The authors would like to thank the MRC-LMB workshop for the production of the enhanced gene gun barrel. We would also like to thank Dr. David R. Hampson at the University of Toronto for the use of equipment and resources.

The use of animals and animal tissues should strictly adhere to ethical committee approval under local rules and regulation. All tissues obtained during this study adhered to the MRC-LMB&#39;s animal experimentation guidelines.
1. Preparation of Materials &#38; Culture Media
<ol>
	<li>Prepare all solutions using Millipore water; use only analytical grade reagents. Prepare and store all reagents at RT (unless indicated otherwise).</li>
	<li>Make 500 ml of HEPES-buffered saline (10 mM HEPES, 120 mM NaCl pH 7.2). Filter through a 0.2 &#181;m sterile filter unit. Store at 4 &#176;C.</li>
	<li>Make 500 ml of Phosphate-buffered saline (PBS; 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Sterilize by autoclaving.</li>
	<li>Prepare neuronal medium by supplementing DMEM (Dulbecco&#8217;s modified eagle&#8217;s medium) with 25 mM HEPES, 10% Fetal calf serum, 30 mM glucose, 1:100 N2 supplement, penicillin-streptomycin 1,100 U/ml; pH 7.2. Filter with a 0.2 &#181;m sterile filter unit. Store at 4 &#176;C.</li>
	<li>Make polyvinylpyrrolidone (PVP) stock solution with 20 mg PVP in 1 ml 100% Ethanol. Aliquot this into 1 ml amounts, freeze and store at -20 &#176;C. Working solution is 0.05 mg/ml, therefore add 10 &#181;l of PVP stock solution to every 4 ml of 100% ethanol.</li>
	<li>Prepare a 0.05 M spermidine stock solution in Millipore water; adjust the pH to 7.2.</li>
	<li>Make a 2% agarose solution in DMEM (2 g electrophoresis grade agarose in 100 ml DMEM in a sterilized screw cap bottle). Microwave this until the agarose has dissolved. Store in the 4 &#176;C fridge for several weeks, if necessary.</li>
</ol>
2. Preparation of Organotypic Slices
<ol>
	<li>Euthanize C57 Black 6 mice of the desired age by CO2 asphyxiation followed by decapitation. Remove the brain using lateral skull cuts starting at the foramen magnum and ending at the olfactory lobes. Gently lift the skull from the rear to expose the brain.</li>
	<li>Cut between the olfactory lobes and the frontal cortex, and rostral to the cerebellum. Gently lift the rostral part of the brain and cut the optic nerves. Finally, remove the brain and place it in a petri dish filled with ice cold HEPES-buffered saline on ice.</li>
	<li>Using a dissecting microscope, peel away the pia using fine forceps; carefully remove blood vessels and meninges around the brain.</li>
	<li>Place the fresh brain into a small recipient mold and cover it with the agarose solution cooled to slightly above RT. Then rapidly cool the mold to 4 &#176;C by placing it on ice. 
	NOTE: Process the slices as rapidly as possible to avoid loss of viability.</li>
	<li>When the agarose has set (5 - 10 min), remove the agarose-embedded brain from the mold (trim if necessary), and then glue it to the vibroslicer platform. Place this into a vibroslicer chamber containing sterile ice cold PBS. Disinfect the vibroslicer chamber before being used with 70% ethanol to minimize subsequent contaminations.</li>
	<li>Cut the brain using a custom built vibroslicer or commercial equivalent. The model concept was to design a tissue slicer that could generate large amplitude and high frequency movements horizontal to the edge of the blade while minimizing vertical vibrations.</li>
	<li>Use the oscillation frequency of 90 Hz, at an amplitude of 1.5 - 2.0 mm, and the cutting blade positioned at an angle of 15&#176; to the horizontal plane; set the mounting block holding the agarose-embedded brain to move at 1.7 mm/min towards the blade. Collect sections (150 &#181;m) into a chamber containing ice cold culture media (DMEM Pen/Strep + 10% FCS). View cutting and manipulation of the tissue slices as a supplementary video in Arsenault and O&#39;Brien13.</li>
	<li>Gently place the slices into cell culture inserts (0.4 &#181;m, 30 mm diameter) in a 6 multi-well tray with the culture media on the outside of the insert, and incubate in a humidified incubator at 37 &#176;C with 5% CO2. 
