We would like to thank William A. Eimer for imaging technical assistance. This work was supported by the Cure Alzheimer's Fund [to R.E.T], the National Institute of Health [R01AG014713 and R01MH60009 to R.E.T; R03AR063271 and R15EB019704 to A.L.], and the National Science Foundation [NSF1455613 to A.L.].

1. Experimental Preparation
<ol>
	<li>Prepare the following reagents: Dulbecco&#39;s phosphate buffered saline (PBS); Triton X-100; 0.2% PBST (PBS + 0.2% Triton X-100); 32% paraformaldehyde (PFA), diluted into 4% before use; silicone elastomer base and curing agent; antifade mounting medium (e.g., ProLong Gold); and fingernail polish.</li>
	<li>Prepare the following equipment: dissection microscope, two sharp forceps and a pair of scissors for microdissection, a number of pins for microdissection, a Petri dish for making the dissection dish, microscope slides and coverslips, a confocal microscope, and a computer with Fiji ImageJ software installed.</li>
</ol>
2. Larvae Collection
<ol>
	<li>Mate UAS-Sox102F-RNAi flies with UAS-GFP, ppk-GAL4, or W1118 wild type flies, respectively. Culture flies in standard conditions at 25 &#176;C.</li>
	<li>In ~5 - 6 days, collect the third instar larvae of UAS-Sox102F-RNAi/ ppk-GAL4, UAS-GFP or UAS-GFP, and ppk-GAL4 /+ control carefully with forceps for dissection.</li>
</ol>
3. Dissection of Larvae
NOTE: All procedures in this section are operated under a microdissection microscope. The magnification is up to the investigators. Try to adjust to the optimal sight view. ~4 - 6X magnification is recommended.
<ol>
	<li>Place a larva on a dissection dish made of silicone elastomer base and a tissue culture Petri dish.</li>
	<li>Pin the larva mouth hook to allow the dorsal side to face up, and then pin the tail of larva. Place the dorsal side in the middle as much as possible to expose the midline, which will be the open-up marker.</li>
	<li>Add 200 &#181;L of PBS to the larva to maintain body moisture.</li>
	<li>Make a cut with a pair of scissors along the dorsal midline between the two tracheas, from caudal to rostral. Make a small cut at each of the four corners of the larva body.</li>
	<li>Place a pin at each of the four corners of the body so the body lies flat.</li>
	<li>Fix the larva body wall in 4% PFA for 25 min at room temperature.</li>
	<li>Wash in PBST for 5 min. Repeat twice more.</li>
	<li>Remove the pins from the tissue. Then transfer the tissue onto a glass slide, cover with antifade mounting medium, and mount with a cover slip. Air dry for 1 h and seal the edges with fingernail polish.</li>
</ol>
4. Imaging Processing
NOTE: Images were taken using an inverted confocal microscope system. The user can photograph the sample using a 20X objective (recommended).
<ol>
	<li>Capture Z-series images. Open the &#34;Capture Z-series&#34; dialog box in the confocal microscope software. Determine the range for capturing the Z series images.
	<ol>
		<li>Click the &#34;Top and Bottom&#34; button. Determine the top and bottom position and input the values to &#34;Bottom:&#34; and &#34;Top:&#34; boxes. Set the &#34;Step&#34; in &#34;Capture Z-series&#34; dialog box as 0.5 &#181;m. Then click the &#34;Run now&#34; button to acquire the Z-series images.</li>
	</ol>
	</li>
	<li>Save the obtained images as *.tiff or *.nd2 files.</li>
	<li>Store the slides in a dark holder at 4 &#176;C after imaging.</li>
</ol>
5. Dendrite Evaluation
Note: GFP protein was co-overexpressed in UAS-GFP and ppk-GAL4 flies in the da neurons for GFP fluorescence imaging analysis. The length, branching, and structure of da neurons in the third instar larvae were quantified. Analysis parameters include length of dendrites (&#181;m), surface area (&#181;m2), total number of branches, and branching structure (%). Figure 1 shows the analysis parameters in detail.
<ol>
