We thank the Moser Lab at the Kavli Institute for Systems Neuroscience and Centre for Neural Computation, Norwegian University of Science and Technology, for the chronic neural recording procedures in rats. This work was supported by NIH grant R21 NS098146, and Human Frontier Science Program Long-Term Fellowship LT000211/2016-L to L. Lu.

1. Stereolithography of 3D Models
<ol>
	<li>Use stereolithographic techniques to print the hyperdrive parts and accessories. Each hyperdrive is comprised of eighteen shuttles, eighteen shuttle bolts, and one each of all other plastic pieces (Figure 1). 
	NOTE: The accessories are not part of the hyperdrive but are necessary for hyperdrive construction.</li>
</ol>
2. Preparation of Accessories (Figure 2).
<ol>
	<li>Prepara...

<ol>
	<li>O'Keefe, J., Recce, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+relationship+between+hippocampal+place+units+and+the+EEG+theta+rhythm.">Phase relationship between hippocampal place units and the EEG theta rhythm.</a> Hippocampus. 3 (3), 317-330 (1993).</li><li>Wilson, M. A., McNaughton, B. L. <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+the+hippocampal+ensemble+code+for+space.">Dynamics of the hippocampal ensemble code for space.</a> Science. 261 (5124), 1055-1058 (1993).</li><li>Gothard, K. M., Skaggs, W. E., Moore, K. M., McNaughton, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+hippocampal+CA1+neural+activity+to+multiple+reference+frames+in+a+landmark-based+navigation+task.">Binding of hippocampal CA1 neural activity to multiple reference frames in a landmark-based navigation task.</a> J Neurosci. 16 (2), 823-835 (1996).</li><li>Gray, C. M., Maldonado, P. E., Wilson, M., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodes+markedly+improve+the+reliability+and+yield+of+multiple+single-unit+isolation+from+multi-unit+recordings+in+cat+striate+cortex.">Tetrodes markedly improve the reliability and yield of multiple single-unit isolation from multi-unit recordings in cat striate cortex.</a> J Neurosci Methods. 63 (1-2), 43-54 (1995).</li><li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neuronal+ensembles.">Large-scale recording of neuronal ensembles.</a> Nat Neurosci. 7 (5), 446-451 (2004).</li><li>Fyhn, M., Hafting, T., Witter, M. P., Moser, E. I., Moser, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grid+cells+in+mice.">Grid cells in mice.</a> Hippocampus. 18 (12), 1230-1238 (2008).</li><li>Skaggs, W. E., 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=EEG+sharp+waves+and+sparse+ensemble+unit+activity+in+the+macaque+hippocampus.">EEG sharp waves and sparse ensemble unit activity in the macaque hippocampus.</a> J Neurophysiol. 98 (2), 898-910 (2007).</li><li>Guo, P., Pollack, A. J., Varga, A. G., Martin, J. P., Ritzmann, 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=Extracellular+wire+tetrode+recording+in+brain+of+freely+walking+insects.">Extracellular wire tetrode recording in brain of freely walking insects.</a> J Vis Exp. (86), (2014).</li><li>Knierim, J. J., McNaughton, B. L., Poe, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spatial+selectivity+of+hippocampal+neurons+during+space+flight.">Three-dimensional spatial selectivity of hippocampal neurons during space flight.</a> Nat Neurosci. 3 (3), 209-210 (2000).</li><li>Leutgeb, S., 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=Independent+codes+for+spatial+and+episodic+memory+in+hippocampal+neuronal+ensembles.">Independent codes for spatial and episodic memory in hippocampal neuronal ensembles.</a> Science. 309 (5734), 619-623 (2005).</li><li>Lansink, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+split+microdrive+for+simultaneous+multi-electrode+recordings+from+two+brain+areas+in+awake+small+animals.">A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals.</a> J Neurosci Methods. 162 (1-2), 129-138 (2007).</li><li>Kloosterman, F., 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=Micro-drive+array+for+chronic+in+vivo+recording:+drive+fabrication.">Micro-drive array for chronic in vivo recording: drive fabrication.</a> J Vis Exp. (26), (2009).</li><li>. Google Patents Available from: <a target="_blank" href="https://www.google.com/patents/US5928143">https://www.google.com/patents/US5928143</a> (1999)</li><li>Redish, A. 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=Independence+of+firing+correlates+of+anatomically+proximate+hippocampal+pyramidal+cells.">Independence of firing correlates of anatomically proximate hippocampal pyramidal cells.</a> J Neurosci. 21 (5), RC134 (2001).</li><li>Schmitzer-Torbert, N., Redish, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+in+the+rodent+dorsal+striatum+in+sequential+navigation:+separation+of+spatial+and+reward+responses+on+the+multiple+T+task.">Neuronal activity in the rodent dorsal striatum in sequential navigation: separation of spatial and reward responses on the multiple T task.</a> J Neurophysiol. 91 (5), 2259-2272 (2004).</li><li>Nguyen, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-drive+array+for+chronic+in+vivo+recording:+tetrode+assembly.">Micro-drive array for chronic in vivo recording: tetrode assembly.</a> J Vis Exp. (26), (2009).</li><li>Chang, E. H., Frattini, S. A., Robbiati, S., Huerta, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+microdrive+arrays+for+chronic+neural+recordings+in+awake+behaving+mice.">Construction of microdrive arrays for chronic neural recordings in awake behaving mice.</a> J Vis Exp. (77), e50470 (2013).</li><li>Vandecasteele, 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=Large-scale+recording+of+neurons+by+movable+silicon+probes+in+behaving+rodents.">Large-scale recording of neurons by movable silicon probes in behaving rodents.</a> J Vis Exp. (61), e3568 (2012).</li><li>Siegle, J. 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=Chronically+implanted+hyperdrive+for+cortical+recording+and+optogenetic+control+in+behaving+mice.">Chronically implanted hyperdrive for cortical recording and optogenetic control in behaving mice.</a> Conf Proc IEEE Eng Med Biol Soc. 2011, 7529-7532 (2011).</li><li>Brunetti, P. 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=Design+and+fabrication+of+ultralight+weight,+adjustable+multi-electrode+probes+for+electrophysiological+recordings+in+mice.">Design and fabrication of ultralight weight, adjustable multi-electrode probes for electrophysiological recordings in mice.</a> J Vis Exp. (91), e51675 (2014).</li><li>. Google Patents Available from: <a target="_blank" href="https://www.google.com/patents/US4575330">https://www.google.com/patents/US4575330</a> (1986)</li><li>Ludvig, N., Potter, P. E., Fox, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+single-cell+recording+and+microdialysis+within+the+same+brain+site+in+freely+behaving+rats:+a+novel+neurobiological+method.">Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method.</a> J Neurosci Methods. 55 (1), 31-40 (1994).</li></ol>

