Name: visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons
Uploaded: 2018-10-30T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Axonal Growth Cone Collapse and Early Amyloid Î² Effects in Cultured Mouse Neurons at JoVE.com

This work was partially supported by research grants from JSPS (KAKENHI 18K07389), Japan, Takeda Science Foundation, Japan, and Kobayashi Pharmaceutical Co., Ltd., Japan.

All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama and were approved by the Committee for Animal Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama (A2014INM-1, A2017INM-1).
1. Collapse Assay
<ol>
	<li>Poly-D-lysine coating
	<ol>
		<li>Coat 8-well culture slides with 400 &#956;L of 5 &#956;g/mL poly-D-lysine (PDL) in phosphate-buffered saline (PBS) an...

The protocol described in this study enabled the observation of early phenomena in axonal growth cones after A&#946;1-42 treatment. A&#946;1-42 induced endocytosis in axonal growth cones within 20 min, and growth cone collapse was observed within 1 h of treatment. This endocytosis was probably mediated by clathrin. By using this protocol, the inhibition of clathrin-mediated endocytosis was confirmed to prevent A&#946;1-42-induced growth cone collapse and axonal degeneration in cultured neurons27. ...

Amyloid-&#946; (A&#946;) deposits are found in the brain of patients with Alzheimer's disease (AD) and are considered a critical cause of AD1 that disrupt neuronal networks, leading to memory impairments2,3,4. Many clinical drug candidates have been shown to effectively prevent amyloid-&#946; (A&#946;) production or remove A&#946; deposits. However, none have succeeded in improving memory function in AD patients5. A&#946; is already deposited in the brain prior to the onset of memory impairments6; therefore, decreasing A&#946; levels in the brains of patients exhibiting memory impairments may be ineffective. A&#946; deposition is present in preclinical AD patients; however, these patients rarely present with neuronal degeneration and memory deficits6. There is a time lag between A&#946; deposition and memory impairments. Therefore, a critical strategy for the prevention of AD is blocking A&#946; toxicity signaling during the early stages of AD, prior to the development of memory deficits. A&#946; deposition induces axon degeneration7,8,9,10,11,12,13, which may lead to a disruption of neural networks and permanent impairment of memory function. Many studies have investigated the mechanisms of A&#946; toxicity; for example, the degenerated axons of AD mice brains have been shown to have increased autophagy14. Calcineurin activation has been reported as a possible mechanism of A&#946;-induced axonal degeneration15; however, the direct trigger of axonal degeneration remains unknown. 
This study focuses on the collapse of axonal endings called growth cones. The collapse of axonal growth cones can be caused by axonal growth repellents, such as semaphorin-3A and ephrin-A516,17,18,19,20. Collapse-like dystrophic axonal endings have been observed in the brains of AD patients21,22. Additionally, a failure of growth cone functioning can provoke axonal degeneration23. However, it is unknown whether A&#946; induces growth cone collapse. Therefore, this study presents a novel protocol to observe the early effects of A&#946; in cultured neurons and investigate A&#946;-induced growth cone collapse.

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			<td height="40" style="height:40px;width:339px;">ddY mice</td>
			<td style="width:133px;">SLC</td>
			<td style="width:109px;"></td>
			<td style="width:539px;"></td>
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			<td height="40" style="height:40px;">poly D lysine</td>
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			<td height="40" style="height:40px;">Culture medium, Neurobasal medium</td>
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			<td>21103-049</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">house serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
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		<tr height="40">
			<td height="40" style="height:40px;">glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
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		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine</td>
			<td>Wako</td>
			<td>074-00522</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">0.05% trypsin</td>
			<td>Gibco</td>
			<td>25300-054</td>
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		<tr height="40">
			<td height="40" style="height:40px;">DNase I</td>
			<td>Worthington</td>
			<td>DP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">soybean trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Filter with 70 &#181;m mesh size, cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
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		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">CO2 incubator</td>
			<td>Astec</td>
			<td>SCA-165DS</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Amyloid &#946;1-42</td>
			<td>Sigma-Aldrich</td>
			<td>A9810</td>
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		<tr height="40">
			<td height="40" style="height:40px;">paraformaldehyde</td>
			<td>Wako</td>
			<td>162-16065</td>
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		<tr height="40">
			<td height="40" style="height:40px;">sucrose&#160;</td>
			<td>Wako</td>
			<td>196-00015</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Aqueous mounting medium, Aqua-Poly/Mount</td>
			<td>polysciences</td>
			<td>18606-20</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope A</td>
			<td>Carl Zeiss</td>
			<td>Axio Observer Z1&#160;</td>
			<td>Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 20x</td>
			<td>Carl Zeiss</td>
			<td>420650-9901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Plan-Apochromat 63x</td>
			<td>Carl Zeiss</td>
			<td>440762-9904</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective, CFI Plan Apo Lambda 40X</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-MAP2 IgG</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-tau-1 IgG</td>
			<td>Chemicon</td>
			<td>MAB3420</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-amyloid &#946; antibody</td>
			<td>IBL</td>
			<td>10379</td>
			<td>clone 11A1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">normal goat serum</td>
			<td>Wako</td>
			<td>143-06561</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bovine serum albumin</td>
			<td>Wako</td>
			<td>010-25783</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">t-octylphenoxypolyethoxyethanol</td>
			<td>Wako</td>
			<td>169-21105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-mouse IgG conjugated with AlexaFluor 594</td>
			<td>Invitrogen</td>
			<td>A11032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-rabbit IgG conjugated with AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">hot plate</td>
			<td>NISSIN</td>
			<td>NHP-M30N</td>
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			<td height="40" style="height:40px;">cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-545-85</td>
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			<td height="40" style="height:40px;">35 mm dish</td>
			<td>IWAKI</td>
			<td>1000-035</td>
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			<td height="40" style="height:40px;">Silicone RTV</td>
			<td>Shin-Etsu</td>
			<td>KE42T</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:339px;">hand punch</td>
			<td style="width:133px;">Roper Whitney</td>
			<td style="width:109px;">No. XX</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorescence membrane probe, FM1-43FX</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ca2+- and Mg2+-free Hanks&#39; balanced salt solution</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transfection solution, Nucleofector solution</td>
			<td>Lonza</td>
			<td>VPG-1001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electroporator, Nucleofector I</td>
			<td>Amaxa</td>
			<td></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Inverted microscope B</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Image software, ImageJ</td>
			<td style="width:133px;">NIH</td>
			<td style="width:109px;"></td>
			<td style="width:539px;">https://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
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visualizing axonal growth cone collapse and early amyloid &#946; effects in cultured mouse neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

