Name: rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection
Uploaded: 2017-06-13T14:00:00
Description: Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

We would like to thank Yoichi Miyahara for many helpful discussions and insights. MA and PG acknowledge funding from NSERC.

All procedures detailed below were approved by McGill University&#39;s Animal Care Committee and conformed to the guidelines of the Canadian Council of Animal Care.
1. Standardization of Neuronal Cultures Using Microfluidic Devices: Device Assembly
<ol>
	<li>Select a suitable microfluidic device for the desired experiment. To connect neurons within the same population, use the Neuro Devices (Figure 1) and to connect neurons in different populations use the Co-Cu...

<ol>
	<li>Chew, D. J., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+long-distance+axon+regeneration+in+the+injured+CNS.">The challenges of long-distance axon regeneration in the injured CNS.</a> Prog. Brain. Res. 201, 253-294 (2012).</li><li>Bradke, F., Fawcett, J. W., Spira, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+a+new+growth+cone+after+axotomy:+the+precursor+to+axon+regeneration.">Assembly of a new growth cone after axotomy: the precursor to axon regeneration.</a> Nat. Rev. Neurosci. 13 (4), 189-193 (2012).</li><li>Aguayo, A. 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=Synaptic+connections+made+by+axons+regenerating+in+the+central+nervous+system+of+adult+mammals.">Synaptic connections made by axons regenerating in the central nervous system of adult mammals.</a> J. Exp. Biol. 153, 199-224 (1990).</li><li>Lamoureux, P., Ruthel, G., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+can+specify+axonal+fate+in+hippocampal+neurons.">Mechanical tension can specify axonal fate in hippocampal neurons.</a> J Cell Biol. 159 (3), 499-508 (2002).</li><li>Goslin, K., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+observations+on+the+development+of+polarity+by+hippocampal+neurons+in+culture.">Experimental observations on the development of polarity by hippocampal neurons in culture.</a> J Cell Biol. 108 (4), 1507-1516 (1989).</li><li>Dotti, C. G., Sullivan, C. A., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Establishment+of+polarity+by+hippocampal+neurons+in+culture.">The Establishment of polarity by hippocampal neurons in culture.</a> J. Neurosci. 8 (4), 1454-1468 (1988).</li><li>Magdesian, M. H., 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=Rapid+mechanically+controlled+rewiring+of+neuronal+circuits.">Rapid mechanically controlled rewiring of neuronal circuits.</a> J. Neurosci. 36 (3), 979-987 (2016).</li><li>Lucido, A. L., 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=Rapid+assembly+of+functional+presynaptic+boutons+triggered+by+adhesive+contacts.">Rapid assembly of functional presynaptic boutons triggered by adhesive contacts.</a> J. Neurosci. 29 (40), 12449-12466 (2009).</li><li>Suarez, F., Thostrup, P., Colman, D., Grutter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+presynaptic+protein+recruitment+induced+by+local+presentation+of+artificial+adhesive+contacts.">Dynamics of presynaptic protein recruitment induced by local presentation of artificial adhesive contacts.</a> Dev. Neurobiol. 73, 1123-1133 (2013).</li><li>General Laboratory Techniques. An Introduction to Working in the Hood. JoVE Science Education Database Available from: <a target="_blank" href="https://www.jove.com/science-education/5036/an-introduction-to-working-in-the-hood">https://www.jove.com/science-education/5036/an-introduction-to-working-in-the-hood</a> (2016)</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell+growth.">Monitoring cell growth.</a> Curr Protoc Immunol. A-3A, (2001).</li><li>Beaudoin, G. M., 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=Culturing+pyramidal+neurons+from+the+early+postnatal+mouse+hippocampus+and+cortex.">Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.</a> Nat. Protoc. 7 (9), 1741-1754 (2012).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev. Biol. 102, 379-389 (1984).</li><li>Lamoureux, P., Buxbaum, R. E., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+outgrowth+of+cultured+neurons+is+not+limited+by+growth+cone+competition.">Axonal outgrowth of cultured neurons is not limited by growth cone competition.</a> J. Cell Sci. 111, 3245-3252 (1998).