Here, we describe the process of constructing a newly developed hyperdrive comprised of eighteen independently movable tetrodes. The drive can be constructed from affordable parts purchased at many available hardware stores, combined with components created by stereolithographic printing. The hyperdrive can be chronically implanted onto a rat&#39;s skull using standard surgical procedures and is capable of recording extracellular neural activity while the animal performs various behavioral tasks.
<p class="jove_conte...

In the field of systems neuroscience, scientists study the neural correlates underlying cognitive processes such as spatial navigation, memory, and decision-making. For these types of studies, it is critical to monitor the activity of many individual neurons during animal behavior. Over the past decades, two important advances have been made to meet the experimental needs for extracellular neural recording in small animals1,2,3. First was the development of the tetrode, a bundle of four microwires used to record neural activity of neurons simultaneously1,2,4. The differential signal amplitudes of activity across the four channels of a tetrode allows for the isolation of individual neuron activity from many simultaneously recorded cells5. In addition, the flexible nature of the microwires allows greater stability of the tetrode minimizing the relative displacement between the tetrode and the target cell population. Tetrodes are now widely used instead of a single electrode for many brain studies in various species, including rodents1,2,6, primates7, and insects8. Second was the development of a hyperdrive carrying multiple independently movable tetrodes, which allows for the simultaneous monitoring of neural activity from larger populations of neurons from multiple recording locations3,9,10,11,12.
The availability of a reliable and affordable multi-tetrode recording device for small animals is limited. The classic hyperdrive, initially developed by Bruce McNaughton13, has been successfully used for neural recordings in freely behaving rats in many labs in the past two decades9,10,14,15. However, for technical reasons, the original components needed to build the McNaughton drive are now very difficult to obtain and are not compatible with recently improved data acquisition interfaces. The other well accepted design of hyperdrive requires the microdrives to be individually handcrafted, which could yield inconsistent results and consume substantial time12. In order to record neural activity from various brain regions in behaving rats, we developed a new hyperdrive using stereolithographic technology. We sought to satisfy the following requirements: (1) the new hyperdrive must allow precise displacement of tetrodes in the brain and provide stable recording from multiple target regions; (2) the new hyperdrive must be compatible with the magnetic quickclip system recently developed to allow easy connection; and (3) the new hyperdrive can be accurately reproduced with materials easily available. Here, we provide a technique for building the 3D-printable hyperdrive containing eighteen independently movable tetrodes, based upon the McNaughton design. In the protocol, we describe the details of the fabrication process of the new hyperdrive, which we have used successfully to record single-neuron action potentials and local field potentials from the postrhinal and medial entorhinal cortices over weeks in a freely behaving rat during natural foraging tasks.

