Name: use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain
Uploaded: 2017-10-29T13:00:00
Description: Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

This work was supported by grants from the National Center for Research Resources (2G12RR03060-26A1); The National Institute on Minority Health and Health Disparities (8G12MD007603-27) from the National Institutes of Health; Professional Staff Congress-City University of New York (PSC-CUNY); and Faculty Research Grant (FRG II) American University of Sharjah. We thank Vidyasagar Sriramoju for introducing us to these methods.

The following protocol follows the animal care guidelines established by the Institutional Animal Care and Use Committee (IACUC) at The City College of New York, which conform to all appropriate state and federal guidelines. Anesthesia is appropriate for ferrets, and should be modified according to species studied.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56547/56547fig1.jpg" /> 
Figure 1: Flowchart outlining the major steps invol...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+alteration+of+synaptic+zinc+in+rat+somatosensory+barrel+cortex.">Experience-dependent alteration of synaptic zinc in rat somatosensory barrel cortex.</a> Somatosens Mot Res. 16 (2), 139-150 (1999).</li><li>Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+selenium+in+the+brain:+a+histochemical+technique+for+light+and+electron+microscopic+localization+of+catalytic+selenium+bonds.">Exogenous selenium in the brain: a histochemical technique for light and electron microscopic localization of catalytic selenium bonds.</a> Histochemistry. 76, 281-293 (1982).</li><li>Danscher, G., Howell, G., Perez-Clausell, J., Hertel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dithizone,+Timm's+sulphide+silver+and+the+selenium+methods+demonstrate+a+chelatable+pool+of+zinc+in+CNS:+a+proton+activation+(PIXE)+analysis+of+carbon+tetrachloride+extracts+from+rat+brains+and+spinal+cords+intravitall+treated+with+dithizone.">The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS: a proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitall treated with dithizone.</a> Histochemistry. 83, 419-422 (1985).</li><li>Gallyas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+myelin+by+means+of+physical+development.">Silver staining of myelin by means of physical development.</a> Neurol Res. 1 (2), 203-209 (1979).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Mir&#243;-Berni&#233;, N., Ichinohe, N., Perez-Clausell, J., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-rich+transient+vertical+modules+in+the+rat+retrosplenial+cortex+during+postnatal+development.">Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development.</a> J Neurosci. 138 (2), 523-535 (2006).</li><li>Ichinohe, N., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+macaque+monkey+amygdala.">Distribution of synaptic zinc in the macaque monkey amygdala.</a> J Comp Neurol. 489 (2), 135-147 (2005).</li><li>Innocenti, G. M., Manger, P. R., Masiello, I., Colin, I., Tettoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+callosal+connections+of+visual+areas+17,+18,+19+and+21+in+the+ferret+(Mustela+putorius).">Architecture and callosal connections of visual areas 17, 18, 19 and 21 in the ferret (Mustela putorius).</a> Cereb Cortex. 12 (4), 411-422 (2002).</li><li>Khalil, R., Levitt, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+histochemistry+reveals+circuit+refinement+and+distinguishes+visual+areas+in+the+developing+ferret+cerebral+cortex.">Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.</a> Brain Struct Funct. 218, 1293-1306 (2013).</li><li>Manger, P. R., Masiello, I., Innocenti, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Areal+organization+of+the+posterior+parietal+cortex+of+the+ferret+(Mustela+putorius).">Areal organization of the posterior parietal cortex of the ferret (Mustela putorius).</a> Cereb Cortex. 12, 1280-1297 (2002).</li><li>Wong, P., Kaas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectonic+subdivisions+of+neocortex+in+the+gray+squirrel+(Sciurus+carolinensis.).">Architectonic subdivisions of neocortex in the gray squirrel (Sciurus carolinensis.).</a> The anatomical record. 291, 1301-1333 (2008).</li><li>Land, P. W., Shamalla-Hannah, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+zinc-containing+cortical+circuits+during+a+critical+period+of+postnatal+development.">Experience-dependent plasticity of zinc-containing cortical circuits during a critical period of postnatal development.</a> J Comp Neurol. 447 (1), 43-56 (2002).</li><li>Czupryn, A., Skangiel-Kramska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+developing+mouse+somatosensory+barrel+cortex.">Distribution of synaptic zinc in the developing mouse somatosensory barrel cortex.</a> J Comp Neurol. 386, 652-660 (1997).</li><li>Timm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Histochemie+der+Schwermetalle.+Das+Sulfid-Silber-Verfahren.">Zur Histochemie der Schwermetalle. Das Sulfid-Silber-Verfahren.</a> Dtsch Z ges gerichtl Med. 46, 706-711 (1958).</li></ol>

The current study employs a histochemical technique based on a modified version of the Danscher (1982) method10, whereby synaptic zinc localization may be detected and visualized in the brain. This method essentially works by injecting the animal with the zinc chelator sodium selenite (Na2SeO3) (15 mg/kg). Following injection, the selenite travels to the brain and binds to free zinc that is localized to presynaptic vesicles of zinc containing neurons. Zinc ions bound to molec...

