This work was supported by funding from the National Institutes of Health (NIH), grants EY026924 and EY014800 (to B.N.), an Unrestricted Grant from Research to Prevent Blindness, Inc., New York, NY to the Department of Ophthalmology and Visual Sciences, University of Utah, and the start-up funds provided to R.E. by the Henry Samueli School of Engineering and the Department of Electrical Engineering at the University of California, Irvine. This method is based on a previous report of a similar method developed in Dr. Tirin Moore&#8217;s lab, published in Noudoost &#38; Moore 2011, Journal of Neuroscience Methods. The authors thank Dr. Kelsey Clark for her comments on the manuscript.

Experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Society for Neuroscience Guidelines and Policies. Protocols for experimental and behavioral procedures were approved by the University of Utah Institutional Animal Care and Use Committee.
1. Construction of the Microinjectrode for Stimulation and Recording (Figure 1a)
<ol>
	<li>Measure the length of the cannula and the probe (in this example a nanosensor). Probe must be longer than the cannula by the length it is to protrude from the cannula tip (depending on probe design) plus approximately 2 cm.</li>
	<li>Under a magnifier or a microscope (~10x magnification), load the probe into the cannula; if possible a back-loading is preferable to protect the tip of the probe. 
	NOTE: This step, performed manually, is challenging. It is recommended to practice with a microelectrode under a magnifying glass before attempting with an actual experimental probe.</li>
	<li>Pass the cannula (containing the probe) through the top ferrule, T-junction, and bottom ferrule.
	<ol>
		<li>If the probe is just a single wire without any attachments, back-load it into the canula and insert the assembly into the T-junction from the bottom ferrule. Top of cannula (flat-end side) should be positioned in the middle of the T-junction, within the bottom but not the top ferrule. The experimental probe or biosensor should protrude above the top of the top ferrule. 
		NOTE: Custom-made ferrules can also be made by drilling a hole in the ferrule plugs using micro drill bits, the size of the hole being based upon the diameter needed for tightening the cannula to the T-junction.</li>
	</ol>
	</li>
	<li>Use the ferrule wrench to tighten the ferrules on the top and bottom of the T-junction. Do not over-tighten. A small piece of tubing can be added to strengthen the electrode support within the top ferrule.</li>
	<li>Solder gold pins to each of the probe terminals (signal, ground, etc.), according to the specifications of the probe.</li>
	<li>Adjust relative position of probe and cannula. Measure the distance that the probe is protruding from cannula under magnification, and adjust manually from the top end (probe can slide freely within ferrules).</li>
	<li>Add epoxy glue between the gold pins and the top ferrule to attach the probe to the ferrule.</li>
	<li>Unscrew the top ferrule to retract probe inside cannula. Visually confirm that the probe is fully within the cannula under magnification.</li>
	<li>Attach the injectrode to the microdrive.</li>
</ol>
2. Construction of the Microinjectrode for Drug Infusion (Figure 1b)
<ol>
	<li>Attach the &#34;non-beveled&#34; or flat-end of the cannula to the bottom of the T-junction using a ferrule. Use the ferrule wrench to tighten the ferrule.</li>
	<li>Attach a small piece of capillary tubing (~1.5 cm) to the top of the T-junction by passing it through the standard ferrule. Tighten with a ferrule wrench.</li>
	<li>Back-load the microelectrode through the capillary tubing, T-junction, cannula and corresponding ferrules.</li>
	<li>Make sure that the back-end of the electrode protrudes less than 1 cm from the back of the capillary tubing, and the tip of the electrode protrudes from the cannula at the desired distance on the bottom side. Electrode position can be manually adjusted from the top-end.</li>
	<li>Solder a gold pin to the microelectrode terminal.</li>
	<li>Add epoxy glue between the gold pin and the top ferrule to attach the microelectrode to the ferrule.</li>
	<li>Unscrew the top ferrule to retract the probe inside cannula. Visually confirm that the microelectrode is fully retracted into the cannula.</li>
</ol>
3. Construction of the Microfluidic Circuit (Figure 2)
<ol>
	<li>Place a breadboard on a stable surface. Place the two three-way valves parallel to the longest sides of the breadboard, about 6 in. apart with one port (the one that is always open) facing each other. Use screws to fix the valves to the breadboard.</li>
	<li>Place a ruler next to the valves (to measure and track movement of fluids inside the capillary tubing).</li>