<table>
	<tbody>
		<tr>
			<td>Welding rod</td>
			<td>Blue Demon</td>
			<td>ER308L-035-01T</td>
			<td>Stainless steel, 0.035&#34; in diameter</td>
		</tr>
		<tr>
			<td>Screw</td>
			<td>McMaster</td>
			<td>91771A060</td>
			<td>Stainless steel, flat head, 0-80&#160;thread,&#160;5/8&#34;&#160;in length</td>
		</tr>
		<tr>
			<td>Screw</td>
			<td>McMaster</td>
			<td>91772A051</td>
			<td>Stainless steel, pan head, 0-80&#160;thread,&#160;5/32&#34;&#160;in length</td>
		</tr>
		<tr>
			<td>Screw</td>
			<td>McMaster</td>
			<td>92196A056</td>
			<td>Stainless steel, socket head, 0-80&#160;thread,&#160;5/16&#34;&#160;in length</td>
		</tr>
		<tr>
			<td>Screw</td>
			<td>McMaster</td>
			<td>92196A055</td>
			<td>Stainless steel, socket head, 0-80&#160;thread,&#160;1/4&#34;&#160;in length</td>
		</tr>
		<tr>
			<td>Screw</td>
			<td>McMaster</td>
			<td>95868A131</td>
			<td>Nylon,&#160; socket head, 2-56&#160;thread,&#160;3/16&#34;&#160;in length, black</td>
		</tr>
		<tr>
			<td>Screw nut</td>
			<td>McMaster</td>
			<td>90730A001</td>
			<td>Stainless steel, narrow hex,&#160; 0-80&#160;thread</td>
		</tr>
		<tr>
			<td>Shoulder screw</td>
			<td>McMaster</td>
			<td>90298A213</td>
			<td>Stainless steel, 8-32 thread, 3/16&#34; in diameter, 1/4&#34; in length</td>
		</tr>
		<tr>
			<td>Cup screw</td>
			<td>McMaster</td>
			<td>92313A105</td>
			<td>Stainless steel, 4-40 thread, 3/16&#34; in length</td>
		</tr>
		<tr>
			<td>Thumb screw</td>
			<td>McMaster</td>
			<td>94323A592</td>
			<td>Nylon, 8-32&#160;thread,&#160;3/8&#34;&#160;in length, black</td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>Apex</td>
			<td>M3X1MMDI</td>
			<td>Neodymium, 3 mm X 1 mm disc</td>
		</tr>
		<tr>
			<td>Metal tubing</td>
			<td>Small Parts</td>
			<td>B00137QHNS</td>
			<td>Stainless steel, 23 gauge, 0.0253&#34; OD, 0.013&#34; ID, 0.006&#34; wall</td>
		</tr>
		<tr>
			<td>Metal tubing</td>
			<td>New England Small Tube</td>
			<td>Custom-made</td>
			<td>Stainless steel, 30 gauge, 0.012/0.0125&#34; OD, 0.007/0.008&#34; ID, full hard</td>
		</tr>
		<tr>
			<td>Heat-shrink tubing</td>
			<td>McMaster</td>
			<td>7856K72</td>
			<td>0.09&#34; ID before shrinking, blue</td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>A-M Systems</td>
			<td>807300</td>
			<td>0.040&#34; ID, 0.085&#34; OD</td>
		</tr>
		<tr>
			<td>Polyimide tubing</td>
			<td>A-M Systems</td>
			<td>823400</td>
			<td>0.0045&#34; ID, 0.0005&#34; wall</td>
		</tr>
		<tr>
			<td>Ground wire</td>
			<td>A-M Systems</td>
			<td>791500</td>
			<td>0.005&#34; bare, 0.008&#34; coated, half hard</td>
		</tr>
		<tr>
			<td>Tetrode wire</td>
			<td>California Fine Wire</td>
			<td>Custom-made</td>
			<td>0.0007&#34; in diameter, platinum-iridium (90%-10%), HML and VG coating</td>
		</tr>
		<tr>
			<td>EIB</td>
			<td>Neuralynx</td>
			<td></td>
			<td>EIB-72-QC-Large</td>
		</tr>
		<tr>
			<td>Gold pins</td>
			<td>Neuralynx</td>
			<td></td>
			<td>large EIB pins</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>Balax</td>
			<td>01302-000</td>
