Name: peering at brain polysomes with atomic force microscopy
Uploaded: 2016-03-16T15:00:00
Description: Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.

The practices used to obtain the mouse tissues were approved by the Body for the Protection of Animals (OPBA) of the University of Trento (Italy), protocol no. 04-2015, as per art.31 Legislative Decree no. 26/2014. All mice were maintained at the Model Organism Facility of the Centre for Integrative Biology (CIBIO), University of Trento, Italy.
Caution: To avoid any RNA degradation of the samples, prepare all buffers using DEPC-treated water for minimizing RNase contamination.
1. Preparation of Polysomes from...

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As the structure of DNA was proven to be of paramount importance to describe the process of transcription and as the organization of chromatin advanced our understanding of transcriptional control of gene expression, it is essential to analyze the organization and structure of polysomes to improve the truthful comprehension of translation and its regulation.
With the described protocol, an optimal density of polysomes gently immobilized on a flat surface, suitable for AFM imaging is obtained. ...

The synthesis of protein is the most energy consuming process in cells1,2. Hence, it is not surprising that protein abundances are mainly controlled at the translational rather than transcriptional level3,4,5. The polysome is the fundamental macromolecular component that converts the mRNA information into functional proteinaceous readouts. Polysomes are thus far recognized as macromolecular complexes where several translational controls converge6-13. Despite hundreds of studies on ribosome structure14- 16, the detailed molecular insights into the dynamics of translation and the topology of polysomes has encountered a limited interest. As a consequence, the organization of the native polysomal ribonucleoprotein complex and its potential effect on translation are still rather obscure issues. Polysomes may hide still unknown ordered and functional organizations, potentially mirroring what nucleosomes and epigenetic controls have represented for the transcription field. Indeed, the investigation of this intriguing hypothesis requires additional studies and new technical approaches. In this line, structural techniques and atomic force microscopy may fruitfully collaborate to unravel new mechanisms for controlling gene expression, similarly to what was achieved for the nucleosome17,18.
Since the discovery of the ribosome14-16, its structure has been extensively characterized in prokaryotes19,20, yeast21 and more recently in human22, providing the molecular description of the mechanisms at the base of protein synthesis. Polysomes were initially recognized on the membrane of the endoplasmic reticulum, forming typical 2D geometrical organizations14. As mentioned before, polysome assembly has not been the object of constant interest as the ribosome structure. In the past, polysomes have been studied essentially by transmission EM-based techniques. Only recently, cryo-EM techniques enabled the 3D reconstruction of purified polysomes from in vitro translation systems23-26, human cellular lysates27 or in cells28. These techniques offered more refined information about the ribosome-ribosome organization in polysomes23, 26-28 and a preliminary molecular description of the contact surfaces of adjacent ribosomes in wheat germ polysomes24. Thus, cryo-EM tomography allows the disclosure of ribosome-ribosome interactions with molecular detail, but it is burdened by extensive post-processing and reconstruction analyses that require heavy computational resources for data handling. Moreover, to obtain the molecular detail of ribosome-ribosome interactions, a highly resolved map of the ribosome is required, and this kind of reference ribosome is available only for a few species. Importantly, cryo-EM tomography is not able to detect free RNA. Therefore, new techniques are required to fully understand the organization of the translation machinery.
Beside cryo-EM, AFM has been also employed as a useful tool for direct imaging of polysomes in lower eukaryotes29-33 and humans27. Compared to EM, AFM requires no sample fixation or labeling. In addition, measurements can be carried out in near physiological conditions and with the unique possibility of clearly identifying both ribosomes and naked RNA strands27. Imaging of single polysomes can be performed relatively quickly, obtaining thousands of images at nano-resolution with little post-processing effort compared to the extensive and heavy post-processing and reconstruction analyses required by cryo-EM microscopy. Consequently, AFM data handling and analysis does not need expensive workstations and high computing power. As such, this technique collects information on polysomal shapes, morphological characteristics (such as height, length and width), ribosome densities, the presence of free RNA and the number of ribosomes per polysome27 with a higher throughput than cryo-EM. In such way, AFM represents a powerful and complementary approach to EM techniques to portray polysomes27.
Here we present a complete pipeline from purification to data analysis where AFM is applied to image and analyze mouse brain polysomes. The proposed protocol focuses on purification issues and on the accurate deposition of polysomes on mica substrates that are used for AFM imaging. In addition to conventional particle analysis that can be easily performed with common software used by the AFM community, an ImageJ34 plugin, called RiboPick, is presented for counting the number of ribosomes per polysome27, 35.