	<li>Load a mixture of 1:1 low viscosity oil and food coloring (marker) into the gastight syringe and place in the Marker pump. Cut one piece of capillary tubing, and use standard ferrules and Luer-lock connectors to connect the syringe to one of the ports on the Input valve. This is the &#34;marker line&#34;.</li>
	<li>Cut a short piece of capillary tubing for the &#34;ruler line&#34;. Use standard ferrules to tighten to the facing ports of the valves.</li>
	<li>Cut two longer pieces of capillary tubing to connect the Output valve to the microinjectrode, and to connect the Drug pump to the Input valve (use standard ferrules). 
	NOTE: The length of these two lines depends on the experimental setup, one must be long enough to reach from the infusion apparatus to the animal, and the other one from the Drug pump to the Input valve. Use a cleaving stone to cut the capillary tubing.</li>
</ol>
4. Mounting the Microinjectrode to the Microdrive (Figure 3)
<ol>
	<li>Make sure the microelectrode/experimental probe is retracted in the cannula prior to mounting. 
	NOTE: The guide tube should be in position in microdrive.</li>
	<li>Attach a custom-made adapter to the microinjectrode.</li>
	<li>Top-load the microinjectrode through the guide tube and secure it to the adapter using screws.</li>
	<li>Measure the microdrive position (depth) at which the microinjectrode protrudes from the guide tube, then retract it ~1 cm to prepare for insertion.</li>
	<li>For microinfusion experiments, connect the &#34;brain line&#34; to the unused T-junction opening of the microinjectrode. Use a standard ferrule and tighten with the ferrule wrench.</li>
</ol>
5. Flushing and Preparation of the Microfluidic System
<ol>
	<li>Position the microdrive with the microinjectrode over a waste beaker.</li>
	<li>Load chlorhexidine (e.g., nolvasan; dissolved at 20 g/L) into the 1 mL gastight syringe and place it in the Drug pump. Turn the flow direction of the valves such that fluid goes from the Drug pump through the valve to the valve line and out the &#34;brain line&#34;.</li>
	<li>Flush the circuit with chlorhexidine using a low flow rate (50-200 &#181;L/min) for a minimum of 10 min. Repeat steps 5.2 through 5.3 with sterile saline and then air. 
	NOTE: It is important to check for leaks at this stage. Gently apply lint-free wipes at the junctions to help reveal any liquid leaks through the ferrules.</li>
	<li>Load the drug in the 500 &#181;L gastight syringe, compress the air and then place in the Drug pump. Flow at 50 &#181;L/min until a few drops flow from the microinjectrode.</li>
	<li>Soak the guide tube in chlorhexidine (dissolved at 20 g/L) for 15 min.</li>
	<li>Turn the direction of the Output valve towards the &#34;flushing line&#34;. Advance the Marker pump until a clear edge of color and oil is observed on the ruler line. Make sure there is always oil between the drug and the color in order to not mix the two water-soluble materials and lose the sharp edge between them. Mark the starting position of this oil/dye line (with a piece of tape or marker).</li>
	<li>Turn the direction of the Output valve towards the brain line.</li>
</ol>
6. Performing Recording or an Infusion Experiment
NOTE: Animal handling steps will vary depending on the lab and experiment. The following steps are to be performed after the necessary surgical set up and preparation has been performed to expose the dura. Following the experiment, all necessary post-procedure steps must be performed in accordance with institutionally approved protocols.
<ol>
	<li>Attach the microdrive to the recording chamber. Lower the guide tube to penetrate the dura. 
	NOTE: The guide tube should not penetrate any further than the dura in order to avoid damaging the cortex.</li>
	<li>Lower the microinjectrode to about 2 mm above the site for recording/injection in the brain.</li>
	<li>Tighten the top ferrule (protruding microelectrode/biosensor) and connect the gold pins to the recording system. Keep advancing the microinjectrode to the target site. 
	NOTE: Remember to include the distance that the microelectrode extends beyond the cannula in the calculations.</li>
	<li>For infusion experiments, use the manual microsyringe pump to move the column of oil by 1 cm every 3 min (~60 nL/min). Once the desired volume has been infused, switch the Output valve towards the flushing line. 
	NOTE: The volume infused will vary based on model species and brain area targeted. Faster flow rates may damage neural tissue.</li>
	<li>When the experiments are complete, retract the microinjectrode within the guide tube (leave the probe protruded). Then remove the microdrive for flushing. Flush the microfluidic system as described in steps 5.1-5.5. to prepare for reuse. 
	NOTE: In our experience, the microinjectrode will last for several uses if proper care is taken. Electrophysiological recording quality drops faster than the capability of injection.</li>
</ol>