			<td>M1.2 thread size</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>2522A811</td>
			<td>0-80 thread size, bottoming</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>2522A771</td>
			<td>0-80 thread size, plug</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>26955A94</td>
			<td>3/8&#34;-24&#160;thread size, bottoming</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>2522A713</td>
			<td>2-56&#160;thread size</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>2522A715</td>
			<td>4-40&#160;thread size</td>
		</tr>
		<tr>
			<td>Tap</td>
			<td>McMaster</td>
			<td>2522A718</td>
			<td>8-32&#160;thread size</td>
		</tr>
		<tr>
			<td>Die</td>
			<td>McMaster</td>
			<td>2576A457</td>
			<td>3/8&#34;-24&#160;thread size, 1&#34; OD</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>30585A82</td>
			<td>Wire gauge&#160;65,&#160;0.035&#34; in diameter</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>30585A83</td>
			<td>Wire gauge&#160;66,&#160;0.033&#34; in diameter</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>30585A87</td>
			<td>Wire gauge&#160;70,&#160;0.028&#34; in diameter</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>30585A88</td>
			<td>Wire gauge&#160;71,&#160;0.026&#34; in diameter</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>30585A91</td>
			<td>Wire gauge&#160;73,&#160;0.024&#34; in diameter</td>
		</tr>
		<tr>
			<td>Drill bit</td>
			<td>McMaster</td>
			<td>8870A23</td>
			<td>3/16&#34;&#160;in diameter</td>
		</tr>
		<tr>
			<td>Dremel disc</td>
			<td>Wagner</td>
			<td>31M</td>
			<td>Diamond coated, 22 mm in diameter, 0.17 mm in thickness</td>
		</tr>
		<tr>
			<td>Steel wire</td>
			<td>Precision Brand</td>
			<td>21212</td>
			<td>0.012&#34; in diameter, full hard</td>
		</tr>
		<tr>
			<td>Steel wire</td>
			<td>Precision Brand</td>
			<td>21007</td>
			<td>0.007&#34; in diameter, full hard</td>
		</tr>
		<tr>
			<td>Steel wire</td>
			<td>A-M Systems</td>
			<td>792700</td>
			<td>0.003&#34; in diameter, half hard</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Loctite</td>
			<td>LT-40640</td>
			<td># 406</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Loctite</td>
			<td>LT-41550</td>
			<td># 415</td>
		</tr>
		<tr>
			<td>Dental acrylic powder&#160;</td>
			<td>Teets</td>
			<td>223-3773</td>
			<td>Coral</td>
		</tr>
		<tr>
			<td>Dental acrylic liquid</td>
			<td>Teets</td>
			<td>223-4003</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of an improved multi-tetrode hyperdrive for large-scale neural recording in behaving rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We used a newly built hyperdrive to obtain trial results. The drive was equipped with tetrodes constructed from &#248; 17 &#181;m (0.0007&#34;), polyimide-coated platinum-iridium (90%-10%) wire. The tips of the tetrodes were plated in platinum black solution to reduce electrode impedances to between 100 and 200 k&#937; at 1 kHz. The hyperdrive was implanted 4.6 mm left of the midline and 0.5 mm anterior to the transverse sinus on the skull of a 550 g, male Long-Evans rat. Additional groun...