In this protocol, A&#946;1-42 was incubated at 37 &#176;C for 7 days before use, because incubation of A&#946;1-42 was needed for producing toxic forms27,28,30,35. After this incubation, aggregated forms of A&#946; were observed (Figure 1A). It has been reported that similar incubation of A&#946;1-42 produced the fibril form of A&#9...

Watch this Scientific Journal Video about Visualizing Axonal Growth Cone Collapse and Early Amyloid Î² Effects in Cultured Mouse Neurons at JoVE.com

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

This method can help answer key questions in the field of Alzheimer disease research, especially the early events involved in growth cone collapse. The main advantage of this technique is to make it possible to visualize and analyze axonal growth cones immediately after amyloid beta treatment. Start by using micro-scissors to mince freshly isolated cerebral cortices from embryonic day-14 mice in neuron culture medium without antibiotics.Collect the tissues and centrifuge the tissues at 87G for three minutes. Following centrifugation, remove the supernatant and add two milliliters of 0.05%trypsin to the pellet. Incubate for 15 minutes at 37 degrees Celsius and mix by tapping every five minutes.After 15 minutes, add four milliliters of medium A to neutralize the trypsin, and mix by tapping. Then centrifuge the tissues at 178 times G for three minutes. After removing the supernatant, add DNAse and soybean trypsin inhibitor solution and incubate for 15 minutes at 37 degrees Celsius with mixing every five minutes as before.When the incubation time is elapsed, add four milliliters of medium A.Mix and centrifuge as before. After removing the supernatant, add four milliliters of medium A and triturate the tissues with a polished Pasteur pipette until no debris is observed. Next, filter the triturated tissues with 70-micron pore-sized mesh.After filtration, count the cells with a hemocytometer and calculate the cell density. Pipette 0.8 times 10 to the fourth cells in medium A into each well of an eight-well culture slide. Then incubate in a humidified atmosphere of 10%CO2 at 37 degrees Celsius.After four hours of culturing, change the culture medium to medium B.The purity of neurons using this method is approximately 75%Begin this assay at least a week in advance by opening a fresh vial of commercially obtained full-length A beta 42 and dissolving the contents in sterile, distilled water to a concentration of 0.5 millimolar. Incubate at 37 degrees Celsius for seven days to induce aggregation and toxicity. Prepare aliquots of aggregated A beta 1-42 and store at minus 80 degrees Celsius until use.On day four of the neuronal culture, treat the wells with 100 microliters of 0.5 micromolar aggregated A beta 1-42 or vehicle solution in medium B for one hour as previously demonstrated. After the hour has elapsed, remove the culture medium and immediately fix the neurons with 4%paraformaldehyde containing 4%sucrose in PBS for one hour at 37 degrees Celsius on a hot plate. Fixation is most important as maintaining the shape of growth cones is critical for this protocol.After fixation, wash the neurons three times with PBS. Then after removing the chamber, mount the neurons with an aqueous mounting medium. Dry the mounting medium at four degrees Celsius for two to four days.Capture the entire area of each well with a 20X dry objective lens on an inverted microscope. Capturing and analyzing the entire area in each well is important for avoiding subjectivity. Classify the longest neurites of each neuron in stage three or four as axons.Axonal growth cones lacking lamellipodia or possessing fewer than three filopodia are considered collapsed growth cones. A beta 1-42 oligomers, shown here before incubation, aggregate after seven days of incubation at 37 degrees Celsius. After treatment with aggregated A beta 1-42, immunostaining with an antibody for the toxic oligomer of A beta shows positive staining in red on A beta 1-42 treated neurons, but not vehicle treated neurons.The early phenomena induced by A beta 1-42 treatment were analyzed after four days in culture. Identified axons were confirmed as such by positive immunostaining for the axonal marker tau-1 shown in red and negative immunostaining for the dendritic marker, MAP-2, shown in green. After one hour of vehicle treatment, growth cones had spread lamellipodia and several filopodia.These were identified as healthy growth cones. Conversely, one hour of A beta 1-42 treatment led to shrunken growth cones, which developed no lamellipodia or filopodia. These were identified as collapsed growth cones.While attempting this procedure, it's important to remember to fix the cells with 4%PFA and 4%sucrose at 37 degrees Celsius to my-nor-idge this fixation protocol in the best. Following this protocol, other method like live-cell imaging and gene transfection can be performed to answer additional questions like when and how the axon growth cones are collapsed and how the collapsed growth cones are recovered.

visualizing-axonal-growth-cone-collapse-early-amyloid-effects

Research

JoVE Journal

Neuroscience

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 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

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.

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 ...

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 ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.