We would like to thank Yue (Pauline) Hu, Rachel Sinit for their technical assistance. The study is supported by NIH grant (PN2 EY016525) and by funding from Down Syndrome Research and Treatment Foundation and the Larry L. Hillblom Foundation.

Surgical and animal procedures are carried out strictly according to the NIH Guide for the Care and Use of Laboratory Animals. All experiments involving the use of animals are approved by UCSD Institutional Animal Care and Use Committee.
1. Plasmid Cloning, Expression and Purification of Mono-biotinylated BDNF (mBtBDNF)
NOTE: Construct pre-proBDNFavi and BirA cDNA into pcDNA3.1 vector and coexpress in HEK293FT cells10. Purify mBtBDNF using Ni-NTA beads according to previously published method of producing mature and biologically active monobiotinylated nerve growth factor (mBtNGF)10.
2. Preparation of Microfluidic Chambers
Microfluidic neuron culturing device makes it possible to fluidically isolate axons from neuron cell bodies. Assemble chambers with freshly coated coverslips right before each dissection. Microfluidic chambers used in this protocol are commercially purchased (see materials and equipment&#8217;s table). Handwash and reused commercially purchased chambers up to 5-6x.
<ol>
	<li>Hand wash microfluidic chambers in 1% Alconox. Rinse three times with Milli-Q water for 30 min each. Hand wash chambers again in 70% ethanol.</li>
	<li>Lay out chambers to dry on Parafilm in the laminar flow hood. Radiate and sterilize both sides of the chambers for 20 min under UV. Store sterilized chambers in a sterile 15 cm dish sealed with Parafilm at room temperature.</li>
	<li>Place 24 x 40 mm No. 1 glass coverslips in a glass container. Soak the coverslips in 35% hydrochloride acid overnight on a rotator. On the following day, rinse the coverslips three times for 30 min each with water.</li>
	<li>Sterilize the coverslips one by one by dipping each coverslip into 100% ethanol and flaming over a Bunsen burner. Store dry coverslips in a sterile petri dish and keep it at room temperature until coating.</li>
	<li>Lay out the coverslips in a 15 cm culture dish. Coat each coverslip with 0.7 ml of 0.01% poly-L-Lysine (PLL) and incubate in the hood at room temperature.</li>
	<li>After 1 hr, rinse the coverslips three times with sterile water. Dry coverslips with vacuum and place each coverslip into a 6 cm culture dish.</li>
	<li>To assemble the microfluidic chamber, place the chamber with microgroove side at the bottom onto the PLL coated coverslip with caution not to touch the microgrooves. Gently pressed down the chamber with a pipette tip to ensure the chamber was tightly sealed.</li>
</ol>
3. Dissection of Neuronal Culture and Plate on Chambers
<ol>
	<li>Place two E17-E18 rat hippocampi (one brain) dissected in a 15 ml conical tube containing 2 ml dissection buffer (HBSS, no calcium, no magnesium, with 1% pen/strep and 10 mM HEPES. Rinse the tissues 3x&#160;with 5 ml dissection buffer each time. Remove the dissection buffer as much as possible and add 900 &#956;l of fresh dissection buffer.</li>
	<li>To digest the tissue, add 100 &#956;l of 10x trypsin (2.5%) into the dissection buffer to make 1x working concentration. Place the conical tube in a 37 &#176;C water bath. After 10 min of digestion, add 100 &#956;l of 10 mg/ml DNase I to a final concentration around 1 mg/ml.</li>
	<li>Using a fire-polished Pasteur glass pipette, gently triturate the tissues by pipetting up and down 5-10x. Right after trituration, quench the trypsin with 2 ml plating medium (Neurobasal with 10% FBS, 2 mM GlutaMax, 2% B27).</li>
	<li>Leave the sample in the hood for 5 min to allow any debris of the tissues to settle down to the bottom. Carefully remove 2 ml of supernatant into a clean sterile 15 ml conical tube and centrifuge at 200 x g for 5 min to pellet the cells. Resuspend the pellet in 50 &#956;l plating media</li>
	<li>Count the cells with hemocytometer. Load 15-20 &#956;l cell suspension (~40,000 cells) into one compartment of the microfluidic chamber. Place the chamber in the incubator for 10 min to allow the cells to attach to the coverslip. After 10 min, add more plating media to fill up both compartments of the chamber.</li>
	<li>On the second day of dissection, completely replace the plating media with maintenance media (Neurobasal with 2 mM GlutaMax, 2% B27) in both the cell body and the axon compartment. Axons from the hippocampal neurons start to cross the microgrooves in day 3 and reach the axon compartment between day 5-7. During this period of time, replace half of the culturing media with fresh maintenance media every 24~48 hr.</li>
</ol>
4. Axonal Transport of QD-BDNF
<ol>
	<li>Prior to live imaging of QD-BDNF axonal transport, deplete BDNF from both the cell body and axon compartments of the microfluidic chamber by thoroughly rinse both compartments with BDNF-free, serum free Neurobasal media every 30 min for 2 hr.</li>
	<li>During BDNF depletion, prepare the QD-BDNF conjugates. Mix 50 nM of mono-biotinylated BDNF dimer with 50 nM QD655-streptavidin conjugates in neurobasal media and incubate on ice for 60 min.</li>
	<li>Remove media in the axon compartment and add 300 &#956;l QD-BDNF with a final concentration of 0.25 nM for 4 hr at 37 &#176;C. To minimize the diffusing of QD-BDNF into the cell body compartment, it is very important to always maintain a higher level of media in the cell body compartment than in the axon compartment. Wash off unbound QD-BDNF after incubation before live imaging.</li>
	<li>Carry out live imaging of QD-BDNF transport using an inverted microscope equipped with a 100X oil objective lens. Warm up the scope and the environmental chamber attached to it to a constant temperature (37 &#176;C) and CO2 (5%). Use a set of Texas red excitation/emission cubes to visualize the QD655 signal.</li>
	<li>Acquire and capture time-lapse images within the middle axons at the speed of 1 frame/sec for a total of 2 min using a CCD camera. Use microgrooves with no axons that have no QD and hence no signal as a control for infiltration.</li>
	<li>Analyze BDNF transport using any image analysis software or NIH ImageJ.</li>
</ol>

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No conflicts of interest declared.