</li><li>Pfister, B. J., Bonislawski, D. P., Smith, D. H., Cohen, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sketch-grown+axons+retain+the+ability+to+transmit+active+electrical+signals.">Sketch-grown axons retain the ability to transmit active electrical signals.</a> FEBS Lett. 580, 3525-3531 (2006).</li><li>Magdesian, M. H., 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=Atomic+force+microscopy+reveals+important+differences+in+axonal+resistance+to+injury.">Atomic force microscopy reveals important differences in axonal resistance to injury.</a> Biophys. J. 103 (3), 405-414 (2012).</li><li>Polleux, F., Snider, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+and+growing+an+axon.">Initiating and growing an axon.</a> CSH Persp. Biol. 2 (4), 001925 (2010).</li><li>Debanne, D., 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=Paired-recordings+from+synaptically+coupled+corticol+and+hippocampal+neurons+in+acute+and+cultured+brain+slices.">Paired-recordings from synaptically coupled corticol and hippocampal neurons in acute and cultured brain slices.</a> Nat. Protoc. 3, 1559-1568 (2008).</li><li>Qi, G., Radnikow, G., Feldmeyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+Morphological+Characterization+of+Neuronal+Microcircuits+in+Acute+Brain+Slices+Using+Paired+Patch-Clamp+Recordings.">Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings.</a> J. Vis. Exp. (95), e52358 (2015).</li><li>Harrison, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+and+development+of+the+nervous+system+studied+by+the+methods+of+experimental+embryology.">On the origin and development of the nervous system studied by the methods of experimental embryology.</a> Proc. R. Soc. Lond. B. , 155-196 (1935).</li><li>Weiss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+patterns:+the+mechanics+of+nerve+growth.">Nerve patterns: the mechanics of nerve growth.</a> Growth 5 (Suppl. Third Growth Symposium). , 153-203 (1941).</li><li>Gray, C., 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=Rapid+neural+growth:+calcitonin+gene-related+peptide+and+substance+P-containing+nerves+attain+exceptional+growth+rates+in+regenerating+deer+antler.">Rapid neural growth: calcitonin gene-related peptide and substance P-containing nerves attain exceptional growth rates in regenerating deer antler.</a> Neurosci. 50 (4), 953-963 (1992).</li><li>Heidemann, S. R., Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension-driven+axon+assembly:+a+possible+mechanism.">Tension-driven axon assembly: a possible mechanism.</a> Front. Cell Neurosci. 9, (2015).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+tension.">Axonal growth in response to experimentally applied tension.</a> Dev. Biol. 102, 379-389 (1984).</li><li>Heidemann, S. R., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+as+a+regulator+of+axonal+development.">Mechanical tension as a regulator of axonal development.</a> Neurotoxicology. 15, 95-107 (1994).</li><li>Heidemann, S. R., Lamoureux, P., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+axonal+development.">Cytomechanics of axonal development.</a> Cell Biochem. Biophys. 27 (3), 135-155 (1995).</li><li>Pfister, B. J., Iwata, A., Meaney, D. F., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J. Neurosci. 24 (36), 7978-7983 (2004).</li><li>Waxman, S. G., Kocsis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The axon: structure, function and pathophysiology. , (1995).</li><li>Cho, E. Y., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate+of+regrowth+of+damaged+retinal+ganglion+cell+axons+regenerating+in+a+peripheral+nerve+graft+in+adult+hamsters.">Rate of regrowth of damaged retinal ganglion cell axons regenerating in a peripheral nerve graft in adult hamsters.</a> Brain Res. 419, 369-374 (1987).</li><li>Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+differences+in+the+growth+rate+of+early+nerve+fibres+related+to+target+distance.">Intrinsic differences in the growth rate of early nerve fibres related to target distance.</a> Nature. 337, 553-555 (1989).</li></ol>

Using standard micromanipulation and innovative microfluidic devices, a new technique was developed to rapidly initiate, elongate and precisely connect new functional neuronal circuits over large distances. Pipette micromanipulation is a common tool in most neuroscience labs4,13. The real challenge to achieving reproducible and reliable results was standardization of healthy, precisely positioned neuronal cultures for the duration of the experiment (which can be ...