<ol>
	<li>Chen, L. T. L., Goffart, L., Sparks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+constructing+microinjectrodes+for+reversible+inactivation+in+behaving+monkeys.">A simple method for constructing microinjectrodes for reversible inactivation in behaving monkeys.</a> Journal of Neuroscience Methods. 107 (1-2), 81-85 (2001).</li><li>Crist, C. F., Yamasaki, D. S. G., Komatsu, H., Wurtz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+grid+system+and+a+microsyringe+for+single+cell+recording.">A grid system and a microsyringe for single cell recording.</a> Journal of Neuroscience Methods. 26 (2), 117-122 (1988).</li><li>Noudoost, B., Moore, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+microinjectrode+system+for+use+in+behaving+monkeys.">A reliable microinjectrode system for use in behaving monkeys.</a> Journal of Neuroscience Methods. 194 (2), 218-223 (2011).</li><li>Zhang, 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=Real-time+simultaneous+recording+of+electrophysiological+activities+and+dopamine+overflow+in+the+deep+brain+nuclei+of+a+non-human+primate+with+Parkinson's+disease+using+nano-based+microelectrode+arrays.">Real-time simultaneous recording of electrophysiological activities and dopamine overflow in the deep brain nuclei of a non-human primate with Parkinson's disease using nano-based microelectrode arrays.</a> Microsystems &amp; Nanoengineering. 4, (2018).</li><li>Altuna, A., 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=SU-8+based+microprobes+for+simultaneous+neural+depth+recording+and+drug+delivery+in+the+brain.">SU-8 based microprobes for simultaneous neural depth recording and drug delivery in the brain.</a> Lab on a Chip. 13 (7), 1422-1430 (2013).</li><li>Noudoost, B., Clark, K. L., Moore, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Distinct+Contribution+of+the+Frontal+Eye+Field+to+the+Visual+Representation+of+Saccadic+Targets.">A Distinct Contribution of the Frontal Eye Field to the Visual Representation of Saccadic Targets.</a> Journal of Neuroscience. 34 (10), 3687-3698 (2014).</li><li>Rajalingham, R., DiCarlo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+Inactivation+of+Different+Millimeter-Scale+Regions+of+Primate+IT+Results+in+Different+Patterns+of+Core+Object+Recognition+Deficits.">Reversible Inactivation of Different Millimeter-Scale Regions of Primate IT Results in Different Patterns of Core Object Recognition Deficits.</a> Neuron. 102 (2), 493 (2019).</li><li>Katz, L. N., Ates, J. L. Y., Pillow, J. W., Huk, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociated+functional+significance+of+decision-related+activity+in+the+primate+dorsal+stream.">Dissociated functional significance of decision-related activity in the primate dorsal stream.</a> Nature. 535 (7611), 285 (2016).</li><li>Esfandyarpour, R., Esfandyarpour, H., Javanmard, M., Harris, J. S., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+biosensor:+A+method+for+direct+label-free+real+time+protein+detection.">Microneedle biosensor: A method for direct label-free real time protein detection.</a> Sensors and Actuators B-Chemical. 177, 848-855 (2013).</li><li>Esfandyarpour, R., Yang, L., Koochak, Z., Harris, J. S., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoelectronic+three-dimensional+(3D)+nanotip+sensing+array+for+real-time,+sensitive,+label-free+sequence+specific+detection+of+nucleic+acids.">Nanoelectronic three-dimensional (3D) nanotip sensing array for real-time, sensitive, label-free sequence specific detection of nucleic acids.</a> Biomedical Microdevices. 18 (1), (2016).</li><li>Bahmani, Z., Daliri, M. R., Merrikhi, Y., Clark, K., Noudoost, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+Memory+Enhances+Cortical+Representations+via+Spatially+Specific+Coordination+of+Spike+Times.">Working Memory Enhances Cortical Representations via Spatially Specific Coordination of Spike Times.</a> Neuron. 97 (4), 967-979 (2018).</li><li>Veith, V. K., Quigley, C., Treue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pressure+Injection+System+for+Investigating+the+Neuropharmacology+of+Information+Processing+in+Awake+Behaving+Macaque+Monkey+Cortex.">A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex.</a> JoVE: Journal of Visualized Experiments. (109), (2016).</li></ol>