	NOTE: For proper tissue viability, be extremely gentle while transferring and manipulating the slices. Also, too much fluid will prevent the organotypic slices from adhering to the membrane of the filter insert. If this happens, remove the excess fluid or plate again.</li>
	<li>Although organotypic slices can be cultured for a few weeks, proceed to transfection no later than 4 days after plating for best transfection yields. 
	NOTE: To avoid glial overgrowth in long term culture conditions, use AraC or serum free medium2.</li>
</ol>
3. Preparation of DNA-coated Micro &#38; Nano-projectiles
<ol>
	<li>Prepare nano-projectiles using 40 nm diameter gold particles. Briefly, add 50 &#181;l of 0.05 M spermidine and 10 &#181;l DNA at 1 mg/ml (pEYFP-N1) to 10 mg of gold particles.
	<ol>
		<li>Add calcium and spermidine in the solution mixture during DNA-gold micro particles preparation in order to aid in the binding of DNA molecules to the gold nanoparticles. 
		NOTE: The amount of DNA used per mg of gold carriers (DLR), should range between 1 and 5 &#181;g DNA per 1 mg of gold. Furthermore, the amount of gold carrier particles that will be shot per cartridge (MLQ), should itself range from 0.1 to 0.5 mg of gold. The DLR and MLQ should be tried and optimized for each user depending on the particular system under investigation as well as the type of particle and gene gun used.</li>
	</ol>
	</li>
	<li>Mix using a vortex while slowly adding 50 &#181;l of 1 M CaCl2 in fractions of 10 - 15 &#181;l. Following 5 min of occasional vortexing, centrifuge at 1,000 x g for 30 sec then remove the supernatant. Resuspend the gold particle pellet in 3.5 ml of 0.075 M PVP.</li>
	<li>Draw this suspension into tubing (2.36 mm internal diameter) using a syringe. Place the tubing in the tubing preparation station. Allow the gold particles to settle and remove the supernatant by aspiration with a syringe. Rotate the tubing to evenly spread the gold particles, which were subsequently dried under a constant nitrogen flow at 5 L per min.</li>
	<li>To create DNA-bullets, cut the tubing using a tubing cutter into 1 cm lengths. Either insert immediately into the gene gun cartridge or keep desiccated (4 &#176;C) until required.</li>
</ol>
4. Biolistic Transfection on Organotypic Slices
<ol>
	<li>Perform the following steps within a sterile laminar flow hood.</li>
	<li>Insert a 9 V battery and an empty cartridge holder into the Helios gene gun.</li>
	<li>Attach the gene gun to the helium tank with the helium hose. Fire 2 - 3 blanks (empty slots) at 50 psi to pressurize the helium hose and the reservoirs in the gun.</li>
	<li>Load the cartridges containing the DNA-coated gold into the cartridge holders and load the holder into the gene gun.</li>
	<li>Unscrew the gene gun spacer and attach the modified gene gun barrel to the gene gun.</li>
	<li>Using sterile forceps, place a filter insert containing the organotypic slice into a sterile plastic dish.</li>
	<li>Remove media from organotypic slices.</li>
	<li>Set the gas pressure at 50 psi. 
	NOTE: Eye protection is essential and ear protection advisable.</li>
	<li>Place the gene gun at the appropriate distances by using the mounting measured to 10 mm. Carefully aim the centre of the barrel over the desired region for genetic delivery.</li>
	<li>After firing, replace the inserts back into their dish with fresh media and return them to normal growth conditions, i.e., 37 &#176;C with 5% CO2 incubator.</li>
	<li>Once finished, close the helium tank, release the pressure from the gene gun and detach the gene gun from the helium tank.</li>
	<li>Remove the cartridge holder and discard the cartridges and clean the gene gun .</li>
	<li>Check the tissue for morphology and for successful penetration by visualizing the slices using an inverted microscope. If using microparticles, evenly disperse the gold; there should be no dense areas of gold particles seen on the slice. Nanoparticles due to their small size can&#39;t be seen as single units by conventional microscopy techniques.</li>
	<li>After the appropriate time interval (usually 1 - 2 days), fix the slices in 4% paraformaldehyde (PFA) for microscopy observation. 
