We thank N. Schwedhelm-Domeyer for excellent technical assistance, C. Hammer&#160;and S. Papiol for help with statistical analyses. Our work is funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Center for Nanoscale Microscopy, and Molecular Physiology of the Brain (CNMPB), G&#246;ttingen, Germany and by the GGNB Junior Group Stipend of the University of G&#246;ttingen.

CGNs can be prepared either from postnatal day (P) 5 mouse pups or P6 rat pups. We follow a protocol, described by Bilimoria and colleagues, which uses a mitotic inhibitor to select for postmitotic CGNs13.
Ethics statement: 
All experiments involving live animals have been conducted according to the animal protocol approved by the &#34;Verbraucherschutz und Lebensmittelsicherheit&#34; of Lower Saxony, Germany.
In vitro assay:
1. Preparation of DNA Plasmid, Media, and Buffers for the Calcium Phosphate Transfection Method
<ol>
	<li>Dissolve plasmid DNA in sterile, endotoxin-free water; DMEM (high glucose); make 2.5 M CaCl2; make 2x HBSS (dissolve 4 g NaCl, 0.1775 g KCl, 0.095 g Na2HPO4 &#8226;7H2O, 0.675 g glucose and 2.5 g HEPES in 250 ml ultrapure water and adjust pH to 7.05, 7.08, and 7.11). Note: When preparing the 2x HBSS solution, test which pH gives best results regarding transfection efficiency of given combination of plasmids.</li>
</ol>
2. Transfection of Cultured Neurons
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig1highres.jpg" src="/files/ftp_upload/51070/51070fig1.jpg" /> 
Figure 1. Flowchart of in vitro axon and dendrite growth assay. Cultured CGNs (24-well plate with glass coverslips), isolated from P6 rat pups, are transfected at DIV 0 or 1 with DNA precipitate containing a fluorescent transfection marker (e.g. GFP). After fixation and immunocytochemistry, neurons are imaged in a blinded manner. Images are imported into ImageJ and processes are measured. Measurements are then processed using a statistical program.
<ol>
	<li>Seed CGNs (20 x 106 per 24-well plate; BME, 10% calf serum, 2 mM Penicillin-Streptomycin-Glutamine (PSG), 25 mM KCl) on nitric acid-washed, polyornithine-coated 12 mm glass coverslips in a 24-well plate with 500 &#956;l of media per well.</li>
	<li>On day in vitro (DIV) 0 (at least 8 hr after plating) or DIV 1, collect growth media and keep at 37 &#176;C. Wash neurons twice with 500 &#956;l of prewarmed DMEM and add 500 &#956;l of DMEM.</li>
	<li>Place neurons in incubator (37 &#176;C, 5% CO2) for 45 min.</li>
	<li>Prepare 40 &#956;l DNA precipitate for each well by mixing: DNA (2-2.5 &#956;g/well, 10% of total DNA should be a transfection marker e.g. GFP to visualize transfected neurons), water (up to 18 &#956;l), add 2 &#956;l of 2.5 M CaCl2, mix well and add 20 &#956;l of 2x HBSS.
	<ol>
		<li>Incubate DNA precipitate for 5 min at RT.</li>
	</ol>
	</li>
	<li>Add DNA precipitate to each well and incubate neurons for 18 min in incubator.</li>
	<li>Remove DMEM/DNA mix and wash neurons twice with 500 &#956;l of prewarmed DMEM.</li>
	<li>Add collected media from step 2.2 back to neurons. If neurons will be in culture for more than 3 days, supplement media with 25 mM glucose at DIV 3 to replenish carbon source.</li>
	<li>After 1-5 days, subject neurons to immunocytochemistry using GFP antibodies.</li>
	<li>Image at least 30 individual neurons per condition in a blinded manner using a fluorescent microscope.</li>
</ol>
3. Measure Axons and Dendrites with NeuronJ, an NIH ImageJ Plugin
Important: Ensure that Images are scaled correctluy by using appropriate pixel:&#956;m ratio depending on magnification and resolution of image.
<ol>
	<li>Convert images to 8-bit with ImageJ: Open image, choose 'Image' -&#62; 'Type' -&#62; '8-bit' -&#62; 'Save' image.</li>
	<li>Run NeuronJ plugin and open the 8-bit image. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot1.jpg" width="500px" /></li>
	<li>Use the 'Add tracings' option to track the axon: click the left mouse button once at the beginning of the axon and move the mouse along the process. Double click on tip of axon if trace matches axonal shape. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot2.jpg" width="500px" /> 
	Note: Should the suggested trace differ from axonal shape, click once on the axonal process to anchor trace, then double click on tip of axon.</li>
	<li>Click on 'Measure tracings', choose the 'Display tracing measurements' option and press 'Run'. Axon measurements are all displayed in a new window. For dendrites, choose the 'Display group measurements option' and press 'Run'. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot3.jpg" width="500px" /> 
	Total dendrite measurements are all displayed in a new window. Save them as a separate file that can be opened in any spreadsheet program.</li>
	<li>Alternatively for manual tracing, use Fiji software: Right click on the 'Straight line' option, choose 'Freehand line', 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot4.jpg" width="500px" /> 
	keep the left mouse button pressed and manually trace the process, press 'Ctrl+M' to measure.</li>
	<li>Calculate average axonal/dendritic length per condition and use appropriate statistical test. 
	 
