An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice
© Stetter et al.; licensee BioMed Central Ltd. 2013
Received: 17 May 2013
Accepted: 9 July 2013
Published: 10 July 2013
Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex.
Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis.
Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging.
Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury.
Keywords2-photon microscopy Fluorescence In vivo imaging Neurons Cranial window Mouse model
Since its introduction in the 1990’s , 2-photon microscopy (2-PM) soon proved its enormous benefit for intravital imaging, especially in the field of neuroscience [2–8]. The possibility of penetrating tissue in depths up to 1 mm [5, 7, 9] and, therefore visualization of neural structures such as neurons, glial cells, and blood vessels led to new insights in developmental and degenerative neurobiology as well as neuronal plasticity after trauma, ischemia and inflammation [3, 10] To obtain high-resolution in vivo images even in deeper areas of the brain (> 500 μm), highly ambitious surgical techniques and even use of fluorescence microendoscopy were developed [11–13]. In addition, combination of high speed, low power 2-PM calcium imaging with patch recodings allow monitoring of spine function  and long term neuronal network activity . The availability of various transgenic mice expressing fluorescent proteins in particular cell types  enables selective observation of neurons, their axons, and dendrites in different layers , while simultaneously monitoring glial cells and blood vessels [4, 10, 17–20]. By creating a permanent entrance to the brain via a cranial window [21, 22] in transgenic mice, even repeated and long-term 2-PM imaging became feasible [7, 8, 23]. The microstructure of neuronal tissue, e.g. dendritic spines and synapses, was shown to be a dynamic, highly delicate process of formation and elimination . These new imaging methods will help us to better understand the role of synaptic plasticity after traumatic head injuries or degenerative disease.
Materials and methods
For all experiments, we used male C57/Bl6 transgenic Thy1-YFP (H) mice expressing yellow-fluorescent protein (YFP) in layer 5 pyramidal neurons. All experiments required an appropriate animal experimentation facility and needed to be conducted in accordance with the laws and regulations of the regulatory authorities for animal care. The animal experiments presented here were approved by and conducted in accordance with the laws and regulations of the regulatory authorities for animal care and use in Lower Franconia (Regierung von Unterfranken, Würzburg, Germany; file number: 54–2531.01-20/07).
Operating microscope (Carl Zeiss AG, Jena, Germany).
Stereotactic frame (TSE, Bad Homburg, Germany, Figure 1).
2-Photon microscope (Figure 2) with multifocal scanhead splitter (TriMScope, LaVision Biotec, Bielefeld, Germany).
Custom-made head holding device (Figure 3).
Surgical instruments and materials
Scalpel No. 15 (Aesculap, Tuttlingen, Germany).
Microsurgical blade (Surgistar #38-6961; Surgistar, Vista CA, USA).
Scissors (delicate curved sharp scissors; Aesculap, Tuttlingen, Germany).
Microdrill with diamond tip (diameter 1.5 - 3 mm; Figure 4).
Cyanoacrylate (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Dental acrylic (Dentsply, York, PA, USA).
Custom-made cover slips (diameter 5 mm, thickness 1 mm).
Low-melting point agarose (1%, Sigma Type III; Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Sterile irrigation (e.g. sodium-chloride 0.9%, B.Braun, Melsungen, Germany).
Forceps (anatomical tips, straight or curved; Aesculap, Tuttlingen, Germany).
Needle holder (Aesculap, Tuttlingen, Germany).
Sterile suture material (Prolene 4.0, Vicryl 4.0; Ethicon, Norderstedt, Germany).
Anesthetics (xylazine/ketamine, isoflurane).
Cottonoids, swabs, gloves, and eye ointment (e.g. dexpanthenol).
Methods and results
In the following section, we describe two different techniques to create a cranial window and illustrate the imaging setup. For surgery, all mice were anaesthetized with intra-peritoneal injection of 0.1 mg/g ketamine (Ketanest-S 25 mg/ml; Pfizer, New York, NY, USA) and 0.005 mg/g xylazine (Rompun 2%; Bayer Health Care, Leverkusen, Germany). The depth of surgical anesthesia was verified before starting surgery and the mouse head was fixed in a stereotactic frame (Figure 1). For in vivo imaging, mice were anaesthetized with isoflurane (Isofluran, Baxter, Deerfild, IL, USA) via a facial mask and the head was restrained in a custom-made head-holding device (Figure 3).
After restraining the mouse head in a stereotactic frame, the scalp was incised in the midline. Periosteum was softly separated from underlying bone with cotton swabs.
In this article, we provide a thorough methodological description of in vivo imaging of neuronal and vascular structures via two types of cranial windows. In experienced hands and with an established setup of two-photon microscopy, this method is a suitable tool for highly ambitious in vivo research, especially in the field of neurotrauma, neurodegenerative disorders, and neurovascular disease. The LaVision system was optimized for our application but the method is applicable for all two photon microscope systems. One of the advantages of the open skull method is that there is only one single surgery compared to multiple re-thinning procedures of the skull (and therefore multiple re-openings of the skin), an easier re-location of the same region of interest, and a higher penetration depth. However, the preparation of the open-skull window is demanding and bears a higher risk of dural tears and cortical injuries due to pressure or direct penetration. In addition, a damaged cover slip or opaque agarose layer could impair imaging results. Xu et al. reported a higher inflammation rate in neuronal tissue in the open-skull window with an increased turnover rate of dendritic spines . Both models allow a “live” view on intracranial structures, not only on the surface of the brain, but even in deeper regions of neuronal tissue, and the possibility of long-term imaging.
The authors thank Prof. Jens Eilers, Department of Physiology, University of Leipzig, for his expert advice. This work was supported by the German Research Council (DFG) SFB581/TP B27, the Interdisciplinary Center for Clinical Research (IZKF), University of Würzburg (TP N-229) and the University of Würzburg in the funding programme Open Access Publishing.
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