<table>
	<tbody>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DNAseI</td>
			<td>Thermo Scientific</td>
			<td>89836</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>RiboLock RNAse Inhibitor</td>
			<td>Life technologies</td>
			<td>EO-0381</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>40718</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma</td>
			<td>T8532</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S5016</td>
			<td>Sucrose gradient preparation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima LE-80K</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor</td>
			<td>Beckman Coulter</td>
			<td>SW 41 Ti</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Polyallomer tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Density Gradient Fractionation System&#160;</td>
			<td>Teledyne Isco</td>
			<td>67-9000-176</td>
			<td>Sucrose gradient fractionation</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>Asylum Research</td>
			<td>Cypher</td>
			<td>Polysome visualization</td>
		</tr>
	</tbody>
</table>

peering at brain polysomes with atomic force microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

Sucrose gradient polysomal profiling of whole mouse brain 
It is possible to purify polysomes from a cellular lysate by polysomal profiling, which separates macromolecules in accordance to their weight and size. With polysomal profiling, cytoplasmic lysates obtained from cultured cells or tissues, such as in this example, are loaded onto a linear sucrose gradient and processed by ultracentrifugation to separate ...

Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

The overall goal of this protocol is to provide a detailed pipeline for the accurate purification and deposition of brain polyribosomes on mica and to obtain thousands of images at the nanoscale resolution without the need for heavy post-processing or 3D reconstruction analyses. This method can help answer key questions in the fields of translation and translational control, such as understanding the impact of ribosome organization within polysomes during the rewiring of gene expression. The main advantages of this technique are that no sample fixation or labeling are required, and measurements can be carried out in near physiological conditions to easily identify the ribosomes and naked RNA stands.Although this method can provide insight into the organization of mammalian polyribosome, it can also be applied to other microorganisms, such as yeast, bacteria, and insect, as well as status involving translational controls of gene expression. Visual demonstration of this method is critical, as handling polysomal sample for absorption of mica and the washing steps are quite tricky and require handling skills and experience. Demonstrating the procedure will be Paola Bernabo, and Lorenzo Lunelli.To begin, collect and freeze brain tissue as described in the accompanying text protocol. Then, pull the frozen tissue from the freezer and bring it to the bench. Place the frozen brain tissue and liquid nitrogen into a pre-chilled mortar and pulverize it using a pestle until it forms a powder.Transfer about 25 milligrams of the brain powder to a cold microcentrifuge tube and immediately add 0.8 milliliters of lysis buffer. Pipette the mixture up and down 25 times quickly to disrupt the cells. Next, centrifuge the tube at 12, 000 x g for one minute at four degrees Celsius to pellet the cellular debris.Transfer the supernatant to a new microcentrifuge tube and keep the tube on ice for 15 minutes. Then, centrifuge the tube for five minutes to pellet nuclei and mitochondria. Next, carefully remove one milliliter from the top of a previously prepared sucrose gradient.Overlay the sucrose drop by drop with the supernatant from the cytosolic lysate. WIth the sample now loaded on top of the gradient, carefully lower the tube and a counterbalance tube into the buckets of a swinging bucket rotor. Centrifuge the gradient for 100 minutes.After ultracentrifugation, leave the tubes in their buckets for 20 minutes at four degrees Celsius, to let the gradient stabilize. Then, carefully remove the sample tube and mount it on the collector device of a density gradient fractionation system. Collect one milliliter fractions while monitoring the absorbance of nucleic acids at 260 nanometers with a UV visible light detector and put them on ice.Next, prepare 30-40 microliter aliquots of the fractions of interest, and keep them on ice. If the samples will not be used right away, store them at 80 degrees Celsius and take steps to limit the number of freeze-thaw cycles. To prepare samples for AFM