	In vivo electroporation:</li>
</ol>
1. Equipment and Preparation of Reagents
<ol>
	<li>You need 30 G needles, spacer (1-2 mm), syringe, dead volume reducer (DVR), electroporator, and tweezertrodes, heating pad or infrared heat lamp, gooseneck lamp and isoflurane.</li>
	<li>Put DVR into needle, then attach needle to syringe, and finally put spacer onto needle (Figure 2). 
	<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig2highres.jpg" src="/files/ftp_upload/51070/51070fig2.jpg" /> 
	Figure 2. Preparation of needle. DVR is cut off a 200 &#956;l pipette tip and placed into the needle to reduce the dead volume. Spacer is derived from a 200 &#956;l loading tip and is placed on the end of the needle to regulate the depth of penetration into the cerebellum to approximately 2 mm. Ruler units: cm</li>
	<li>Dissolve DNA in PBS/0.03% Fast Green. Note: As a transfection marker, it is advantageous to use a fluorescent protein that is under a neuron-specific promoter (e.g. Synapsin) to visualize neurons only. 25% of the total plasmid amount should be the plasmid encoding the transfection marker.</li>
	<li>Make 70% ethanol.</li>
	<li>Mix equal volumes of OCT and 30% sucrose dissolved in PBS.</li>
	<li>Fill syringe with 4 &#956;l of DNA (4 &#956;g/&#956;l of plasmid DNA in PBS/0.03% Fast Green).</li>
</ol>
2. IVE of Rat Pups
Flowchart of IVE: see Figure 3
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig3highres.jpg" src="/files/ftp_upload/51070/51070fig3.jpg" /> 
Figure 3. Flowchart of in vivo electroporation. P4 rat pups are anaesthetized with Isoflurane and plasmid DNA encoding a fluorescent transfection marker (e.g. GFP) is injected into the cerebellum, followed by exposure to 5 electrical pulses. Five days later, isolated GFP-positive cerebella are sectioned and subjected to immunohistochemistry. Images are captured using a confocal microscope and analyzed using Imaris software. Data are processed with a statistical program.
<ol>
	<li>Use P4 rat pups from albino strain (Wistar or Long Evans).</li>
	<li>Anesthetize pups (one after another) with isoflurane in small box (e.g. P1000 pipette tip box) with 200 &#956;l of isoflurane (soaked in tissue) for 1-2 min until pup is no longer moving. Take care that pups do not get into contact with Iiquid isoflurane. Monitor time closely as individual pups respond differently to anesthesia.</li>
	<li>Sterilize back of pup&#8217;s head with 70% ethanol.</li>
	<li>Fix head of pup between thumb and index finger and use gooseneck lamp to locate cerebellum of albino pup. The transverse sinus sharply demarcates the midbrain (superior and inferior colliculus) from the cortical hemispheres (Figure 3). The cerebellum is located adjacent to midbrain and appears in a darker shade. Use a permanent marker to indicate the cerebellum with a dot. Important: Keep pup in fixed position! Note: Should anesthesia wear off during this procedure, expose pup to isoflurane prior to injecting the DNA.</li>
	<li>Insert needle (Figure 3) and slowly inject 3 &#956;l of DNA into the cerebellum.</li>
	<li>Let the DNA solution diffuse for 30-60 sec.
	<ol>
		<li>Place head of pup between tweezertrodes so that the minus pole makes contact with the back of the head (cerebellar region) and that the plus pole contacts the opposite side of the head (Figure 3).</li>