In this study, we report the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. By conjugating the protein to quantum dot streptavidin, and using a microfluidic chamber, the method allows one to detect axonal transport of BDNF in primary neurons with single molecule sensitivity, in real-time and with spatial and temporal resolutions. The tools used herein provide a means by which to study the molecular machines that mediate ...

Neurons are highly polarized cells whose long and often highly elaborated processes are fundamental for establishing and maintaining the structure and function of neural circuits. The axon plays a vital role in carrying cargoes to and from synapses. Proteins and organelles synthesized in the cell soma need to be transported through axons to reach the presynaptic terminal to support neuronal function. Correspondingly, signals received at distal axons need to be transduced and conveyed to the soma. These processes are essential for neuronal survival, differentiation, and maintenance. In that axonal transport in some neurons must be conducted through distances more than 1,000 times the diameter of the cell body, the possibility is readily envisioned that even small deficits could markedly impact neuronal and circuit function.
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, is present in many brain regions, including hippocampus, cerebral cortex, and basal forebrain. BDNF plays a crucial role in cognition and memory formation by supporting the survival, differentiation, and function of neurons that participate in cognitive circuits. BDNF binds to its receptor, the tyrosine kinase TrkB, at the axon terminal where it activates TrkB-mediated signaling pathways including the mitogen activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), phosphatidylinositol-3-kinase (PI3K) and phospholipase C-gamma (PLC&#947;). The proteins that participate in these signaling pathways are packaged onto endocytic vesicular structures to form the BDNF/TrkB signaling endosome1-6 that are then retrogradely transported to the neuronal soma.
The microfluidic culture chamber is a very useful platform for studying axonal biology under normal conditions as well as in the setting of injury and disease7,8. By isolating axons from the cell bodies, the device has allowed one to study transport specifically in axons8-10. The PDMS based microfluidic platforms with 450 &#956;m microgroove barriers used in this study were commercially purchased (see Materials&#160;table). To examine BDNF transport, we developed a novel technology to produce monobiotinylated BDNF (mBtBDNF). We took advantage of the biotin acceptor peptide, AP (also known as AviTag). It is a 15 amino acid sequence that contains a lysine residue that can be specifically ligated to a biotin by the Escherichia coli enzyme biotin ligase, BirA. We fused the AviTag to the C-terminus of the mouse pre-proBDNF cDNA by PCR (Figure 1A). The construct was cloned into the mammalian expression vector, pcDNA3.1 myc-his vector. We also cloned the bacterial BirA DNA into the pcDNA3.1 myc-his vector. The two plasmids were transiently co-transfected into HEK293FT cells to express both proteins. BirA catalyzed the ligation of biotin specifically to the lysine reside within the AviTag at the C-terminus of BDNF at a 1:1 ratio to produce monobiotinylated BDNF monomer. Biotinylated, mature BDNF with a molecular mass of ~18 kDa was recovered and purified from the media using Ni-resin (Figure 1C). The biotinylation of BDNF was complete, as judged by the inability to detect unmodified BDNF by immunoblotting (Figure 1D). Streptavidin conjugated quantum dots, QD 655, were used to label mBtBDNF to make QD-BDNF. The presence of the AviTag did not interfere with activity of BDNF as the mBtBDNF was able to activate phosphorylated TrkB (Figure 1E) and stimulate neurite outgrowth (Figure 1F) to the extent of recombinant human BDNF (rhBDNF). Immunostaining shown that QD-BDNF colocalized with TrkB in hippocampal axons, indicating that QD-BDNF is bioactive (Figure 1G). To study the BDNF transport, QD-BDNF was added to distal axon compartment of microfluidic cultures containing rat E18 hippocampal neurons (Figure 2A). QD-BDNF retrograde transport within axons was captured by real-time live imaging of the red fluorescent tag (Supporting videos&#160;S1, S2). By analyzing the kymograph generated, QD-BDNF was observed to be transported retrogradely at a moving velocity of around 1.06 &#956;m/sec (Figure 3A). GFP or mCherry-tagged BDNF have been used to track axonal movement of BDNF. The major drawbacks are that they are not bright enough for single molecule studies. Also, the presence of both anterograde and retrograde BDNF movements makes it difficult to evaluate whether or not the retrogradely transported BDNF was in a neurotrophin/receptor complex.
In this video, we demonstrate the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons. The ultrabrightness and excellent photostability of quantum dots makes it possible to perform long-term tracking of BDNF transport. These techniques can be exploited to enhance studies of axonal function in AD, HD, and other neurodegenerative disorders.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Production and Purification of Biologically Active Mono-biotinylated BDNF
The expression vector of BDNF fused with an AviTag sequence (GGGLNDIFEAQKIEWHE) was created according to a previously published protocol10. The molecular mass of the full length fusion protein was predicted to be ~32 kDa (<a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a>) Monobiotinylated mature BDNF with a predicted molecular mass of 18 kDa (...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.0K Views.  University of California, San Diego. Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons. 

Video: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

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Chapters in this video

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

Mechanical Manipulation of Neurons to Control Axonal Development

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System