	<li>Length of Dendrites 
	NOTE: The length of dendrites is the sum of all dendrites labeled in tracer plugin.
	<ol>
		<li>Setup and run Fiji ImageJ (https://fiji.sc/)20&#160;software. Then split images into separate channels, if there are several channels of images. 
		&#8203;NOTE: The image in this protocol only has one channel of GFP.</li>
		<li>Run &#34;Image | Stacks | Z project&#34; to get a Z projection; a new window will appear with the name &#39;Z-projection.&#39;&#160;Click &#34;max intensity&#34; for projection type, and organize numbers between start slice and stop slice depending upon individual preferences. Click &#34;OK.&#34;&#160;A Z-projection image will appear presenting the dendrite projection clearly.</li>
		<li>Trace the neurites.
		<ol>
			<li>Select &#34;Plugins | Segmentation | Simple Neurites Tracer&#34; in the window to trace the dendrites. Find the neuron soma, and then click one point at a dendrite starting from the cell body, and another point at the tip of this dendrite. 
			NOTE: The program will connect the two points with a blue line.</li>
			<li>Press &#34;Y&#34; in the plug-in window if the path is clear; the dendrite path is traced until the endpoints of the selected path in the visible images. Click &#34;complete path&#34; on the same plugin window; the segment is completed (Figure 2). 
			NOTE: Some dendrites ended farther from the starting points, in which the pathway was divided into smaller segments. The segments were combined to provide a complete path for the tracer.</li>
		</ol>
		</li>
		<li>After a path is completed, go back to a new point where the branches are. The new path can be built on; the &#34;All paths&#34; window box will show the length values of all the paths (Figure 3). Save the tracing file, and continue with unfinished traces as shown in Figure 2.</li>
		<li>Export the dendrite length data by clicking, &#34;File | Export as *.cvs&#34; in the &#39;Plugin&#39; window. Then sum up all the path lengths, and export the data to a data analysis/spreadsheet software. (Figure 3).</li>
	</ol>
	</li>
	<li>Surface Area of the da Neurons
	<ol>
		<li>Select the Freehand drawing tool in the &#39;Fiji ImageJ&#39; window. Trace the path, and then connect the endpoints. From the &#39;Analyze&#39; menu, select &#34;Set Measurements | Area.&#34;&#160;Click &#34;measure&#34; from the &#39;Analyze&#39; menu. The result appears in a new box with the value for the area selected (Figure 4). Copy it to the data analysis software.</li>
	</ol>
	</li>
	<li>Total Number of Branches and Branching Structure of the da Neurons
	<ol>
		<li>Calculate the total number of branches by opening &#34;Analysis&#34; and then &#34;Render | Analyze Skeletonized Paths&#34; in the plugin window of tracing (Figure 5). 
		NOTE: The structure of the arbor is the sum of the levels of branches. The path was defined between the cell body and the dendrite tips, and one path can include multiple branches, such as the primary arbors are the dendrites from the neuron cell body. The secondary arbors are branches from the primary and so on with the tertiary, quaternary, and quinary arbors. The structure of dendrite branching is separated depending on the levels of branches. For instance, the primary % is the number of branches divided by total number of branching, and so on.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Dendrites that innervate the epidermis are the input regions of neurons, and their morphologies determine how information is received and processed by individual neurons. Development dendrite morphology reflects gene modulation of dendrite organization. The Drosophila larval da neuron of the peripheral nervous system is an important model for studying dendrite development because of: 1) the functional similarity with mammals11,12; 2) four class distincti...