Watch this Scientific Journal Video about Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats at JoVE.com

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

The overall goal of this procedure is to construct a recording device with multiple independently adjustable electrodes for the examination of brain activity in freely behaving rats. So this method is very exciting because it will help answer several questions related to special navigation, decision making, and other cognitive processes. The main advantage of this hyperdrive is that it provides reliable neuro recording in the brain using materials that are easy to obtain.Although this method was designed to be used specifically with rats, it can be used with any species of similar or larger head sizes such as non-human primates, thereby allowing investigations of many species. Those new to this method might struggle with the nuance involved in the preparation of the steps cause there's a certain level of experience and accuracy that's required to develop these tricks that can make a successful drive. Begin by expanding the holes in the 3D printed core using different sized drill bits.Use a diameter of 0.61 millimeter for the 12 ground wire through holes. For the 18 tetrode through holes, first use a 0.66 millimeter bit and then widen with a 0.71 millimeter bit. A 0.84 millimeter diameter bit is used for the 18 guide rod blind holes.Next, thread the two through holes on top of the core and the remaining eight blind holes with a 0.80 tap. Use a bottoming tap for the blind holes. Create external threads at the base of the core using a 3/8-24 die.Adjust the die properly so the hyperdrive nut will fit over the new threads. Depending on the desired number of ground wires, insert multiple six millimeter long segments of 23 gauge metal tubing into the ground wire holes in the core gluing them if necessary. File the outer ends of the ground wire cannulas until they are flushed with the core and then clean the cannulas with a 0.30 millimeter diameter steel wire.Next, fully insert 18 08015.88 millimeter long flathead screws head down into the slots in the core. Do not bend the screws or damage the threads during this process. Using the rod positioning complex into the core station, position 18 17 millimeter segments of 0.89 millimeter diameter welding rod over the guide rod holes in the core and hammer them down about five millimeters until flushed with the screws.Correct the positions of the welding rods and screws if necessary then tighten the central shoulder screw and the surrounding six screws in the rod positioning complex to secure the outward directions of the rods in the core. Screw the nut onto the core with the rod positioning complex and fit the core into the hyperdrive holder to allow easier positioning under a stereoscope. Fill the slots with dilute dental cement to secure the screws to the core.Fill two to three slots at a time before the dental cement gets too thick. Scrape away any excess dental cement on the core to maintain a proper fit with the shield then allow to air dry for 15 minutes. Next, glue the screws and rods into the core with thin super glue and allow to dry for 15 minutes.Clean and expand the two outer holes in the 3D printed shuttle with drill bits. Use a 0.61 millimeter diameter bit for the smaller hole and a 0.89 millimeter bit for the larger hole. Insert the shuttle bolt into the bolt holder base ensuring the proper orientation.Then close the bolt holder lid. Hold the bolt holder tightly and thread slowly through the hole in the lid with a 0.80 tap. Tap two or three times until smooth.Then insert the shuttle bolt into the shuttle from the side with the smaller opening. Place the shuttle-shuttle bolt complex upside down in the microdrive assembly station base. Next, smooth both ends of a 15 millimeter segment of 23 gauge metal tubing, then position the tubing over the 0.61 millimeter diameter hole guided by the slot in the station lid.Hammer the cannula into the hole until the upper end is flushed with the station lid. Remove the outer half of the upper tip of the cannula with a sanding wheel. Clean the cannula with a 0.30 millimeter diameter metal wire.Then glue the cannula onto the shuttle using thin super glue making