Injuries to the adult central nervous system (CNS) may lead to permanent disability due to multiple mechanisms that limit axonal regrowth1. Following injury, many CNS axons do not form a new growth cone and fail to mount an effective regenerative response2. Furthermore, damage and scar tissue surrounding CNS lesions significantly inhibit axonal growth1,2,3. Current therapies to promote CNS regeneration after injury have focused on enhancing the intrinsic growth potential of the injured neuron and on masking the inhibitors of axonal extension associated with myelin debris and the glial scar1,3. Despite this, the capacity to regenerate long axons to distant targets and to form appropriate functional synapses remains severely limited4,5,6,7.
In the present work, microbeads, pipette micromanipulation, and microfluidic devices are used to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. Previous work has shown that poly-D-lysine-coated beads (PDL-beads) induce membrane adhesion followed by the clustering of synaptic vesicle complexes and the formation of functional presynaptic boutons8. It was also shown that when the PDL-bead is mechanically pulled away after presynaptic differentiation, the synaptic protein cluster follows the bead, initiating a new neurite9. The following procedure exploits this fact along with the ability to culture embryonic hippocampal neurons of rats into organized regions on a coverslip using polydimethylsiloxane (PDMS) microfluidic devices to precisely rewire a neuronal circuit.
These PDMS microfluidic devices are non-toxic, optically transparent and consist of two chambers connected by a system of microchannels. Once assembled on a coverslip, each device serves as a mold to guide neuronal growth and maintain healthy neuronal cultures on precise patterns for longer than 4 weeks in vitro.
Here, a framework is presented in which to investigate the limits of extension and functionality of the new neurite. New, functional neurites are created and positioned to controllably (re)wire neuronal networks. The extension rates achieved are faster than 20 &#181;m/min over millimeter-scale distances and functional connections are established. These results show, unexpectedly, that the intrinsic capacity of these neurites for elongation is much faster than previously thought. This proposed mechanical approach bypasses slow chemical strategies and enables controlled connection to a specific target. This technique opens new avenues for the in vitro study of novel therapies to restore neuronal connectivity after injury. It also enables the manipulation and rewiring of neuronal networks to investigate fundamental aspects of neuronal signal processing and neuronal function in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Co-culture devices</td>
			<td style="width:104px;">Ananda Devices</td>
			<td style="width:119px;"></td>
			<td style="width:397px;">Commercially available at http://www.anandadevices.com</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Neuro devices</td>
			<td style="width:104px;">Ananda Devices</td>
			<td style="width:119px;"></td>
			<td>Commercially available at http://www.anandadevices.com</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">No. 1 Glass Coverslip 25 mm Round</td>
			<td style="width:104px;">Warner Instruments</td>
			<td style="width:119px;">64-0705</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">35mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated</td>
			<td style="width:104px;">MatTex Incorporation</td>
			<td style="width:119px;">P35G-0-20-C</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">35mm cell culture dish, Non-Pyrogenic, Sterile</td>
			<td style="width:104px;">Corning Inc</td>
			<td style="width:119px;">430165</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">95mmx15mm Petri Dish, Slippable Lid, Sterile Polystyrene</td>
			<td style="width:104px;">Fisherbrand</td>
			<td>FB0875714G</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">50mL Centrifuge tubes with printed graduations and flat caps</td>
