This work was supported by the American Heart Association (Post-doctoral fellowship to AYS) and the National Institutes of Health (MH085499, EB003832, and OD006831 to DK). We thank Beth Friedman and Pablo Blinder for comments on the manuscript.

1. Preparing for Surgery i
<ol>
 <li>Clean the surgical tools by sonicating in a mixture of Maxizyme and Surgical Milk in an ultrasonic cleaner. Autoclave the surgical tools before each experiment.</li>
 <li>Ensure that all necessary reagents and disposables are available. A list of reagents and disposables is provided in Table 2. Reagents and disposables that come in contact with exposed tissue should be sterile, when possible.</li>
 <li>Induce anesthesia. Typical anesthetics suitable for survival studies a...

<ol>
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(43), e2059-e2059 (2010).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+neural-hemodynamic+coupling+and+the+hemodynamic+response+function+in+the+awake+rat.">Investigating neural-hemodynamic coupling and the hemodynamic response function in the awake rat.</a> Neuroimage. 32, 33-48 (2006).</li><li>Shih, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> Journal of Cerebral Blood Flow and Metabolism. , (2011).</li><li>Kobat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+multiphoton+microscopy+using+longer+wavelength+excitation.">Deep tissue multiphoton microscopy using longer wavelength excitation.</a> Optics Express. 17, 13354-13364 (2009).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4, 1128-1144 (2009).</li><li>Xu, H. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Srinivasan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+the+quantitative+study+of+cerebrovascular+physiology.">Optical coherence tomography for the quantitative study of cerebrovascular physiology.</a> Journal of Cerebral Blood Flow &#38; Metabolism. 31, 1339-1345 (2011).</li><li>Hu, S., Wang, L. 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Two-photon imaging through a PoRTS window requires transmission through the thinned bone and the dura, which attenuates the laser light and adds optical aberrations at greater depths 8. However, despite this drawback, imaging depths up to 250 &mu;m below the pial surface can be achieved with 900 nm excitation. Greater imaging depths may in principle be possible with longer excitation wavelengths 13. A major advantage of this method is the absence of cortical inflammation that might exist transiently...

<table border="1">
<tbody>
 <tr>
 <td> Agent</td>
 <td>Route of delivery</td>
 <td>Dose for mouse</td>
 <td>Duration</td>
 <td>Notes</td>
 <td>Source</td>
 <td> Ref Ref</td>
 </tr>
 <tr>
 <td>Pentobarbital (Nembutal) </td>
 <td>IP</td>
 <td>90 &mu;g/g</td>
 <td>15-60 min</td>
 <td>Narrow safety margin. Work up to proper dose of anesthesia slowly. Supplement 10 % of induction dose as required.</td>
 <td>036093; Butler Schein</td>
 <td>7</td>
 </tr>
 <tr>
 <td>Ketamine (Ketaset)
 mixed with
 Xylazine (Anased)</td>
 <td>IP</td>
 <td>60 &mu;g/g (K)
 