sure not to glue the shuttle bolts to the shuttle. Prepare at least 18 microdrives. Test the microdrives on the microdrive rack.Make sure that the shuttle bolt can rotate smoothly in the shuttle and that the entire microdrive moves freely along the length of the threaded rod. To insert guide cannulas into the hyperdrive core, begin by removing the heat shrink tubes and slide a four millimeter segment of silicone tubing along the bundle to the soldered-unsoldered border. Wedge the slit in the hyperdrive spacer to widen the central hole allowing the spacer to slip around the silicone tube.Remove the wedge when the spacer sits at the center of the silicone tube. Next, organize the positions of the guide cannulas in the bundle by placing 10 centimeter long segments of 0.18 millimeter diameter metal wire through each cannula into a specific tetrode hole in the hyperdrive core preventing any crossover of the wires or cannulas in the process. Bend the end of the wires to hold them in place.Push the cannulas through their respective holes in the core being careful to avoid bending or crossing between them until the free end of each cannula is at least two millimeters outside the upper end of the tetrode hole. Secure the spacer by screwing the nut onto the core being careful to prevent the spacer from rotating. Apply a drop of very dilute dental cement from the top of the core onto the junction of the cannulas to secure their relative positions.Cut the guide wires from the soldered end of the bundle and remove them from the cannulas by retracting from the free end. Load the microdrives slowly and carefully onto each threaded rod of the core. Confirm that the 23 gauge microdrive cannula goes into the tetrode hole smoothly, the 30 gauge guide cannula goes into the 23 gauge microdrive cannula smoothly, and the shuttle bolt turn smoothly along the threaded rod.Screw the microdrives down to one to 1.5 millimeters above the lower end of the threaded rods. Then cut 18 pieces of polyamide tubing into 43 millimeter segments which is the length of the guide cannula bundle plus seven millimeters. Clean each tube with a 0.8 millimeter diameter steel wire.Invert the core, insert the polyamide tubes carefully into the guide cannulas from the soldered end and push them all the way in. Then flip the core upright and glue the upper end of the polyamide tube onto the microdrive cannula with thick super glue. Place the core upside down and let the glue dry for 15 minutes.After drying, cut the extra polyamide tubing at the upper end leaving 0.5 to one millimeter outside the microdrive cannula. To assemble the ground wires, first cut lengths of 25 to 30 millimeters from coated steel wire. Then strip two millimeters of the plastic insulation from both tips of the wires and insert one end of each into both sides of a six to eight millimeter long 30 gauge cannula.Flatten this end of each cannula to secure the connection to their respective wires. Finally, insert the round end of the 30 gauge cannula into the upper end of the ground wire cannula in the core and press to make the insertion tight. Then complete assembly of the multi-tetrode hyperdrive according to the instructions in the written protocol.This scatter plot shows the relationship between peak to peak amplitudes of spikes recorded from two electrodes of a tetrode located in the post rhinal cortex. Each dot corresponds to one spike. Clusters of spikes are likely to originate from the same cell.Four clusters are color coded as seen here. The mean spike waveforms from all four tetrode channels of the color coded cells shown in the previous scatter plot are seen here. Traces of local field potential in the theta frequency range recorded simultaneously from four different tetrodes located in the medial entorhinal cortex when the rat was freely foraging are seen here.While attempting this procedure, it's important to work with precision throughout the entire procedure to ensure that all parts fit together properly. Also use care and wear proper PPE when working with the demo tool to avoid injury. After watching this video, you should have a good understanding of how to construct these improved hyperdrives carrying multiple tetrodes for chronic neuro recordings in behaving rats.