Several methods are currently available to perform simultaneous drug delivery and electrophysiology. Our system is intended to have the flexibility to be used for recordings either independently or in combination with drug injection, and to have the ability to precisely place any fragile experimental probe, such as a nanosensor or a microelectrode, protected from any damage, through the dura mater and neural tissue. The system allows precise control of drug infusion volumes with the naked eye (17 nL precision shown in previous studies in our lab3).
There are more specialized systems for pressure injection with smaller diameters12. Those systems allow for multiple recording sites, but the complex setup of software and hardware required for control of the system carries higher costs for each of the components, and has less flexibility to interface with experimental probes that are not yet commercialized on a large scale. Moreover, our injectrode does not require a chronic implant and provides a great degree of flexibility: compatible with biosensors to measure chemical and electrophysiological signals, and capable of infusing drugs as well, with the potential to measure the effect of localized drug infusions on these responses.
The design allows the experimental probe to be protruded after dura penetration in order to avoid damage to the structure of the probe. This feature allows for the multifunctionality of the device, to penetrate the dura without risking damage of any experimental probe such as nanometer-scale nanosensors10. However, there is a limitation of the length that can be protruded, restricted by the number of turns of the ferrule, limited to ~1 mm for the standard ferrules. There is minimal tissue damage due to the small cannula diameter (228 &#181;m).
In the experiment we showed, the system was used to perform controlled delivery of muscimol for reversible inactivation of FEF, simultaneously with either electrical stimulation or extracellular recording (single neuron, local field potential) using a microelectrode. This experiment in FEF requires microstimulation of the FEF to confirm saccade vectors prior to inactivation, and the drug was infused to study working memory during reversible FEF inactivation. It is unlikely that a recording from the same isolated single neuron can be maintained before and after the drug injection; however, we were able to record local field potentials before and after infusion. Here, we show an experiment combining injection, recording, and electrical stimulation.
Once it is set up, the method is very reliable and robust. However, due to precipitation of small molecules (e.g., salt) within the small tube and ports, a thorough flushing is required after each experiment in order to keep the microfluidics free of obstructions and leaks. Due to the simplicity of the entire circuit, each component can be replaced independently for easy troubleshooting.
Although the method was demonstrated in the FEF area in a non-human primate, the principle can be applied to any other brain area where some combination of electrical stimulation, recording, and drug injection are desired, in species of rodent size or larger.

