This work was supported by Burroughs Wellcome Career Award in the Biomedical Sciences (A.K.M), The Whitehall Foundation Research Grant (A.K.M), Sloan Foundation Fellowship (A.K.M), NIH EY019277 (A.K.M), and an NEI funded, Vision Training Grant, EY013319 (E.A.K.).

<ol>
 <li>To visualize neurons in the intact brain using two-photon microscopy, a preparation where neurons are labeled with fluorescent markers is used. In the experiments presented here we use the GFP-M transgenic mouse line which labels layer 5 pyramidal cells1. Layer 5 pyramidal cells project dendritic processes into superficial layers, allowing the visualization of dendrites and dendritic spines up to a depth of 300 &mu;m below the level of the pia. An alternate approach is to label cells using viral markers (see Lowery et al., submitted to JOVE).</li>
 <li>Sterilize the workspace using 70% ethanol and autoclave tools for aseptic surgery. Cover operating surface with clean dressing.</li>
 <li>Anesthetize mice with a Fentanyl cocktail (fentanyl 0.05 mg/kg; midazolam 5 mg/kg; metatomadin 0.5 mg/kg) followed by a quarter dose of Avertin (0.0075 mg/ml) using IACUC approved procedures. Pretreat mice with the analgesic, buprenorphine (0.5 mg/kg) administered subcutaneously (SQ). Monitor the level of anesthetic plane by testing reaction to tail/toe pinch.</li>
 <li>Maintain animal at 37.5&deg;C using heating blanket with attached rectal thermometer.</li>
 <li>Apply ophthalmic ointment to eyes to prevent the eyes from drying out. Hood eyes with small sheet of paper to protect eyes from light source during surgery.</li>
 <li>Using scissors, remove hair from the back of head (behind ears) to the eyes.</li>
 <li>Clean the top of animal's head with sequential application of 70% ethanol, followed by betadine scrub and betadine solution.</li>
 <li>Make an L-cut incision behind the ears and up along either the right or left side of the midline (depending on which hemisphere will be imaged) to the eyes. Fold the skin over and away from surgery site. Do not remove skin as the skin will be sutured following the imaging session.</li>
 <li>Using fine forceps gently pull the skin up and away from your area of interest.</li>
 <li>Gently scrape away the periosteum from the exposed area of the skull with clean forceps or microsurgical blade. Using cotton swab, apply a small amount of 10% Ferric Chloride to the skull to completely dry the periosteum membrane and ensure that it has been removed completely. It is important that the skull is completely dry to ensure proper adhering of the glue (see 11). Using forceps or microsurgical blade, gently scrape away dried periosteum membrane.</li>
 <li> Apply a thin layer of cyanoacrylate glue around the imaging window of the stainless steel head plate (see Figure 1) and affix the plate to the skull.</li>
 <li>Using the wooden end of a cotton-swab stick, apply glue to inside seams of the imaging window; making sure to fill all gaps between the imaging plate and curvature of skull. </li>
 <li>Place a drop of glue accelerator into the window. Quickly wipe away excess glue accelerator with a cotton swab or kim wipe. The glue should be completely solidified before drilling.</li>
 <li>Begin to thin the skull in the imaging window using a dental drill affixed with a 0.7 mm stainless steel burr. Alternate drilling with occasional application of 0.9% sterile saline to avoid generating too much heat on the skull. </li>
 <li>Thin a 4x4 mm window on the skull using small strokes across the skull surface with the dental drill. Test the thinness of the skull with blunt end forceps. The skull surface will indent slightly with gentle pressure. The optimal skull thickness for imaging is between 10 and 30 &mu;m.</li>
 <li>When the skull is thin and the window cleaned of all debris, place saline on the skull. Photograph the imaging window with a digital camera mounted to a dissecting scope. Photography and use of the brain vasculature will aid in locating the position to place the imaging plate for subsequent imaging sessions. </li>
 <li>Transport the animal to the 2-photon rig. The animal should remain on the heating blanket (with rectal probe) during the entire imaging session. Note: while under sedation, the animal's core body temperature will fluctuate greatly in the absence of a heating unit resulting in cortical damage or possibly death.</li>
 <li>A two-photon microscope with a Mai Tai laser, a 10W solid state pump for maximum power and a modified Olympus Fluoview confocal unit is used. The system is optimized for deep tissue imaging and external detectors are installed as close to the objective as possible to allow maximal detection of fluorescence from the brain. An Olympus LUMPlan fl/IR 20X/0.95NA objective lens is used. 0.9% saline is used during imaging for objective submersion.</li>
 <li>Using this microscope, and low digital zoom (x1) identify an area containing brightly labeled neurons.</li>
 <li>Using a digital camera affixed to the microscope eye piece, take a photograph of the imaging area. Alternatively, sketch out the blood vasculature pattern. This is necessary to return to the same imaging location at a subsequent time point.</li>