	NOTE: Fixing in PFA is not always desired. For live cell imaging avoid this step.</li>
</ol>
5. Fixing and Visualization of Brain Slices
<ol>
	<li>After biolistic transfection, wash slices twice in PBS for 2 min.</li>
	<li>Fix slices by incubating in freshly made, ice cold 4% PFA in PBS for 20 min.</li>
	<li>Wash slices twice in PBS for 2 min.</li>
	<li>Gently cut round the membrane supports using a scalpel, without disturbing the slices mounted on them. Use forceps to place each membrane support/slice on a microscope slide. Rest the organotypic slices on top of the membrane on the glass slide.</li>
	<li>Add one drop of mounting media on top of the slice and add a coverslip gently without any force directly in contact with the organotypic slice.</li>
	<li>Secure the coverslip with a thin layer of nail varnish.</li>
	<li>View the slices on an appropriate microscope.</li>
</ol>
6. Confocal Applications
<ol>
	<li>Visualize the slices by using an upright scanning laser confocal microscope with a 60X/1.4 numerical aperture (NA) oil immersion objective lens. 
	NOTE: The most problematic feature of the confocal microscope is the pinhole size (set at 1 AU) which is capable of isolating and collecting a plane of focus, thus eliminating the out of focus &#39;haze&#39; normally seen with a fluorescent sample. Fine details are often obscured by this haze and cannot be detected in a nonconfocal microscope.</li>
	<li>Set the Argon laser to an excitation wavelength of 488 nm and optimize for collection of emitted light in the 500 to 520 nm band. Typically, collect images at 1,024 x 1,024 pixels and with stacks of z-sections at 0.5 &#956;m intervals to encompass the full width of each nerve cell that was analyzed.
	<ol>
		<li>To observe the morphology of the nucleus, counter stain these cells with DAPI (excitation wavelength set at 405 nm and emission light collected between 420 - 4 40 nm). Finally, for examining red fluorescence, set the laser at 561 nm for excitation and 565 - 620 nm for emission.</li>
	</ol>
	</li>
	<li>Typically, acquire images by using a 4X scan zoom that covered an area of 57.6 &#956;m2; image sizes were in the region of 1,024 x 1,024 pixels. Record stacks of z-sections in 0.5 &#956;m intervals and projections of 5 - 8 frames combined.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+the+central+nervous+system+after+intracerebroventricular+injection+of+adeno-associated+viral+vectors+in+neonatal+and+juvenile+mice.">Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice.</a> Human Gene Therapy Methods. 24, 205-213 (2013).</li><li>Arsenault, J., 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=Botulinum+protease-cleaved+SNARE+fragments+induce+cytotoxicity+in+neuroblastoma+cells.">Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.</a> Journal of Neurochemistry. 129, 781-791 (2014).</li><li>Arsenault, J., 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=Stapling+of+the+botulinum+type+A+protease+to+growth+factors+and+neuropeptides+allows+selective+targeting+of+neuroendocrine+cells.">Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells.</a> Journal of Neurochemistry. 126, 223-233 (2013).</li></ol>

Dr. John A. O'Brien holds patents for the improved gene gun barrel. US patent number 10/380,452 and European patent Number 01974517.3.

The protocol describes approaches to deliver dyes and genetic materials into adult organotypic slices by using an enhanced gene gun. Essentially, the types of dyes and the variety of possible genes make this method multifaceted and amenable to answer a wide range of biological questions. The regioselective delivery method used, in this case to specific brain region, opens new avenues for experimentation as heterologous gene modulation of localized areas within a biologically relevant architecture hasn&#39;t been easily feasible before. We have previously determined that this method can be used on adult organotypic slices, where mature synapses are fully formed, as it showed improved cell survival and heterologous gene expression for many weeks13. With the advent of new and improved culture conditions for mammalian brains as well as other tissues, the use of regioselective genetic modulations will become increasingly useful for a wide variety of biochemical analyses.
