |
Loctite 454 | Fisher Scientific | NC9194415 | cyanoacrylate adhesive gel |
Micropipette Puller | Sutter Instruments | Equipment | |
Multiphoton Microscope | Equipment | ||
Nitrogen | Matheson | NI M200 | Gas |
Oxygen | Matheson | OX M250 | Gas |
Picospritzer | Parker | intracellular microinjection dispense system | |
Pipette Tips | Rainin | 17014340 | Surgery Supply |
Rodent Hair Trimmer | Wahl | Equipment | |
Saline (0.9% Sodium Chloride) | Med Vet International | RX0.9NACL-30BAC | Surgery Supply |
Scalpel Blades, Size 11 | Integra | 4-111 | Surgery Tools |
Scissors | World Precision Instruments | 503667 | Surgery Tools |
Stereotaxic Instrument | Stoelting | Equipment | |
Sugi Sponge Strips (sponge strips) | Kettenbach Dental | 31002 | Surgery Supply |
SURGIFOAM (gel foam) | Ethicon | 1972 | Surgery Supply |
Syringe with 26 G Needle | BD | 309625 | Surgery Supply |
Tamoxifen | Sigma Aldrich | T5648-1G | Reagent |
Ti:Sapphire Laser | Coherent | Equipment | |
Transfer Pipettes | Fisher Scientific | 13-711-9AM | Surgery Supply |
Water Blanket | Fisher Scientific | Equipment | |
Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL | Fresenius Kabi USA | Drug |
To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.
To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.
To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.