 <li>Obtain a low magnification stack (x20 objective, 512x512 pixel; 5 &mu;m step) through the entire visible extent of the brain (usually spanning ~200 &mu;m) showing the extent of dendritic ramification. Note: this low magnification image will be used to locate the imaging area at subsequent time points as both blood vessels and dendrites maintain their structure in young and adult animals.</li>
 <li>Using imaging software (Fluoview, v. 5.0, FV300), increase digital magnification 8-fold. Adjust the X,Y coordinates (pan coordinates) if necessary and collect a high magnification stack which will show detailed dendritic morphology including the locations and structure of dendritic spines. Take detailed notes regarding each stack collection, i.e. pan coordinates, the range of collection, the z-step size and the number of steps in your stack. These notes will be used when returning to this dendrite or axon at a subsequent time point.</li>
 <li>Following imaging, remove the head plate by gently separating plate from skull surface. Using forceps, remove any residual glue remaining on skull and wipe skull surface with 0.9% sterile saline. Suture the skin over the skull together using #6 suturing thread. Wipe area with 70% ethanol. To alleviate pain during post-operative recovery, administer 0.1 mg/kg Buprenorphine analgesic, SQ.</li>
 <li>Place animal in clean cage under a heat lamp until it wakes up and is mobile. Return animal to original cage.</li>
 <li>To reimage animal at a later time point (our imaging time points can be anywhere from 2 days to many months apart), remove stitches or reopen the skin on head using aseptic methods as above. Use the digital image of brain vasculature (taken on first surgery date) to locate the original imaging location. Reaffix stainless steel headplate (steps 10-12). Use a microsurgical blade to gently thin away any bone that might have re-grown between imaging time points. If necessary, thin with dental drill. Clear imaging window of debris and apply a drop of 0.9% sterile saline.</li>
 <li>Carry animal to two-photon microscope and locate original imaging field. Use the digital photograph taken during first imaging session for assistance.</li>
 <li>Open the Fluoview program and open the original low magnification image. Affix a transparency sheet to the computer screen and sketch the major blood vessel pattern using a transparency pen.</li>
 <li>Open the Live image and realign the blood vasculature to the sketched pattern on the transparency film.</li>
 <li>To reimage the 8x zoomed in structures, open up the desired collected stack from the previous imaging session. Sketch the structure (i.e. axonal, dendritic, glial) onto a transparency film, affixed to the computer screen. </li>
 <li>Zoom in 8x from the Live Image and set the pan coordinates to the first day imaging parameters. Depending on the accuracy of aligning the low magnification location, it may be necessary to also adjust the angle of the image slightly. Once the desired structure is found, match up the structure to your sketched high magnification pattern. Collect the z-stack with the identical step size and number of z-stack images for subsequent analysis. </li>
 <li>Repeat steps 29 and 30 until all the high magnification images are re-collected. </li>
 <li>When imaging is complete, repeat steps 22-24. Re-imaging can be repeated at up to 2 separate time points.</li>
 <li>Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Vivarium and Division of Laboratory Animal Medicine of the University of Rochester School of Medicine and Dentistry, in full accreditation by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and are in compliance with state law, federal statute and NIH policy.</li>
</ol>
Representative Results
In GFP-M expressing mice, a subset of layer 5 pyramidal cells and corresponding dendritic processes can easily be visualized using 2-photon laser scanning microscopy (2PLSM)1. Here we demonstrated a thinned-skull preparation in which the skull over the desired imaging location is thinned to ~10-30 &mu;m. Performed correctly, our protocol allowed us to clearly visualize cortical dendrites and spines in the intact visual cortex for acute or chronic analysis (Figure 2).
<img src="/files/ftp_upload/2060/2060fig1.jpg" alt="Figure 1" /> Figure 1. Custom head and base plate. (A) Top view of custom made head and base plate used for thinned-skull surgery preparation. (B) Side view of custom-made head and base plate. (C,D) Detailed schematic and image of head plate.
<img src="/files/ftp_upload/2060/2060fig2.jpg" alt="Figure 2" /> Figure 2. Representative results from 2PLSM following thin skull preparation. (A) Dendrites imaged in vivo in the visual cortex of a transgenic GFP-M mouse using two-photon microscopy. Image is a projection of single image obtained every 5 &mu;m from the pia to a final depth of 175 &mu;m. The boxed area in A represents the magnified region in (B) and (C) imaged on day 0 (D0) and four days later (D4). (D,E) Higher magnification of dendritic branch boxed in B and C demonstrating stable spines (white arrow heads), lost/retracting spines (red arrow heads) and new spines (green arrow heads). Scale bars= A. 200 &mu;m, B,C. 20 &mu;m, D,E. 5 &mu;m.