 10 &mu;g/g (X) (mix in same syringe)</td>
 <td>20-30 min</td>
 <td>Xylazine is co-injected as a muscle relaxant and analgesic. Supplement only Ketamine at 50% of induction dose as required.</td>
 <td>(K) 010177, (X) 033198; Butler Schein</td>
 <td>7</td>
 </tr>
 <tr>
 <td>Isoflurane (Isothesia) </td>
 <td>Inhalation</td>
 <td>4% mean alveolar concentration (MAC) for induction;
 1-2% MAC for maintenance</td>
 <td>4-6 h. </td>
 <td>Provided in mixture of 70% oxygen and 30% nitrous oxide. Prolonged anesthesia leads to slow recovery.</td>
 <td>029403; Butler Schein</td>
 <td>26</td>
 </tr>
 </tbody>
 </table>
 Table 1. Suggested anesthetic agents for survival studies.
 <table border="1">
 <tbody>
 <tr>
 <td>ITEM</td>
 <td>COMPANY</td>
 <td>CATALOG # / MODEL</td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Butler Schein</td>
 <td>6906950</td>
 </tr>
 <tr>
 <td>Buprenorphine (Buprenex) </td>
 <td>Butler Schein</td>
 <td>031919</td>
 </tr>
 <tr>
 <td>Fluorescein isothiocyanate dextran, 2 MDa molecular weight</td>
 <td>Sigma</td>
 <td>FD2000S</td>
 </tr>
 <tr>
 <td>Isopropyl alcohol</td>
 <td>Fisher</td>
 <td>AC42383-0010</td>
 </tr>
 <tr>
 <td>Lactated Ringer's Solution</td>
 <td>Butler Schein</td>
 <td>009846;</td>
 </tr>
 <tr>
 <td>Lidocaine solution, 2 % (v/v) </td>
 <td>Butler Schein</td>
 <td>002468</td>
 </tr>
 <tr>
 <td>Saline </td>
 <td>Butler Schein</td>
 <td>009861</td>
 </tr>
 <tr>
 <td>Surgical Milk</td>
 <td>Butler Schein</td>
 <td>014325</td>
 </tr>
 <tr>
 <td>Texas Red dextran, 70 kDa molecular weight</td>
 <td>Invitrogen</td>
 <td>D1864</td>
 </tr>
 <tr>
 <td>Maxizyme</td>
 <td>Butler Schein</td>
 <td>035646</td>
 </tr>
 <tr>
 <td>DISPOSABLES</td>
 </tr>
 <tr>
 <td>Carbide burrs, 1/2 mm tip size</td>
 <td> Fine Science Tools</td>
 <td>19007-05</td>
 </tr>
 <tr>
 <td>Cottoned tip applicators </td>
 <td>Fisher Scientific</td>
 <td>23-400-100</td>
 </tr>
 <tr>
 <td>Cover Glass, no.&nbsp;0 thickness </td>
 <td>Thomas Scientific</td>
 <td>6661B40</td>
 </tr>
 <tr>
 <td>Cyanoacrylate glue </td>
 <td>ND Industries</td>
 <td>31428 H04308</td>
 </tr>
 <tr>
 <td>Gas duster</td>
 <td>Newegg</td>
 <td>N82E16848043429</td>
 </tr>
 <tr>
 <td>Grip cement powder </td>
 <td>Dentsply</td>
 <td>675571</td>
 </tr>
 <tr>
 <td>Grip cement solvent</td>
 <td>Dentsply</td>
 <td>675572</td>
 </tr>
 <tr>
 <td>Insulin syringe, 0.3 mL volume with 29.5 gauge needle </td>
 <td>Butler Schein</td>
 <td>018384</td>
 </tr>
 <tr>
 <td>Nut and bolt to secure the head </td>
 <td>Digikey</td>
 <td>Nut, H723-ND; bolt, R2-56X1/4-ND</td>
 </tr>
 <tr>
 <td>Opthalmic ointment </td>
 <td>Butler Schein</td>
 <td>039886</td>
 </tr>
 <tr>
 <td>Scalpel blades </td>
 <td>Fisher Scientific</td>
 <td>12-460-448</td>
 </tr>
 <tr>
 <td>Screws, self-tapping #000 </td>
 <td>J.I. Morris Company</td>
 <td>FF000CE125</td>
 </tr>
 <tr>
 <td>Silicone aquarium sealant</td>
 <td>Perfecto Manufacturing</td>
 <td>31001</td>
 </tr>
 <tr>
 <td>Tin oxide powder</td>
 <td>Mama's Minerals</td>
 <td>EQT-TINOX</td>
 </tr>
 <tr>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Glass scribe</td>
 <td>Fisher Scientific</td>
 <td>08-675</td>
 </tr>
 <tr>
 <td>Dissecting microscope </td>
 <td>Carl Zeiss</td>
 <td>OPMI-1 FC</td>
 </tr>
 <tr>
 <td>Electric powered drill</td>
 <td> Foredom or Osada</td>
 <td>K.1020 (Foredom) or EXL-M40 (Osada)</td>
 </tr>
 <tr>
 <td>Electrical razor</td>
 <td>Wahl</td>
 <td>Series 8900</td>
 </tr>
 <tr>
 <td>Forceps, Dumont no. 55 </td>
 <td>Fine Science Tools</td>
 <td>11255-20</td>
 </tr>
 <tr>
 <td>Feedback regulated heat pad</td>
 <td>FHC</td>
 <td>40-90-8 (rectal thermistor, 40-90-5D-02; heat pad, 40-90-2-07)</td>
 </tr>
 <tr>
 <td>Isoflurane vaporizer </td>
 <td>Ohmeda</td>
 <td>IsoTec4</td>
 </tr>
 <tr>
 <td>Pulse oximeter</td>
 <td>Starr Life Sciences</td>
 <td>MouseOx</td>
 </tr>
 <tr>
 <td>Screwdriver, miniature </td>
 <td>Garret Wade</td>
 <td>26B09.01</td>
 </tr>
 <tr>
 <td>Stereotaxic frame</td>
 <td>Kopf Instruments</td>
 <td>Model 900 (with mouse anesthesia mask and non-rupture ear bars)</td>
 </tr>
 <tr>
 <td>Surgical scissors, blunt end </td>
 <td>Fine Science Tools</td>
 <td>14078-10</td>
 </tr>
 <tr>
 <td>Ultrasonic cleaner </td>
 <td>Fisher Scientific</td>
 <td>15-335-30</td>
 </tr>
 </tbody>
 </table>
 Table 2. List of specific reagents, disposables and equipment.

a polished and reinforced thinned-skull window for long-term imaging of the mouse brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

a-polished-reinforced-thinned-skull-window-for-long-term-imaging

Research

JoVE Journal

Neuroscience

26.6K vistas.  University of California, San Diego. Se presenta un método para formar una ventana de imagen en el cráneo del ratón que se extiende milímetros y es estable durante meses sin inflamación del cerebro. Este método es muy adecuado para estudios longitudinales de flujo de sangre, la dinámica celular, y células / estructura vascular utilizando microscopía de dos fotones.

Video: A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

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 ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...