Electrophysiology and drug injection methods are widely used in neuroscience to study neuronal activity and behavior, in vivo, in rodents and primates. Over the last three decades, improvements of the early injectrode models allowed a more precise and less invasive technique, and simultaneous recording and drug injection at specific brain sites1,2,3. For primates in particular, the ability to precisely deliver small volumes with minimal tissue damage is critical if the technique is to be used for the study of advanced cognitive functions that require highly trained animals. Recent advances include chronic electrophysiological and chemical measurements in combination with stimulation using implanted probes4, and combined recording and microfluidic drug delivery has recently been piloted in rodents5. The injectrode system described here allows electrophysiological recording, stimulation, and precise drug delivery, and it has already been successfully implemented in multiple primate labs6,7,8.
The increasing availability of delicate, specialized sensors, such as nanosensors9,10 with neuroscience applications, demands a reliable method for getting the probe through the dura mater without damaging the fragile nanoscale devices or microelectrode tips.
We designed a microinjectrode system that overcomes the technical challenges of combining these methods using readily available, low-cost components, and facilitates two main functions: (i) The ability to place a fragile experimental probe, such as a microelectrode or nanosensor, through the dura mater and neural tissue, protected from any damage. This functionality allows placement of the experimental probe at targeted locations, delivered by using the cannula as a guide through the neural tissue. (ii) The ability to use a microelectrode to perform experiments combining electrophysiology recordings and electrical stimulation with drug injection.
Our system uses a guide tube to penetrate the dura, along with a cannula which functions both for drug delivery (when using the system for microinfusion) and provides additional protection for the microelectrode or nanosensor (both when passing through the dura and neural tissue). This system can be easily constructed with widely commercially available components, which are inexpensive and easy to find. We minimize penetration damage by using a small diameter cannula (outer diameter OD = 235 &#181;m, inner diameter ID = 108 &#181;m).
Here we present step-by-step instructions for the microinjectrode construction and configuration of the microfluidic system. We explain the steps needed for use of the microinjectrode, either independently or coupled to the microfluidic system for drug injection. A similar approach can be applied with any fragile experimental probe, such as a nanosensor9,10. The probe can be front- or back-loaded into the cannula (depending on design), and will be protected from damage when penetrating the dura and neural tissue. We provide example data from an in vivo experiment with non-human primates, in which we used a tungsten microelectrode to perform electrical stimulation, and subsequently injected muscimol in the frontal eye field (FEF) while the animal performed a memory guided saccade (MGS) task.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-port manual valves</td>
			<td>LabSmith</td>
			<td>Manual 3-Port Selector Valve (MV201-C360)</td>
			<td><a href="https://products.labsmith.com/mv201-manual-3-port-selector-valve/#.XNYEC9NKh26">https://products.labsmith.com/mv201-manual-3-port-selector-valve/#.XNYEC9NKh26</a></td>
		</tr>
		<tr>
			<td>Cannulae</td>
			<td>Vita Needle Company</td>
			<td>304 Stainless steel tubing, Outer Diameter 228&#956;m, Inner Diameter 165&#956;m</td>
			<td><a href="https://www.vitaneedle.com/assets/files/Vita_Needle_Master_Tubing_Gauge_Chart.pdf">https://www.vitaneedle.com/assets/files/Vita_Needle_Master_Tubing_Gauge_Chart.pdf</a></td>