As we shall highlight here and previously mentioned by others24, many different factors could readily affect the accuracy and reliability of biolistic delivery. Firstly, the preparation of the gold particle linked to dyes or DNA must be adequately balanced to prevent aggregation and to facilitate the cartridge cargo expulsion. Calcium and spermidine in the DNA-gold micro particles solution can aid the binding of DNA molecules to the gold nanoparticles. The ability for the nanoparticles to penetrate the cells under reduced pressure was previously determined17. Secondly, the gene gun must be mounted as steady as possible with the correct angle and distance. A reliable pressure gauge is also necessary for reproducibility, to maintain tissue integrity, and to properly accelerate the gold particles to their intended targets. Indeed, the distance, orientation, and stability of the equipment are also a crucial factors for the targeting and precision of regioselective delivery. As these brain structures are very small, minor variations in the firing angle could easily render the entire procedure less reliable. And finally, the preparation and maintenance of viable organotypic slices have been fraught with difficulties for many decades yet abundant and reliable protocols for different animals, brain regions, ages and cocultures are currently available2,4,25,26. These procedures should be user-optimized before submitting the tissues to biolistic procedures. This will more readily permit the user to unbiasedly ascertain the impact of the biolistic particles and the effect of the heterologous gene on their own cultures. To this end, the use of fluorescent genes such as EYFP and EGFP can both permit a novel user to test the targeting precision and also follow the cellular viability through heterologous gene expression over a sustained period of time13. Many of the critical steps for reliable and reproducible use of this protocol are highlighted in the following three paragraphs.
For efficient transfection, the DNA concentration must be appropriate. Concentrations that are too low would hinder the transfection yield while concentrations that are too high can cause agglomeration of the nanoparticles. Aggregates of gold can dramatically reduce transfection efficiency, cause uneven distributions, and can lead to increased cytotoxicity and oxidative stress. Problems with coating efficiency are likely due to the expiration of the spermidine solution (should be replaced every 2 to 3 months). For an adequate biolistic spread, the labeling must be even. A non-homogeneous labeling of the gold nanoparticle/DNA suspension may be due to the PVP solution. It should also be replaced every 2 to 3 months. Coating of the gold nanoparticles can be seen on agarose gel electrophoresis as a higher MW band27.
Each biological system under investigation as well as each different instrument used might require small changes in gas pressure to reach optimized levels of transfection and biolistic spread. The critical parameter is the pressure of the helium pulse required to strip the micro- or nano-carriers from the plastic cartridge and propel them into the tissue. A high gas pressure will affect cell survival, because it will create shock waves across the target and disrupt or detach the tissue from the matrix17. Aiming the gene gun barrel and having the apparatus exactly perpendicular to the tissue is important for accurate biolistic delivery. The selected area should be placed in the direct center of the barrel at precisely 10 mm from the outer aperture. This results in a 3 mm diameter of gold particle delivery around the epicenter. The gene gun barrel should be cleaned with 70% ethanol after each use. To protect the tissue from large particle aggregates, the barrel also contains a nylon mesh that should be replaced regularly to maintain optimum performance. In order to replace the mesh unscrew the cap then insert a new nylon mesh between the cap and the O-ring.
Fluorescently labeled cells should be visible at least 1 - 2 days after transfection around the epicenter. Absence or misalignment of labeling can result from inadequate preparation and instability of the gene gun mounting. The observations of cells using confocal microscopy depends on a number of factors: the wavelength of the excitation/emission light, pinhole size, NA of the objective lens, refractive index of components in the light path, depth of the tissue, and the alignment of the instrument. These factors should be optimized for each system under investigation.