imaging, first cover a washed mica sheet with 200 microliters of one millimolar nickel ii sulfate and incubate the sheet for three minutes at room temperature.Then remove the solution, dry the surface with compressed air, and place the petri dish on ice. Next, gently add the entire aliquot from the fraction of interest drop by drop on the mica. Using a 100 or 200 microliter tip, spread the sample over the entire surface of the mica, and incubate the sample on ice for three minutes.Then, cover the mica sheet drop by drop with 200 microliters of cold buffer AFM and incubate the sample on ice for one hour. Maintaining the sample at 40 degrees and using cold buffer that were prepared in RNase-free water are important to preserve polyribosome organization. Following incubation, carefully remove the buffer and wash the mica three to four times with 200 microliters of cold buffer AFM to remove any excess sucrose.Then, wash the mica three times with cold washing solution and drain the excess water from the mica using paper. The complete removal of sucrose from the absorbant samples is crucial now that you have severed both ribosome and naked RNA in the polysome. Leave the sample to dry under the chemical hood with the top of the petri dish partially opened.After two hours, close the petri dish. The sample can now be stored at room temperature. Attach the prepared sample to the sample holder of the AFM using double sided tape.Then, insert the sample holder in the AFM stage in accordance with the manufacturer's instructions. Following calibration, approach the sample until the cantilever tip engages the surface. Select a scan area of two by two microns, a resolution of at least 512 x 512 pixels, choose live background subtraction mode, and select a Z-scale of 20-25 nanometers.Then, begin image acquisition. Inspect the image, looking for the presence of round objects characterized by a height between 10 and 15 nanometers when acquiring images in the air. Next, adjust the set point and feedback parameters until sharp objects are visualized.The background should appear relatively flat in good samples with some two to four nanometer high objects. Once the image looks good, acquire several two by two micron scans at different sample areas. After the deposition of sucrose aliquots on mica, AFM provides accurate size descriptions of single polysomes that appear as clusters of tightly packed ribosomes.Taking a closer look at one of the ribosomal peaks using cross-section analysis reveals the height of ribosomal peaks are around 14 nanometers. This matches what was previously observed for human ribosomes and polysomes after air-drying. In the image on the right, a macro was used to identify ribosome location and mark each ribosome with a red circle.Using this information, the frequency distribution of the number of ribosomes per polysome can be analyzed. This experimental distribution was fitted with a Gaussian curve that was centered at 5.8 ribosomes per polysome, with a standard deviation of 1.3. Once mastered, this technique from brain pulverization to polyribosome images acquisition can be done in less than eight hours if it is performed properly.While attempting this procedure, it is important to remember to keep the sample on ice and use cold buffers for the sample deposition on the mica. Following this procedure, and using the ImageJ plugin of a developer to make available in your paper, you can precisely count the number of ribosome per polysome. After each development, this technique will pave the way to understand the kinetics of polysome formation and identifying the changes in polysome organization and their different cellular or tissue conditions.After watching this video you should have a good understanding of how to obtain thousands of polysome images for systematically analyzing and studying their organization.

peering-at-brain-polysomes-with-atomic-force-microscopy

Research

JoVE Journal

Neuroscience

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. The frequency of play fighting can be measured by counting the number of playful nape attacks occurring per unit time. However, playful defense, as it can only occur in response to attack, is necessarily a contingent measure that is best measured as a percentage (#attacks defended/total # attacks X 100%). How a particular attack is defended against can involve one of several tactics, and these are contingent on defense having taken place; consequently, the type of defense is also best expressed contingently as a percentage. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.

Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.