		</tr>
		<tr>
			<td>Cleaving stone</td>
			<td>Molex</td>
			<td>Cleaving stone 1&#34; x 1&#34; (part No. 1068680064)</td>
			<td>Highly recommended to follow method for cleaving capillary tubing: https://www.cmscientific.com/info_sheets/cleaving_procedure.pdf</td>
		</tr>
		<tr>
			<td>Clorhexidine diacetate</td>
			<td>Walmart</td>
			<td>Nolvasan solution disinfectant (AAP311)</td>
			<td>Used for microfluidic circuit flushing, dissolved at 20 g/L</td>
		</tr>
		<tr>
			<td>Custom adapter</td>
			<td>Custom provider</td>
			<td>-</td>
			<td>Custom machined adapter to connect microinjectrode to hydraulic microdrive</td>
		</tr>
		<tr>
			<td>Driver</td>
			<td>LabSmith</td>
			<td>T7 TORX driver for installing breadboard screws (LS-TORX Driver)</td>
			<td><a href="https://products.labsmith.com/ls-torx-driver/#.XO8sndNKh25">https://products.labsmith.com/ls-torx-driver/#.XO8sndNKh25</a></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>LabSmith</td>
			<td>Two-part high-strength epoxy adhesive (LS-EPOXY) for metal and plastic bonding</td>
			<td><a href="https://products.labsmith.com/ls-epoxy-12ml-epoxy-adhesive/#.XO8t89NKh24">https://products.labsmith.com/ls-epoxy-12ml-epoxy-adhesive/#.XO8t89NKh24</a></td>
		</tr>
		<tr>
			<td>Ferrule</td>
			<td>LabSmith</td>
			<td>One-Piece Fitting (C360-100) for connecting capillary, thru hole sized for 360&#956;m OD capillary</td>
			<td><a href="https://products.labsmith.com/one-piece-fitting#.XNYEaNNKh24">https://products.labsmith.com/one-piece-fitting#.XNYEaNNKh24</a></td>
		</tr>
		<tr>
			<td>Ferrule plug</td>
			<td>LabSmith</td>
			<td>One-Piece Plug (C360-101) for use in any -C360 port</td>
			<td><a href="https://products.labsmith.com/one-piece-fitting-plug/#.XNYFl9NKh24">https://products.labsmith.com/one-piece-fitting-plug/#.XNYFl9NKh24</a></td>
		</tr>
		<tr>
			<td>Ferrule wrench</td>
			<td>LabSmith</td>
			<td>1/8&#34; hex wrench for installing one-piece fittings and plugs (LS-HEX 1/8&#34; Hex Wrench)</td>
			<td><a href="https://products.labsmith.com/ls-hex-1-8-hex-wrench/#.XO8sqtNKh24">https://products.labsmith.com/ls-hex-1-8-hex-wrench/#.XO8sqtNKh24</a></td>
		</tr>
		<tr>
			<td>Gastight syringe</td>
			<td>Hamilton Company</td>
			<td>500&#956;L gastight syringe model 1750 (81220) and 1mL gastight syringe model 1001 (81320)</td>
			<td><a href="https://www.hamiltoncompany.com/laboratory-products/syringes/81220#top">https://www.hamiltoncompany.com/laboratory-products/syringes/81220#top</a></td>
		</tr>
		<tr>
			<td>Gold pins</td>
			<td>Aim-Cambridge</td>
			<td>Male gold plated crimp-on connector pin (40-9856M)</td>
			<td><a href="https://www.masterelectronics.com/aim-cambridge-cinch-connectivity-solutions/409856m-10109145.html">https://www.masterelectronics.com/aim-cambridge-cinch-connectivity-solutions/409856m-10109145.html</a></td>
		</tr>
		<tr>
			<td>Lint-free wipes</td>
			<td>Kimberly Clark</td>
			<td>Kimtech Science Kimwipes Delicate Task</td>
			<td>Lint-free wipes, used to identify leaks in the system</td>
		</tr>
		<tr>
			<td>Liquid food color</td>
			<td>McCormick &#38; Co.</td>
			<td>Water based, black liquid food color (52100581873)</td>
			<td><a href="https://www.mccormick.com/spices-and-flavors/extracts-and-food-colors/food-colors/black-food-color">https://www.mccormick.com/spices-and-flavors/extracts-and-food-colors/food-colors/black-food-color</a></td>
		</tr>
		<tr>
			<td>Low viscosity oil</td>
			<td>Clearco Products Co.</td>
			<td>Pure Silicone Fluid Octamethyltrisiloxane with a viscosity of 1cSt at 25&#176;C (PSF-1cSt)</td>
			<td><a href="http://www.clearcoproducts.com/pure-silicone-super-low-viscosity.html">http://www.clearcoproducts.com/pure-silicone-super-low-viscosity.html</a></td>
		</tr>
		<tr>
			<td>Luer-Lock connector</td>
			<td>LabSmith</td>
			<td>Luer-Lock Adapter (C360-300), female fitting for connecting Luer Lock syringe to 360&#956;m capillary tubing</td>