Recent advances in biolistics were exploited for the success of this method. The gene gun barrel used in this study was modified in order to reduce the pressure necessary as well as funnel the biolistic scatter pattern into a more constrained and specific area17,19. Other barrel modifications have tried to restrict the biolistic spread and reduce the outflow pressure28 yet this custom designed gene gun barrel designed by Dr. O&#39;Brien is a single component fitted to the standard Helios Gene gun. Subsequently, sub-micrometer gold particles were used to reduce the invasiveness of micrometer particles that we had previously determined caused cellular damage and ultimately, loss of sustained heterologous gene expression. These changes to the biolistic procedure improved the overall feasibility and reproducibility of the method13. Other improvements on slice preparation such as in-depth troubleshooting of the slicing procedure, using a DMEM-Agarose matrix to preserve the brain, and numerous other useful advances in organotypic brain slice preparation previously reported13 enabled us to explore the use of adult slices that have developed mature synaptic networks.
In our study we mainly used dyes and fluorescent heterologous genes to visualize cell morphology as a readout on transfection efficiency and an ability to image the morphological characteristics of neurons in mouse brain tissues. The signal to noise ratio of the stochastic delivery within a defined radius enabled us to completely isolate a single cell&#39;s dentritic harbor within its native context. As numerous efforts are used to investigate these morphological characteristics, mapping &#39;connectomics&#39; or to label single cells for various analyses, this method could easily be combined with existing protocols such as electrophysiology and neurite measurements29,30. Evidently, combinations of fluorescent heterologous genes could enable a much more robust delineation of single cells as the stochastic distribution of fluorescent proteins, and their combination would determine the color of the individual cells31,32. The use of cell specific promoters such as synapsin and glial fibrillary acid protein upstream of the heterologous gene exon can also restrict the expression to subpopulations33. Indeed, the unending variety of genetic material could also be delivered in a regioselective manner by gold nanoparticles using this same procedure. The total transfected regions could thus be analyzed with traditional methods, such as dissection and submitting that region to western immunoblotting to analyze the localized effect of the heterologous gene.
Improvements in organotypic slice procedures will also permit wider use of brain tissues for long-term experimentation as well as permit the use of other tissues and cocultures and would facilitate transfection procedures. Lipid and polymer transfection have been previously reported to affect membrane homeostasis, internalization, protein permeability34 and frequently show very low efficiency to transfect terminally differentiated neurons. Cell specific cargo targeting is also amenable only to cells that express the cell surface receptors needed for internalization, and although this method can be highly selective, it can lack targeted regioselectivity35. Viral vectors otherwise are often difficult and costly to produce, require stringent regulations and have on occasion demonstrated safety concerns. Biolistic delivery thus has an unparalleled ability to regioselectively transfect neurons and other cell types within viable tissues. As gold is non toxic, further advances in the use of biolistics could pave the way for biomedical uses such as transdermal pharmaceutical delivery, noninvasive in vivo gene delivery, as well as localized nonviral gene therapies.

Originally the biolistic technique, a turn-of-phrase for &#8220;biological ballistics&#8221;, was established for particle-mediated gene transfer into plant cells1. This physical method of cell transformation accelerates micro- or nanoparticles at high velocity to overcome the physical barriers of the impermeable cell membranes in order to deliver cargos such as DNA or dyes. Because it does not depend on specific ligand-receptors and/or the biochemical properties at the cell surface membranes, particle-mediated gene transfer can be readily applied to a variety of biological systems such as organotypic brain slices.
Using organotypic slices have advantages over other in vitro platforms since they maintain many anatomical and biochemical properties that are pertinent to in vivo biology2-4. The slices mostly conserve the local architectural characteristics from where they have originated and preserve neurochemical activity and connectivity of the synapses. The use of brain slices for basic research, and in pharmaceutical endeavors, has concomitantly increased with the number of possible biotechnological manipulations to measure and monitor the neurobiological behaviors of the brain in an in vivo like context3,5-7. The major advantages for using organotypic slice-based assay systems is that it provides easy experimental control and allows precise manipulations of extracellular environments.