<ol>
	<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 (1), 41-51 (2000).</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> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Majewska, A. K., Newton, J. R., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+synaptic+structure+in+sensory+cortical+areas+in+vivo.">Remodeling of synaptic structure in sensory cortical areas in vivo.</a> J Neurosci. 26 (11), 3021-3029 (2006).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat Protocols. 5 (2), 201-208 (2010).</li><li>Xu, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nat Neurosci. 10 (5), 549-551 (2007).</li></ol>

No conflicts of interest declared.

Chronic imaging using two-photon microscopy is becoming an increasingly popular technique to study morphological changes triggered during plasticity2-5. Here we demonstrate a thinned-skull preparation to follow identified dendritic spines in the intact mouse brain on different imaging days. In this protocol, the skull is left intact, causing minimal damage to the cortex and resulting in very low levels of neuroinflammation which may alter brain function6. This allows the animal to be imaged immedia...

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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.

Watch this Scientific Journal Video about Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation at JoVE.com

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

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 ...

chronic-imaging-mouse-visual-cortex-using-thinned-skull

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JoVE Journal

Neuroscience

13.8K vistas.  University of Rochester. En este material de vídeo y complementaria, se muestra un protocolo para la crónica In vivo Imágenes del cerebro intacto con una preparación diluida-cráneo.

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

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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.

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 ...

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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 ...

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed as candidate sites for misrouting. Since a correlation between the number of lateral geniculate nucleus (LGN) relay neurons and LGN size has been shown, and based on previously reported reductions in LGN volumes in human albinism, we suggest that fiber projections from LGN to the primary visual cortex (V1) are also reduced. Studying structural differences in the visual system of albinism can improve the understanding of the mechanism of misrouting and subsequent clinical applications. Diffusion data and tractography are useful for mapping the OR (optic radiation). This manuscript describes two algorithms for OR reconstruction in order to compare brain connectivity in albinism and controls.An MRI scanner with a 32-channel head coil was used to acquire structural scans. A T1-weighted 3D-MPRAGE sequence with 1 mm3 isotropic voxel size was used to generate high-resolution images for V1 segmentation. Multiple proton density (PD) weighted images were acquired coronally for right and left LGN localization. Diffusion tensor imaging (DTI) scans were acquired with 64 diffusion directions. Both deterministic and probabilistic tracking methods were run and compared, with LGN as the seed mask and V1 as the target mask. Though DTI provides relatively poor spatial resolution, and accurate delineation of OR may be challenging due to its low fiber density, tractography has been shown to be advantageous both in research and clinically. Tract based spatial statistics (TBSS) revealed areas of significantly reduced white matter integrity within the OR in patients with albinism compared to controls. Pairwise comparisons revealed a significant reduction in LGN to V1 connectivity in albinism compared to controls. Comparing both tracking algorithms revealed common findings, strengthening the reliability of the technique.

This manuscript describes deterministic and probabilistic algorithms for white matter (WM) reconstruction, used to examine differences in optic radiation (OR) connectivity between albinism and controls. Although probabilistic tractography follows the true course of nerve fibers more closely, deterministic tractography was run to compare the reliability and reproducibility of both techniques.

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...