Dendrites, which are the branched projections of a neuron, cover the field that encompasses the neuron&#39;s sensory and synaptic inputs from other neurons1,2. Dendrites are an important component of synapse formation and play a critical role in integrating synaptic inputs, as well as propagating the electrochemical stimulation in a neuron. Dendritic arborization (da) is a process by which neurons form new dendritic trees and branches to create new synapses. The development and morphology of da, such as branch density and grouping patterns, result from multi-step biological processes and are highly correlated to neuronal function. The goal of this protocol is to provide a method for quantitative analysis of neuronal dendritric arborization complexity in Drosophila. 
The complexity of dendrites determines the synaptic types, connectivity, and inputs from partner neurons. Branching patterns and the density of dendrites are involved in processing the signals that converge onto the dendritic field3,4. Dendrites have the flexibility for adjustment in development. For instance, synaptic signaling has an effect on dendrite organization in the somatosensory neuron during the developmental phase and in the mature nervous system5. The establishment of neuronal connectivity relies on the morphogenesis and maturation of dendrites. Malformation of dendrites is associated with impaired neuronal function. Studies have shown that the abnormality of da neuron morphogenesis might contribute to the etiologies of multiple neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and Lou Gehrig&#39;s disease/Amyotrophic lateral sclerosis (ALS)6,7,8. Synaptic alterations appear in the early stage of AD, in concert with the decline and impairment of neuron function7,8. However, the specifics of how dendrite pathology contributes to pathogenesis in these neurodegenerative diseases remains elusive.
The development of dendrites is regulated by genes that encode a complex network of regulators, such as the Wnt family of proteins9,10, transcription factors, and ligands on cell surface receptors11,12. Drosophila da neurons consist of four classes (Class I, II III, IV), of which class IV da neurons have the most complex branching patterns and have been employed as a powerful experimental system for better understanding morphogenesis13,14. During early morphogenesis, overexpression and/or RNAi silencing of genes in class IV da neurons result in changes in branching patterns and dendrite pruning13. It is important to develop a practical method for quantitative analysis of the neuronal dendritic arborization.
We have previously shown that silencing of the Drosophila ortholog of SOX5, Sox102F, led to shorter dendrites of da neurons and reduced complexity in class IV da neurons15. Here, we present the procedure of quantitative analysis for the neuronal dendritic arborization complexity (NDAC) in Drosophila. This protocol, adapted from the previous described methodology, provides a brief method to assay the development of da sensory neurons. It illustrates the robust image labeling and the da neuron in the third instar larval body wall16,17,18,19. It is a valuable protocol for researchers who wish to investigate the NDAC and developmental differences in vivo.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Phosphate buffered saline(PBS)</td>
			<td style="width:223px;">Gibco Life Sciences</td>
			<td style="width:197px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TritonX-100</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:363px;">Paraformaldehyde(PFA)</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:197px;">15714-S</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:29px;width:363px;">Sylgard 184 silicone elastomer base and curing agent</td>
			<td style="width:223px;">Dow Corning Corportation</td>
			<td style="width:197px;">3097366-0516;3097358-1004</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:363px;">ProLong Gold Antifade Mountant</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:197px;">P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Fingernail polish&#160;</td>
			<td style="width:223px;">CVS</td>
			<td style="width:197px;">72180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Stereo microscope</td>
			<td style="width:223px;">Nikon</td>
			<td style="width:197px;">SMZ800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Confocal microscope</td>
			<td style="width:223px;">Nikon</td>
			<td style="width:197px;">Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Petri dish</td>
			<td style="width:223px;">Falcon</td>
			<td style="width:197px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Forceps</td>
			<td style="width:223px;">Dumont</td>
			<td style="width:197px;">11255-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Scissors&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-5611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Insect Pins&#160;</td>
			<td style="width:223px;">Roboz Surgical Instrument Co</td>
			<td style="width:197px;">RS-6082-25</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:363px;">Microscope slides and cover slips</td>
			<td style="width:223px;">Fisher Scientific</td>
			<td style="width:197px;">15-188-52</td>
			<td></td>
		</tr>
	</tbody>
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quantitative analysis of neuronal dendritic arborization complexity in drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

The dendrites of da neurons were labeled by co-overexpressing GFP (UAS-GFP; ppk-GAL4) in the da neural soma and dendritic arbors for GFP fluorescence imaging analysis. The morphology of da neuron dendrites was imaged by an inverted confocal microscope (Figure 2).
The dendrites of da neurons were traced using Fiji ImageJ software. The file was used to estimate the dendrite length (<strong class="xfig...

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Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

quantitative-analysis-neuronal-dendritic-arborization-complexity

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.0K Views.  Mass General Institute of Neurodegenerative Disease. This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Video: Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

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 ...

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 ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

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 ...

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 ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

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 Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

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Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Analyzing Dendritic Morphology in Columns and Layers

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method