			<td style="width:104px;">VWR</td>
			<td style="width:119px;">89039-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL Polypropylene Conical Tube, 17x120mm style, Non Pyrogenic, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Neurobasal Medium</td>
			<td style="width:104px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">B-27 Supplement (50X), serum free</td>
			<td style="width:104px;">B-27 Supplement (50X), serum free</td>
			<td style="width:119px;">17504044</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Pennicilin, Streptomyocin, Glutamine</td>
			<td style="width:104px;">Thermo Fisher Scientific&#160;</td>
			<td>11995-065</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">200uL Pipettors</td>
			<td style="width:104px;">VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-20uL Pipettors</td>
			<td style="width:104px;">Aerosol Resistant Tips</td>
			<td style="width:119px;">2149P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Falcon 3mL Transfer Pipettes [Non-sterile]</td>
			<td>BD Falcon</td>
			<td>357524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Gibco</td>
			<td style="width:119px;">15023-021</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">7365-45-9</td>
			<td>Extracellular solution/Beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">7647-14-5</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">7447-40-7</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">10043-52-4</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">7786-30-3</td>
			<td>Extracellular solution</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">#5 Dumont Dumostar Tweezers 11cm</td>
			<td style="width:104px;">World Precision Instruments</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools</td>
			<td>Braun, Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine Hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Micro particles based on polystyrene, 10 um</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">72986</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Borosilicate tubes</td>
			<td style="width:104px;">King Precision Glass, Inc.</td>
			<td style="width:119px;">14696-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Horizontal Pipette Puller</td>
			<td style="width:104px;">Sutter Instruments</td>
			<td style="width:119px;">Brown-Flaming P-97</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Micromanipulators, PCS-5000 Series</td>
			<td style="width:104px;">SD Instruments</td>
			<td style="width:119px;">MC7600R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml Syringe</td>
			<td style="width:104px;">BD Luer-Lok</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:104px;">Olympus&#160;</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">UIS2, LUCPLFLN 40X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD Camera</td>
			<td style="width:104px;">Photometrics</td>
			<td style="width:119px;">Cascade II: 512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s (1x) L-15 Medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Typsin-EDTA (0.05%), Phenol red</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (1x) Dulbecco&#39;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>Rat Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (1x) Hank&#39;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td>
			<td>Life Technologies</td>
			<td>14170-112</td>
			<td>Rat Dissection</td>
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rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Embryonic rat hippocampal neurons are cultured in microfluidic devices to enable precise positioning of cells, PDL-beads and micromanipulators. The first step is to properly assemble the microfluidic device on a glass coverslip or dish. It is essential that the microfluidic device be well attached to the substrate to avoid cells exiting the chambers and moving under the parts of the device that should be sealed (Figure 1a). To maintain healthy cultures for se...

Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

The overall goal of this procedure is to initiate new neurites, rapidly extend them, and precisely connect them to target cells. This procedure is only possible by using cells grown in a controlled and reproducible environment. This method allows us to answer key questions in neuroscience.What limits the growth rate of neurons? How fast can they grow? Do mechanical clues play a role?We can also study how synapses are formed, how do neurons find a target cell? How do synapses age, mature? And thirdly, we can start building systematically neural networks, study how neurons compute.The main advantages of this technique are that it enables easy manipulation, experimental reproducibility, and ultimately a high degree of control at the single-cell level. To begin this procedure, clean and prepare the desired number of sterile cover slips. Next, coat each cover slip with 0.5 to 1 milliliters of 100 microgram per milliliter PDL for at least two hours at room temperature.After that, wash the cover slips twice with water and remove all the liquid. Let them dry in a sterile environment for 5 to 10 minutes or until the surface is completely dry. Then, place the microfluidic devices with the patterns facing up under the UV light in a biosafety cabinet for 10 minutes.After 10 minutes, place a microfluidic device with the pattern facing down in contact with the clean cover slip. Use the tweezers to softly press the device so it adheres to the glass. To fill the single population device with medium, add 50 microliters of complete cell medium to the right upper well.And then add another 50 microliters of complete cell medium to the well diagonal to it. To fill the multiple populations device with medium, add 30 microliters of complete NBM to one row of wells. Make sure the medium flows between the wells.Then, add 50 microliters of medium to each of the remaining wells. Place the devices in a bigger plate with an open dish of autoclaved water, and place the dish in the incubator for one to two hours while preparing the cell culture. Now, remove the medium from the upper wells of the microfluidic devices without emptying the wells.Then, add 20 microliters of the concentrated cell solution into the top right well of the microfluidic device, and check in the microscope how cells flow inside the single population microfluidic device. To plate cells in the multiple populations device, add 20 microliters of the concentrated cell solution into each of the top wells. Under the microscope, check if the cells are inside the chambers.Afterwards, place the devices in the incubator for 15 to 30 minutes to promote cell attachment to the substrate. After the cells attach to the substrate, add 40 microliters of NBM to the two top wells of the single population device. This will help remove the air bubbles from the micro channels.Add 20 microliters of MBM to the top wells of the multiple populations device, the same wells where the cells were injected. The media should protrude slightly to form a positive meniscus, and give the wells a muffin top aspect. One to two days before the microfluidic device's removal, add two milliliters of pre-warmed NBM to each sample dish and flood the chambers.Then, keep the devices in the incubator. Subsequently, use sterile tweezers and a pipette tip to remove the microfluidic devices from the cover slips leaving a patterned configuration of neurons. In this procedure, and 40 to 60 microliters of the prepared PDL coated beads to the cell culture.Then, return the sample to the incubator for one hour to promote the formation of synaptic contacts. Subsequently, install the sample in an experimental setup such that the cells can be assessed from above by two micropipettes, and also be accessed optically. In this configuration, a CCD camera is located on the side port of the microscope for image capture.Connect each pipette filled with saline to a one milliliter syringe, then perfuse the sample with physiological saline solution. Next, under the microscope, check the gap between the two isolated neuronal populations to insure that the two populations are not naturally connected. Select a PDL bead that is not attached to the neuron in the field of view.Align the bead with a micropipette tip by focusing on the bead and then on the micropipette. Subsequently, bring the tip as close as possible to the bead by monitoring it through the microscope. Then, apply negative pressure to the pipette to pick up the bead, and maintain the negative pressure throughout the experiment.Now, select a PDL bead attached to a neuron in the field of view, and attach it to the second micropipette using suction. Pull the PDL bead neuron complex by slowly moving either the micromanipulator or the sample stage at 0.5 micrometers per minute for 1 micrometer, and pause for five minutes to allow neurite initiation. Pull the PDL bead neuron complex by slowly moving it laterally at 0.5 micrometers per minute over two micrometers, then pause for five minutes to allow neurite elongation.After successful initiation and neurite extension for the first five micrometers, continuously pull the neurite at 20 micrometers per minute. To connect the neurons, select a region rich in neurites. Use other beads to gauge the pipette tip height above the cover slip, and lower the PDL bead neurite complex so that it physically contacts the neurites.Leave the PDL bead neurite complex in contact with the target neurite while manipulating the second micropipette. Then, lower the second pipette with the second PDL bead on top of the newly formed neurite about 20 micrometers from the first bead, and use the second PDL bead to push the new neurite filament towards the target cell. Next, hold both beads in place for at least one hour.Verify the absence of focal swelling, a thickening of the neurites contacting the bead, with the microscope. During this time, slowly change the medium of the sample from physiological saline to pre-warmed carbon dioxide equilibrated NBM. Release the bead from the second pipette by releasing the suction.If the new neurite remains attached, release the first bead as well. Afterward, carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. You can use whole cell patch clamp recordings to investigate the electrical functionality of the micromanipulated connection.Shown here are the isolated neuronal populations separated by a 100 micrometer gap in PDMS microdevices. Pair to patch clamp recordings were performed in whole-cell configuration, and the mechanically induced connections were analyzed, and compared with those in naturally connected neuronal populations, and non-connected populations. The electrical responses after pre-synaptic action potential recorded from the neurons connected naturally and by micromanipulation are significantly higher and temporarily correlate with the pre-synaptic activity.Once mastered, this technique can be performed in eight hours if done properly, and while attempting this procedure, it is important to remember to have healthy and well-patterned long term cell cultures to guarantee fast and reproducible results. Following this procedure different methods can be used to verify the electrical functionality of the micromanipulated connection. After watching this video, you should have a good understanding of how to grow cell populations in microfluidic chambers, and use pipette manipulations to initiate, extend, and connect neurites.

rewiring-neuronal-circuits-new-method-for-fast-neurite-extension

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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

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

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

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...