		<li>Subject pup to 5 electrical pulses. Adjust voltage to weight of pups to ensure good electroporation efficiency without compromising their survival (Table 1).
		<table border="0">
			<tbody>
				<tr>
					<td>Weight</td>
					<td>Voltage</td>
					<td>Pulse</td>
					<td>Interval</td>
				</tr>
				<tr>
					<td>8-9 g</td>
					<td>160 V</td>
					<td>50 msec</td>
					<td>950 msec</td>
				</tr>
				<tr>
					<td>9-10 g</td>
					<td>165 V</td>
					<td>50 msec</td>
					<td>950 msec</td>
				</tr>
				<tr>
					<td>&#62; 10 g</td>
					<td>170 V</td>
					<td>50 msec</td>
					<td>950 msec</td>
				</tr>
			</tbody>
		</table>
		Table 1. Electroporation of P4 rat pups.</li>
		<li>Let pups recover on heated pad or underneath an infrared lamp. Return pups to dam. Important: Make sure that heating source does not inflict any burns.</li>
	</ol>
	</li>
	<li>Sacrifice pups 5 days after electroporation by placing them in CO2 followed by decapitation.
	<ol>
		<li>Isolate the cerebella and screen for GFP-positive ones cerebella using a fluorescent microscope.</li>
	</ol>
	</li>
	<li>Fix cerebella in 4% PFA O/N at 4 &#176;C, then incubate in 30% sucrose at 4 &#176;C until cerebella sink to bottom of tube.</li>
	<li>Embed cerebella in OCT/30% sucrose and cut 40 &#956;m coronal sections using a cryostat. Note: Section each cerebellum in a blinded manner.</li>
	<li>Subject sections to immunohistochemistry using the GFP antibody. Counterstain with nuclear dye (DAPI or Hoechst 33258) and determine localization of at least 200 transfected neurons per animal.</li>
	<li>For an in-depth analysis, subdivide the IGL into halves, resulting in an upper IGL facing the ML and a lower IGL facing the white matter and count GFP-positive neurons residing in each half. Note: Count GFP-positive neurons of each section in a blinded manner.</li>
</ol>
3. Measuring Dendrite Length, Acquire the Images of the Section in x, y, z Plane Using a Confocal Microscope
Note: for example, use 40 images for a 40 &#956;m section with a z-stwp of 1 &#956;m.
<ol>
	<li>Open image series in the software, Imaris, to generate a 3D image of the dendrites.</li>
	<li>Click on 'Surpass' mode to view the neuron in 3D. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot5.jpg" width="500px" /></li>
	<li>Select 'Add new filament' and click 'Skip automatic creation' to start semiautomatic tracing. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot6.jpg" width="500px" /> 
	Note: Analyze each 3D image in a blinded manner</li>
	<li>Select 'Draw' tab and 'AutoPath'. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot7.jpg" width="500px" /></li>
	<li>Move the mouse cursor on the cell body and Shift+mouse right click to select the cell body. Note: Autocalculation by software may require a few minutes. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot8.jpg" width="500px" /></li>
	<li>Add paths to the filament (dendrite) using Shift+mouse left click. Note: Paths can be visualized in real time. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot9.jpg" width="500px" /></li>
	<li>Go to filament statistics window and click on 'Detailed', 'Specific values' and 'Filament dendrite length (sum)' for sum of total dendrite length. 
	<img alt="screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot10.jpg" width="500px" /></li>
	<li>Use appropriate statistical tests to analyze data.</li>
</ol>