Fruitfully, organotypic slice culture systems have been established from a variety of brain regions such as, but not restricted to, the cortex, spinal cord, and cerebellum8-10. Furthermore, a number of cocultures have been demonstrated, which allow the assessment of intercellular communication across distal brain regions as well as between neurons and pathological cells11,12. Many protocols have already been established to successfully culture organotypic slices and can maintain long-term viability and many recent studies now utilize the membrane interface methods and various modifications to it13. This principle maintains the organotypic slices at the interface between the medium and the incubator&#39;s humidified atmosphere by placing the slices on a porous membrane filter. The medium can thus provide sufficient nutrients to feed the organotypic slices via capillary motion. Typically slices have been prepared from early postnatal animals (3 - 9 days old; P3 - 9). However, brain tissues from these slices display a high level of cellular plasticity and have an inherent resistance to mechanical stresses, which is helpful to obtain viable cultures, yet mature synapses and neuroanatomical circuitry have not fully developed in vivo until 2 to 3 weeks of age14. For example, previous observations had shown that hippocampal slices obtained from P0 - 1 neonates, although highly viable following preparation, gradually lost some morphological characteristics. Essentially, they were shown to be unsuitable for long-term cultures suggesting immature cells were more likely to de-differentiate compared to organotypic cultures from older animals15,16. For this reason our method has been optimized for adult organotypic brain slices at which maturation and architectural development have reached their terminal stages13,17-21. Nevertheless, this method is also suitable for neonate and juvenile organotypic slices.
Once the viable organotypic slices have been produced the entire plate containing the slices can be brought to the biolistic mount and submitted to regioselective delivery and transfection. Proper mounting of the gene gun (as described in Figure 1), oriented 90&#176; at a distance of 10 mm directly over the slices (from the aperture to the tissue), permits the rapid biolistic delivery of the 40 nm gold particle coated cargos. These cargos such as dyes and fluorescent DNA vectors, as well as any gene of interest, are readily delivered into the viable slices with ease. This method thus describes the protocol necessary to deliver, in a regioselective manner, cargo coated gold nanoparticles into viable organotypic brain slices. The gene gun barrel and optimal mounting can be seen in Figure 1. For further information regarding the improved barrel and nanoparticle ballistics, please see O&#39;Brien, et al.17.
Subsequent refinements are also indicated for the user to optimize conditions such as the proper amount of gold delivered per target area, defined as Microcarrier Loading Quantity (MLQ), and to determine the amount of DNA loaded per mg of gold, defined as DNA Loading Ratio (DLR). Prior to precipitating DNA onto gold particles and loading them into the Tefzel tubing that comprises the actual gene gun &#39;cartridges&#39;, it is necessary to calculate the amount of DNA and gold required for each transfection which can differ slightly among tissues and conditions (ratio should be maintained between 1 mg gold and 1 &#181;g DNA to 1:5). It is vital to prepare the proper proportion of MLQ to DLR as otherwise DNA coated gold particles could adhere together forming larger than expected agglomeration, which could reduce the overall homogeneity of the biolistic spread as well as increase tissue damage and cytotoxicity.
This method shows the recent improvements in biolistic delivery, which have been tested and determined to be amenable for neonatal, juvenile and adult organotypic brain slices. Furthermore, by exploiting the gene gun barrel&#39;s reduced biolistic spread, the user is now able to selectively transfect a punctual region in the brain. Following the appropriate incubation time, the expression of the fluorescent proteins, which reaches its maximum rates between 24 and 48 hr, can be visualized by epifluorescence and confocal microscopy. These fluorophores permit the morphological analysis and localization of individual cells within biologically relevant structures. However, the regioselective modulation of the organotypic slices by other heterologous genes can also be monitored by any other tests amenable to these preparations. Possible working strategies for the localized gene delivery are presented in Figure 2.