			<td><a href="https://products.labsmith.com/luer-lock-adapter-assembly#.XO81MtNKh24">https://products.labsmith.com/luer-lock-adapter-assembly#.XO81MtNKh24</a></td>
		</tr>
		<tr>
			<td>Micro drill bits</td>
			<td>Grainger</td>
			<td>Micro drill bit, 0.23mm (414H85)</td>
			<td><a href="https://www.grainger.com/category/machining/drilling-and-holemaking/drill-bits/machining-drill-bits/micro-drill-bits">https://www.grainger.com/category/machining/drilling-and-holemaking/drill-bits/machining-drill-bits/micro-drill-bits</a></td>
		</tr>
		<tr>
			<td>Microelectrode</td>
			<td>FHC</td>
			<td>Metal microelectrode, tungsten with epoxy insulation</td>
			<td><a href="https://www.fh-co.com/category/metal-microelectrodes">https://www.fh-co.com/category/metal-microelectrodes</a></td>
		</tr>
		<tr>
			<td>Oil hydraulic micromanipulator</td>
			<td>Narishige Group</td>
			<td>Oil Hydraulic Micromanipulator with guide tube attached (MO-96)</td>
			<td>http://products.narishige-group.com/group1/MO-96/chronic/english.html</td>
		</tr>
		<tr>
			<td>Polymicro Capillary Tubing</td>
			<td>Molex</td>
			<td>Polymicro Flexible Fused Silica Capillary Tubing (TSP150375), Outer Diameter 375&#181;m, Inner Diameter 150&#181;m</td>
			<td><a href="https://www.molex.com/webdocs/datasheets/pdf/en-us/1068150024_CAPILLARY_TUBING.pdf">https://www.molex.com/webdocs/datasheets/pdf/en-us/1068150024_CAPILLARY_TUBING.pdf</a></td>
		</tr>
		<tr>
			<td>Programmable syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>Standard Infuse/Withdraw Pump, programmable (70-2213)</td>
			<td><a href="https://www.harvardapparatus.com/standard-infuse-withdraw-pump-11-pico-plus-elite-programmable-syringe-pump.html">https://www.harvardapparatus.com/standard-infuse-withdraw-pump-11-pico-plus-elite-programmable-syringe-pump.html</a></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Empire</td>
			<td>Stainless steel 6&#34; Stiff ruler (27303)</td>
			<td><a href="http://www.empirelevel.com/rulers.php">http://www.empirelevel.com/rulers.php</a></td>
		</tr>
		<tr>
			<td>Screw set</td>
			<td>LabSmith</td>
			<td>Valve mounting screw set (LS-SCREWS .25), thread-forming screws (2-28 x 1/4&#8221;) to mount valves to breadboard</td>
			<td><a href="https://products.labsmith.com/ls-screws-25#.XO8widNKh24">https://products.labsmith.com/ls-screws-25#.XO8widNKh24</a></td>
		</tr>
		<tr>
			<td>Standard Breadboard</td>
			<td>LabSmith</td>
			<td>4&#34; x 6&#34; platform (LS600), with 0.25&#34; hole spacing for mounting fluid circuit</td>
			<td><a href="https://products.labsmith.com/standard-breadboard/#.XO8xDdNKh24">https://products.labsmith.com/standard-breadboard/#.XO8xDdNKh24</a></td>
		</tr>
		<tr>
			<td>Sterile saline (sodium chloride) 0.9%.</td>
			<td>Baxter</td>
			<td>0.9% Sodium Chloride sterile</td>
			<td>Sterile Intravenous Infusion</td>
		</tr>
		<tr>
			<td>Sterile syringe filters</td>
			<td>Millipore Sigma</td>
			<td>MilliporeSigma&#8482; Millex&#8482;-GP Sterile Syringe Filters with PES Membrane (SLGPM33RS)</td>
			<td><a href="https://www.fishersci.com/shop/products/emd-millipore-millex-sterile-syringe-filters-pes-membrane-green-4/slgpm33rs">https://www.fishersci.com/shop/products/emd-millipore-millex-sterile-syringe-filters-pes-membrane-green-4/slgpm33rs</a></td>
		</tr>
		<tr>
			<td>Stoelting manual microsyringe pump</td>
			<td>Stoelting Company</td>
			<td>Manual infusion/withdrawal pump (51222)</td>
			<td><a href="https://www.stoeltingco.com/manual-infusion-withdrawal-pump-2649.html">https://www.stoeltingco.com/manual-infusion-withdrawal-pump-2649.html</a></td>
		</tr>
		<tr>
			<td>T-junction</td>
			<td>LabSmith</td>
			<td>Interconnect tee (C360-203) for combining flow streams, for use with 360&#956;m OD capillary tubing</td>
			<td><a href="https://products.labsmith.com/interconnect-tee#.XO8z8dNKh24">https://products.labsmith.com/interconnect-tee#.XO8z8dNKh24</a></td>
		</tr>
	</tbody>
</table>