Histological stains have traditionally been used to aid in the identification of cortical areas in various species by revealing differences in architectonic features. The combined use of histochemical techniques such as for Nissl substance, cytochrome oxidase (CO) reactivity, or myelin can prove fruitful as they reveal similar areal boundaries in the adult brain. However, these histochemical stains do not always adequately reveal clear boundaries between cortical areas and layers in the immature brain.
In the central nervous system, zinc has several critical functions that include stabilizing DNA structure, acting as an enzyme cofactor, participating in numerous regulatory functions, and acting as a neuromodulator through its presence in synaptic vesicles1. Synaptic zinc is unique as it can be visualized using histological methods, whereas protein-bound zinc cannot be visualized2. This feature has been exploited to reveal the synaptic zinc pattern in different cortical regions, and synaptic zinc histochemistry has been used in a number of studies. A subset of glutamatergic neurons in the cerebral cortex contain zinc in the presynaptic vesicles within their axon terminals3,4. Histochemical studies have revealed a heterogeneous distribution of synaptic zinc in the cerebral cortex5,6,7. There appears to be a different areal and laminar distribution of histochemically reactive zinc in different cortical regions (e.g., visual versus somatosensory cortex), or layers (e.g., zinc levels in the supragranular and infragranular layers of primary visual cortex are substantially higher than in thalamocortical input layer IV with relatively low synaptic zinc levels)5,8,9. The heterogeneity in synaptic zinc staining observed in the cortex is especially advantageous as it facilitates areal and laminar identification.
Here we present a detailed description of a synaptic zinc histochemical procedure, which is a modified version of Danscher&#39;s 1982 method10. This method utilizes selenite injected intraperitoneally (IP) into animals as a chelating agent. The selenite travels to the brain to react with pools of free zinc found in vesicles of a subset of glutamatergic synapses in the brain. This reaction yields a precipitate that can be enhanced subsequently by silver development2,10,11.
This procedure reveals laminar and areal patterns of synaptic zinc staining; densitometric analysis may be used to assess these patterns both qualitatively and quantitatively in the adult and immature brain to study effects of other interventions, such as sensory, environmental, pharmacological, or genetic manipulations. Moreover, one may also want to assess potential developmental changes in the distribution of synaptic zinc in other cortical or subcortical structures in other model systems. The quantitative information that densitometric analysis provides in this method can be advantageous for following brain development over time. This protocol provides a companion to other immuno- and histochemical markers to reveal laminar and areal boundaries.

<table>
	<tbody>
		<tr>
			<td>Euthasol (Euthanasia solution)</td>
			<td>Henry Schein</td>
			<td>710101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma-Aldrich</td>
			<td>214485</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketaved)</td>
			<td>Henry Schein</td>
			<td>48858</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Xylazine (Anased)</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma-Aldrich</td>
			<td>G9752-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>W302600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma-Aldrich</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>Sigma-Aldrich</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td>(Type III, from equine heart)</td>
		</tr>
		<tr>
			<td>Catalse</td>
			<td>Sigma-Aldrich</td>
			<td>C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Domino</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used for dehydration prior to slide mounting</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Brain Research Laboratories</td>
			<td>#3660-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted unsubbed slides</td>
			<td>Brain Research Laboratories</td>
			<td>#3875-FR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>American Optical Company</td>
			<td>860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Adope Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
			<td></td>
			<td>For densometric measurements</td>
		</tr>
		<tr>
			<td>Plastic tray</td>
			<td>Any standard plastic tray may be used</td>
			<td></td>
			<td>to immerse slides in developer solution</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Any standard hotplate may be used</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

The major steps involved in this protocol to stain brain sections for synaptic zinc are presented in a flowchart in Figure 1. The protocol can be divided into three phases: 1) Perfusion and tissue collection, 2) Tissue preparation and staining, and 3) Zinc histochemistry. Briefly, during the first phase of the protocol, the animal is anesthetized and injected IP with the appropriate dose of sodium selenite. After a sufficient time period (ideally 60 - 90 min)...

Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

The overall goal of this method is perform histochemical staining on ferret frozen brain sections to illustrate utilization of synaptic Zinc staining patterns in distinguishing different regions and layers in the developing and adult brain. This method can help answer key questions such as the qualitative and quantitative differences in synaptic Zinc staining pattern among different brain regions and layers. The main advantage is that this technique is simple and reliable allowing one to identify different regions and layers in the developing and adult brain based on unique staining patterns.Although this method has been utilized to provide insight in demarcating areas and layers in the feral visual cortex it can also be used to differentiate layers in different brain regions of rodents, cats, or primates. To prepare 200 milliliters of the developer's solution, add 40 grams of gum arabic slowly to 120 milliliters of hot water and keep stirring it with a glass rod to dissolve completely. Then it let it cool for few minutes, then filter it through six to eight layers of gauze cloth in a funnel.Next add 5.04 grams citric acid and 4.7 grams sodium citrate in 20 milliliters of distilled water and dissolve to make the citrate buffer. Then add 1.7 grams hydroquinone to 30 milliliters of heated distilled water. Add 0.22 grams silver lactate to 30 milliliters of water to make silver lactate solution.In the following order, mix the gum arabic, citrate buffer, hydroquinone, and finally, silver lactate solutions to prepare the developer's solution and keep it in the dark. To start this procedure, inject a dose of 15 milligrams per kilogram body weight of sodium selenite solution, a Zinc chelator, intraperitoneally into the anesthetized ferret. Then wait for 60 to 90 minutes for the sodium selenite solution to reach the brain.Then inject an overdose of 100 milligrams per kilogram phenobarbital intraperitoneally to euthanize the animal and wait for several minutes for euthasol to work. Perform transcardial perfusion on the euthanized ferret first with normal saline solution for one minute, and then with 4%paraformaldehyde for 20 minutes. Then use a fixative with sucrose to complete the total fixation procedure in one hour.Use a scalpel to make a mid-line incision starting from the nose to neck to expose the skull. Delicately remove the brain and then use a scalpel blade to separate the hemispheres. Then cut and separate the posterior part from the rest of the brain and post-fix for two to four hours.Let the brain sink by keeping the tissue in 30%sucrose solution in 0.1 molar phosphate buffer. After the brain sinks, use a freezing sliding microtome to make semi-tangential 40 micrometer thick sections through the visual cortex. Use a paintbrush to collect all the brain sections and place them in a tackle box filled with phosphate buffered saline.Then, quickly mount one or two series of brain sections on egg-white coated slides. Keep the slides at room temperature overnight to dry. After the slides have dried, immerse them in absolute alcohol for 15 minutes.Then wait an hour to let the slides dry completely at room temperature. Quickly submerge the sections in 1%gelatin solution for 10 seconds then drain carefully. Keep the sections at room temperature to dry overnight.Use a plastic tray to arrange the slides side by side. Then pour over the developing solution and make sure that the slides are completely submerged in it. Remember to check the sections every 30 minutes for the development of the reaction.If the slides have been stained appropriately, remove the slide-mounted sections from the tray and keep the slides on a slide rack. Use a large glass-staining dish to wash the slides inside the slide rack under warm running water for 10 minutes to wash the gelatin coat and the outer silver deposit. After drying the slides at room temperature, dehydrate them for five minutes in 100%ethanol and clear for five minutes in xylene.Finally, place a cover slip with mounting media on the slide. Use a bright-field microscope to examine the sections and take pictures of the areas of interest. Select Zinc stained sections from photomicrographs of regions of interest to randomly choose cortical columns of approximately 450 micrometer width.In each region of interest, choose an appropriate number of sample columns from several different brain sections. Then transfer the column images to an image processing software. In the software, use the rectangular section tool to encompass the entire cortical column.Next utilize the invert tool to contrast reverse the image. Then use the plot profile tool to make a two-dimensional graph of the pixel intensities along the line to produce optical density profiles from the images. Finally, use the plot profile options to convert the plot profile graph to a vertical profile.Then click on plot profile once more. Photomicrographs of semi-tangential Zinc stained sections of juvenile ferret brain show the characterization of over-stained and under-stained tissues. Over-stained tissue develops very dark brown color with inadequate laminar variation and densely stained white matter.In contrast, under-stained tissues develop light-brown color with minimally stained white matter. Photomicrographs of semi-tangential sections show synaptic Zinc in cytochrome oxidase staining in visual cortical areas of an adult ferret. Synaptic Zinc staining intensity is high in the supragranular and infragranular layers of areas 17 and 18, while layer four stains lightly.Conversely, layer four, of area of 17 and 18, is heavily stained when stained for cytochrome oxidase reactivity. Columnar photomicrographs show laminar distribution of synaptic Zinc in all the cortical layers with their complementary normalized optical density values. To reflect changes in synaptic Zinc staining according to cortical depth, normalized plots of optical density values were generated at each visual cortical area.The trough present in plot profiles shows low synaptic Zinc staining in layer four of areas 17 and 18. This procedure can help explain if there are any developmental changes and staining intensity in particular brain regions over time. This technique proved useful for neuroanatomy researchers to explore how sensory, environmental, pharmacological, or genetic manipulations may affect the distribution of synaptic Zinc staining in the adult and the immature brain in a range of species.Thanks for watching. Good luck with your experiments.

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Research

JoVE Journal

Neuroscience

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

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 ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.