<table border="0" cellpadding="0" cellspacing="0" width="656">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:173px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Helios gene gun system</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2431</td>
			<td>Gene gun and tube prep station</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HEPES&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>83264-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:141px;">Sigma</td>
			<td>S7653-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">KCl</td>
			<td style="width:141px;">Sigma</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">Na2HPO4</td>
			<td style="width:141px;">Sigma</td>
			<td>S7907-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:141px;">Sigma</td>
			<td>P9791-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2 &#181;m sterile filter unit</td>
			<td style="width:141px;">Millipore</td>
			<td>SCGPU05RE</td>
			<td>to filter media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:141px;">Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal calf serum</td>
			<td style="width:141px;">Gibco</td>
			<td>10270-098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">D-Glucose</td>
			<td style="width:141px;">Sigma</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N2</td>
			<td style="width:141px;">Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Penicillin/Streptomycin</td>
			<td style="width:141px;">Sigma</td>
			<td>p4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:141px;">Sigma</td>
			<td>856568-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Ethanol&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>277649-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Spermidine</td>
			<td style="width:141px;">Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Agarose</td>
			<td style="width:141px;">Bio Rad</td>
			<td>161-3100EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vibroslicer</td>
			<td style="width:141px;">Laica</td>
			<td style="width:119px;">VT1200</td>
			<td>We used a custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gene gun mount</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Built in house at U of T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Humidified cell culture incubator</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gold nanoparticles</td>
			<td style="width:141px;">cytodiagnostics</td>
			<td style="width:119px;">G-40-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pEYFP-N1</td>
			<td style="width:141px;">Clontech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing cutter</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tefzel tubing</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Barrel</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made by the LMB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture insert</td>
			<td style="width:141px;">Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:141px;">Sigma</td>
			<td>76240 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>for convenience</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Confocal microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>user defined for usage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glass slide</td>
			<td style="width:141px;">VWR</td>
			<td>2588-CA48323-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microcover glass</td>
			<td style="width:141px;">VWR</td>
			<td colspan="2">2448-CA48366-067-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vectashield mounting media</td>
			<td style="width:141px;">Vector laboratories</td>
			<td>H-1400&#160;&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

regioselective biolistic targeting in organotypic brain slices using a modified gene gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Organotypic slice viability can be monitored with lactate dehydrogenase activity, propidium iodide labeling, and dUTP staining as was previously reported13,22. Evidently, the viability and integrity of the slices are important for the long-term sustained expression of the delivered genetic cargo. Following biolistic delivery into the organotypic slices, the expression of the fluorescent heterologous gene was monitored by confocal microscopy. The tightness of the biolistic spread using the modified barrel can be seen in Figure 3A. Figure 3B shows a representative image of the transfected hippocampus region using DNA coated gold nanoparticles. Figure 4 shows representative pictures of transfected adult mouse organotypic slices. As can be seen in Figure 4A, a Purkinje neuron with a remarkable dendritic harbor found at the interface of the Purkinje layer and the molecular layer of the cerebellum shows pronounced labeling following transient biolistic transfection with pEGFP-N1. Figure 4B shows a higher magnification of these dendrites where spines can be observed. Figure 4C shows a pyramidal cell found in the CA1 region of the hippocampus following biolistic delivery of pDSredFP coated gold nanoparticles.
Dyes instead of fluorescent encoded DNA cargos can also be used in a similar manner to visualize morphological characteristics in organotypic slices. DiO, which labels the plasma membranes, can more rapidly delineate individual cell morphologies or can be used in conjunction with a DNA coated biolistic delivered to the same region to confirm the regioselection. As can be seen in Figure 5A a neuron stained with DiO in the hippocampus also shows long dendrites with numerous spines. An arrow indicates the axon which is identified by the presence of synaptic boutons in comparison to the dentritic spines on the other neurites. Figure 5B shows another example of the CA1, which highlights numerous hippocampal projections with a DAPI counter stain to label the nucleus. Figure 5C shows a higher magnification of these types of parallel dendrites with numerous spines.
Evidently, these images illustrate morphological characteristics following cellular labeling. Nothing precludes the same methodology from being applied to deliver any number of exogenous genes coated onto the gold nanoparticles. To permit a visual confirmation of successful transfection, a single plasmid can be constructed to co-express both the gene of interest and a fluorescent reporter protein on the same vector.