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CaMKIIbeta+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nat. Neurosci. , (2011).</li><li>Jia, Y., Zhou, J., Tai, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+promote+cerebellar+granule+neuron+survival.">TRPC channels promote cerebellar granule neuron survival.</a> Nat. Neurosci. 10, 559-567 (2007).</li><li>Huynh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isoform-specific+SnoN1-FOXO1+repressor+complex+controls+neuronal+morphogenesis+and+positioning+in+the+mammalian+brain.">An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.</a> Neuron. 69, 930-944 (2011).</li><li>de la Torre-Ubieta, L., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+neuronal+polarity+and+morphogenesis+in+the+mammalian+brain.">Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain.</a> Neuron. 72, 22-40 (2011).</li><li>Calissano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+insulin-like+growth+factor+I+exerts+a+trophic+action+and+confers+glutamate+sensitivity+on+glutamate-resistant+cerebellar+granule+cells.">Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8752-8756 (1993).</li></ol>

The authors declare&#160;no competing financial interests.

Advantages and limitations of the described in vitro and in vivo methods:
Cultured CGNs from mouse and rats are equally well suited for morphological analyses. Owing to the bigger size of a rat cerebellum, the yield of CNGs from rat pups exceeds that of mouse pups 3-4x. Aside from CGNs, cortical and hippocampal neurons can be used as culture system as well. The calcium phosphate method results in a low (0.01-5%) transfection efficiency, which is desired to analyze the morphol...

The cerebellum is an excellent system to study mechanisms of axon growth and migration. The cerebellum has been the subject of anatomical studies since the dawn of neuroscience1. Modern microscopy and immunohistochemical techniques have significantly expanded and refined the initial discoveries by Santiago, Ramon, and&#160;Cajal2-4. Mouse genetics and molecular studies uncovered essential growth and transcription factors in the control of cerebellar development, which led to greater understanding of crucial events required for proper wiring of different types of neurons including cerebellar granule neurons (CGNs)5-7.
The cerebellum is a derivative of rhombomere 1 of the developing hindbrain8.&#160; The rhombic lip, which is part of the roof of the 4th ventricle, gives rise to cerebellar granule neuron progenitors, which will constitute the most numerous neuronal population in the adult cerebellum9. Following rostral migration, they settle in the cerebellar anlage. Here, mitosis of granule neuron precursors leads to the dramatic expansion of the external granular layer (EGL), which takes place postnatally in rodents. From the EGL, neurons start migrating inward through the molecular layer (ML), past the Purkinje cell layer to ultimately take up residence in the internal granular layer (IGL2). During this migratory process, they acquire a bipolar shape with two axons extending into the ML. Upon further migration, the cell body migrates away from the axons and the two processes fuse to form one bifurcated, T-shaped axon10. Subsequently, these axons fasciculate and are referred to as parallel fibers. Having settled in the IGL, CGNs grow dendrites, which form dendritic claws to establish synapses with mossy fibers. To examine fundamental processes in the developing cerebellum, a combined in vitro and in vivo approach allows for reliable results and conclusions.
CGNs are not only the most numerous neurons of the cerebellum but of the entire brain and can be cultured to high purity11-13. In culture, this highly homogeneous neuronal population becomes rapidly postmitotic and acquires a polar morphology with easily identifiable axons and dendrites. Cultured CGNs have proven to be extremely useful to study various aspects of neuronal development including progenitor proliferation, differentiation, axonal and dendrite development, neuronal migration, apoptosis and electrophysiological properties (14-19 and many others). The use of genetic manipulation has expanded the versatility of cultured CGNs and allowed for further mechanistic insight into the aforementioned events. Transfection of cultured neurons using low-efficiency calcium phosphate or lipophilic methods followed by immunocytochemistry with polarity markers or software-supported analysis facilitates the assessment of e.g. the morphology of individual neurons in a dense neuronal culture. With this approach, the role of proteins of interest in axon or dendrite growth can be studied20-25,26-28. This culture system however is less useful to analyze neuronal migration as migration is very limited in high-density cultures and would require cocultures. The in vitro analysis of axon and dendrite growth also allows for the examination of interconnected proteins of a signaling pathway using combinations of RNA interference (i), over-expression or small molecules.
To establish the relevance of the protein of interest in axon and dendrite growth regulation or neuronal migration, the in vivo electroporation (IVE) technique allows for the analysis in the developing cerebellar cortex. Owing to the fact that cerebellar development in rodents extends way into the first two postnatal weeks, the cerebellum represents an accessible brain structure for genetic manipulations to examine developing axons and dendrites, neuronal migration, synaptogenesis and apoptosis20-24,29,30,26,27,31-34. In addition, this model system is also useful for other aspects of neuronal development that require the intact cerebellar cortex such as axon pathfinding, wiring and connectivity of neurons and neuron-glia interactions Taken together, this protocol provides in vitro and in vivo techniques to tackle a complementary approach regarding neuronal morphogenesis and migration.

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			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
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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.

To analyze the morphology of CGNs in response to different culturing conditions, we transfected the neurons on DIV 0 as described above. After transfection, we placed one set of neurons into full medium (BME, 10% calf serum, 2 mM PSG, 25 mM KCl) and another set into minimal medium containing insulin (BME, 25 mM glucose, 2 mM PSG, 10 &#956;g/ml insulin). We subjected the neurons to immunocytochemistry using the GFP antibody at DIV 1, 2, and 3, followed by measuring axons and dendrites for set 1 and axons only for set 2. O...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

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.

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 ...

genetic-manipulation-cerebellar-granule-neurons-vitro-vivo-to-study

Research

JoVE Journal

Neuroscience

13.6K Views.  Max Planck Institute of Experimental Medicine. 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.

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

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...