microinjectrode system for combined drug infusion and electrophysiology

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

We performed injection of a GABAa agonist (muscimol) for reversible inactivation of the frontal eye field (FEF), while the animal performed a memory guided saccade task11. In this task, the animal fixates and a peripheral visual target is presented. The animal maintains fixation while remembering the target location, and once the fixation point disappears, executes a saccadic eye movement to the remembered location to receive a reward. The microinjectrode was built according to instructions in Figure 1b. The infusion volume for the example experiment was 850 nL. Behavioral performance on the memory guided saccade (MGS) task at various locations and times relative to the muscimol infusion is shown in Figure 4. The largest performance deficits were observed at 2 to 3 h post-infusion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60365/60365fig1a.jpg" /> 
Figure 1: Step by step fabrication of microinjectrode. (a) Configuration for use independent of microfluidic system. Cannula and probe are measured in order to confirm that the tip of the probe can be protruded at the desired length (e.g., 150 &#181;m). The probe is front loaded into the cannula. The cannula is passed through the T-junction and attached on the bottom side, with the flat end in the middle of the T-junction; the back end of the probe continues through the top ferrule. The microinjectrode is finalized by soldering gold pins on each of the probe terminals and adding glue between them and the top ferrule for stability. Connection to the acquisition system depends upon the design of the probe. In this example, our probe is a nanosensor with three leads. (b) Configuration for use with microfluidic system. To couple the microinjectrode to the microfluidic system, a piece of capillary tubing is used for the top side of the T-junction. The probe can be front or back loaded. The microfluidic line is then plugged to the third T-junction opening. In this example we used a microelectrode. See the zoomed picture of the tip of a cannula in which the microelectrode was protruded by tightening the top ferrule. See the Table of Materials for a list of items used in construction. <a href="https://www.jove.com/files/ftp_upload/60365/60365fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60365/60365fig2a.jpg" /> 
Figure 2: Microfluidic system. The two-valve configuration allows control of flow direction towards the microinjectrode or towards the flushing line for troubleshooting. The circuit relies on two 3-port valves connected using capillary tubing and standard ferrules. Gastight syringes are used to carry and inject the infusion drug and the marker. A programmable syringe pump allows for automatic flushing of the system and loading of the drug. A manual microsyringe pump allows for controlled injection and visualization. <a href="https://www.jove.com/files/ftp_upload/60365/60365fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60365/60365fig3.jpg" /> 
Figure 3: Mounting of microinjectrode to a hydraulic microdrive with and without injection capacity. Step 4.1: A custom-made adapter allows for attachment of the microinjectrode to the microdrive. A single screw attaches the adapter to the microdrive; two screws secure microinjectrode to the adapter. The top ferrule should be unscrewed at least 2 turns in order to protect the tip of the microelectrode/experimental probe when loading the microinjectrode in the guide tube of the microdrive. Step 4.3: Insert microinjectrode into guide tube from the top. Step 4.4: If performing microinfusion, plug the drug line to the third T-junction opening using a plastic ferrule. <a href="https://www.jove.com/files/ftp_upload/60365/60365fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60365/60365fig4a.jpg" /> 
Figure 4: Memory guided saccade task during muscimol infusion in FEF. (a) The microinjectrode was placed in the right hemisphere, FEF area. (b) Behavioral performance during an MGS task in which eight targets are placed peripherally. We ran 4 blocks of the MGS task, before and at three times after injection. Polar plot shows performance (eccentricity) at each of these times (color), for different locations relative to the fixation point (angle on polar plot). Performance clearly decreased in the left visual hemifield 2 h after injection (blue trace, left half of polar plot). (c) Saccade traces for 8 peripheral memory locations before (left) and after muscimol injection in the FEF (right, 1 and 3 h post-infusion). Saccade accuracy in the left visual hemifield (left half of polar plots) decreased after muscimol injection. Scale in degrees of visual angle (dva). <a href="https://www.jove.com/files/ftp_upload/60365/60365fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microinjectrode System for Combined Drug Infusion and Electrophysiology at JoVE.com

Microinjectrode System for Combined Drug Infusion and Electrophysiology

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes ...

microinjectrode-system-for-combined-drug-infusion

Research

JoVE Journal

Neuroscience

6.6K Views.  University of Utah. We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

Video: Microinjectrode System for Combined Drug Infusion and Electrophysiology

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug.
The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors.
Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders.
A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.

A validated computerized battery of neuropsychological and neurophysiological tests is used to study pharmacodynamic effects on the central nervous system of newly developed drugs in early phase development. To demonstrate the test battery, the acute effects of mecamylamine and the reversal of these effects by two agonist drugs are described.

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...