<img alt="Figure 1" src="/files/ftp_upload/52148/52148fig1highres.jpg" /> 
Figure 1.&#160;Gene gun barrel and mounting.&#160;(A) The improved barrel developed by Dr. O&#39;Brien at the MRC-LMB that has a restricted biolistic spread that minimizes tissue damage by lowering the required pressure for particle acceleration. (B) The side view of an optimized gene gun mounting developed by Dr. Henderson and Dr. Andras Nagy at the University of Toronto. The mounting holds the gene gun at precisely 90&#730; perpendicular to the organotypic slices while permitting the user to accurately control the distance of biolistic delivery. For optimal biolistic conditions the barrel aperture should be placed directly over the region of interest at a 10 mm distance from the organotypic slices. (C) Back view of the gene gun mounting. The region of interest to be transfected needs to be placed directly underneath the barrel aperture. Targeting can be confirmed with dye (diolistic) or fluorescent protein tranfection (biolistic). <a href="https://www.jove.com/files/ftp_upload/52148/52148fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52148/52148fig2highres.jpg" /> 
Figure 2.&#160;Organotypic slice preparation and regioselective targeting.&#160;(A) A DMEM embedded adult mouse brain ready for slicing. (B) The mould was glued onto the vibroslicer mounting with the blade ready to cut a slice. (C) Regioselective heterologous gene expression strategies for biolistic delivery into coronal organotypic brain slices. Images prepared from pictures obtained from the Allen Brain Atlas anatomical reference atlas23. Left panel shows two different brain regions targeted as an example. To restrict the gene expression (1) to the cortical area of the left hemisphere. To restrict the heterologous gene expression (2) to the hypothalamus region of the brain. Middle panel shows that the expression of two heterologous genes (3 and 4) can be isolated and compares the hippocampus of two hemispheres in the same organotypic slice, essentially eliminating any bias from using different sections and/or granting the possibility to address distal crosstalk from the effects of those genes. Right panel shows the possible overlap of transgenic delivery to express the first gene (5) in a precise cortical region while expressing another gene (6) in a disparate yet overlapping cortical area. This permits to analyze the effect of genetic modulation of each heterologous gene alone and also in combination within the same slice. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52148/52148fig3highres.jpg" /> 
Figure 3.&#160;Biolistic scatter pattern seen on filter paper and low magnification image.&#160;(A) The improved barrel was fired onto filter paper to determine the spread of gold particles. Left side shows the distribution of the improved barrel while the right panel shows the normal barrel. Black bar: 1 cm. Image taken from O&#39;Brien, et al.17. (B) Low magnification image showing the stochastic transfection patter within the 3 mm biolistic spread within the hippocampal region of 6 week old mice. White bar: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52148/52148fig4highres.jpg" /> 
Figure 4.&#160;Biolistic delivery of fluorescent protein encoded DNA into organotypic slices of 6 week old mice.&#160;(A) Inverted confocal images of a fixed organotypic slice of the cerebellum of a pEGFP-N1 coated gold particles transfected Purkinje cell. Image was taken at 40X magnification. Black bar: 30 &#181;m. (B) Shows a 60x magnification of another Purkinje cell&#39;s dentritic harbor to highlight the spines. Black bar: 5 &#181;m. (C) Shows a confocal image of fixed slices taken from the hippocampal region at 60x magnification of four DSredFP transfected pyramidal cells. Black bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52148/52148fig5highres.jpg" /> 
Figure 5.&#160;Diolistic delivery of membrane labeling fluorescent dyes into organotypic slices.&#160;(A) shows the live imaging of a hippocampal neuron labeled with DiO under 20X magnification. The dentritic spines can clearly be observed as well as the axon as identified by the absence of dentritic spines and the presence of synaptic boutons indicated by a white arrow. White bar: 30 &#181;m. (B) Pronounced staining of the hippocampal region labeled with DiO and counterstained with DAPI. White bar: 30 &#181;m. (C) Higher magnification (60x) of the dentritic spines found in the CA1 region of the hippocampus. White bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52148/52148fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun at JoVE.com

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

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Research

JoVE Journal

Neuroscience

16.2K ビュー.  University of Toronto. Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices. 

ビデオ: Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...