construction-an-improved-multi-tetrode-hyperdrive-for-large-scale

Research

JoVE Journal

Neuroscience

11.1K 次观看.  Baylor College of Medicine. The overall goal of this procedure is to construct a recording device with multiple independently adjustable electrodes for the examination of brain activity in freely behaving rats. So this method is very exciting because it will help answer several questions related to special navigation, decision making, and other cognitive processes. The main advantage of this hyperdrive is that it provides reliable neuro recording in the brain using materials that are easy to obtain.Although this ...

视频: Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several...

hybrid microdrive system with recoverable opto-silicon probe and tetrode for dual-site high density recording in freely moving mice

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple...

chronic implantation of multiple flexible polymer electrode arrays

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other...

large-scale recording of neurons by movable silicon probes in behaving rodents

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy...

construction of microdrive arrays for chronic neural recordings in awake behaving mice

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive...

a wireless, bidirectional interface for in vivo recording and stimulation of neural activity in freely behaving rats

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

the td drive: a parametric, open-source implant for multi&#45;area electrophysiological recordings in behaving and sleeping rats

Author Spotlight: Deciphering Memory and Learning Through Neural Implants for Multi&amp;#45;Region Brain Studies

Here, we present a unique, 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously.

The TD Drive: A Parametric, Open-Source Implant for Multi&#45;Area Electrophysiological Recordings in Behaving and Sleeping Rats

micro-drive array for chronic in vivo recording: tetrode assembly

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats.  In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

recording large-scale neuronal ensembles with silicon probes in the anesthetized rat

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local...

extracellular wire tetrode recording in brain of freely walking insects

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving...

odelay: a large-scale method for multi-parameter quantification of yeast growth

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and...

recordings of neural circuit activation in freely behaving animals

Recordings of Neural Circuit Activation in Freely Behaving Animals

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed...

recording brain electrical activity in behaving mice using multi-shank linear silicon probes

Subsequently, apply dental cement to fix the base of the probe to the anchoring screw in the skull. Right after cement application, slowly move the probe to the target depth. Advance the last 200 micrometers after applying the cement to reduce lateral movement of the probe and ensure minimal tissue damage in the target...

Recording Brain Electrical Activity in Behaving Mice Using Multi-Shank Linear Silicon Probes

This video outlines a protocol for implanting a multi-shank linear silicon probe into the mouse brain to measure local field potentials. The procedure involves securing the probe to a stereotaxic unit, positioning it accurately over the target brain region, inserting it into the brain, and securing it with dental cement. After implantation, the assembly is protected with a casing. Following recovery, the setup allows for recording local field potentials, providing insights into neuronal...

an integrated method for crafting flexible and convenient electrophysiological optrodes for multi-region in vivo recording

Author Spotlight: Unraveling Neural Communication and Circuit Interactions in Health and Disease

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling...

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

electrode fabrication and implantation in aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four  in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia...

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

recording spatially restricted oscillations in the hippocampus of behaving mice

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving...

design and construction of a cost effective headstage for simultaneous neural stimulation and recording in the water maze

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming...

multi-unit recording methods to characterize neural activity in the locust (schistocerca americana) olfactory circuits

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

large-scale construction of uas-cdna/orf plasmids by crispr-based modular assembly (crisprmass)

Large-Scale Construction of UAS-cDNA/ORF Plasmids by CRISPR-Based Modular Assembly (CRISPRmass)

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.