Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

NOTE: All procedures were performed in accordance with the directives of the Canadian Council for the Protection of Animals and the Ethics review board of the Universit&#233; de Montr&#233;al.
1. Assembly of the Recording-injection Pipette
<ol>
	<li>Pull an approximately 7 cm long glass capillary (1 mm outer diameter) using a pipette puller.</li>
	<li>Break the tip of the capillary and check the aperture under a light microscope. Confirm that the inner diameter is between 30 &#181;m to 40 &#181;m.</li>
	<li>Insert a 7 cm long silver wire into the glass capillary with approximately 1 cm protruding from the non-tapered end of the glass pipette.</li>
	<li>Bend the excess filament orthogonally to the glass capillary.</li>
	<li>Apply a droplet of flexible plastic adhesive on the shaft of a 30 G hypodermic needle.</li>
	<li>Insert a 30 G hypodermic needle in the glass pipette according to the schematics presented in Figure 1.</li>
	<li>Add a second coating of glue to ensure a proper seal from the junction between the glass pipette and the hypodermic needle.</li>
	<li>Leave the pipette to dry with the tip facing upward for about 12 hr to ensure proper curing of the glue. The finished result is shown in Figure 2.</li>
</ol>
NOTE: This procedure is done on acute experiments and sterilization of the pipette tip is not required.
2. Animal Preparation
<ol>
	<li>Place the rat in an anesthesia box.</li>
	<li>Induce anesthesia using 4% isoflurane for 5 to 10 min.</li>
	<li>Place the animal on a stereotaxic table with heating pad and rectal probe to maintain a body temperature of 37 &#176;C. Use a nose cone to maintain anesthesia with 2% isoflurane. Secure the rat&#8217;s head using ear bars and teeth holder.</li>
	<li>Apply ophthalmic ointment or eye drops, which include 1% Atropine drops to aid in pupil dilation.&#160; To prevent dryness, apply lubricating drops approximately every 30 min.</li>
	<li>Shave the head and clean it with 10% povidone-Iodine.</li>
	<li>For local anaesthesia, inject 0.5 ml of 2% lidocaine under the scalp in 2-3 locations by lifting up the skin and inserting the tip of the needle.</li>
	<li>Confirm adequate level of anesthesia by performing a toe pinch and observing the lack of movement. In addition, monitor the heart rate to ensure that it is within normal values (300 to 400 beats/min).</li>
	<li>Incise the scalp in a straight line along the median with a #10 scalpel blade to expose both the coronal and sagittal sutures.</li>
	<li>Reveal the Lambda and Bregma points by pushing aside the tissue that is covering the cranium with a surgical spatula.</li>
	<li>Level the cranium so that Bregma and Lambda positions are on the same plane.</li>
	<li>To set the reference point, use a stereotaxic device with a mounted glass tube to set it right above Bregma. This will be the &#8220;zero&#8221; for the antero-posterior and medial-lateral measurements coordinates.</li>
	<li>Set the point of interest by moving the stereotaxic mount to the required coordinates, note the stereotaxic coordinates and draw a square around the target area that marks where the craniotomy will be performed.</li>
	<li>Use a surgical drill with a sterilized drill along the marked square slowly without pressure to slowly remove the bone material. Be careful not to drill too long in the same area, as it will produce heat and cause lesions on the cortex.</li>
	<li>When the bone delimiting the craniotomy has become sufficiently thin, carefully remove the cranial section with tweezers to expose the cortex.</li>
	<li>Frequently irrigate the exposed cortex with artificial cerebral spinal fluid to prevent tissue desiccation. 
	NOTE: Dura mater removal is unnecessary on the rat as the tip of the injectrode is sturdy enough to penetrate.</li>
</ol>
3. Filling and Mounting of the Injection System
<ol>
	<li>Fill the 5-10 &#181;l microsyringe by aspiration with mineral oil.</li>
	<li>Fill the hypodermic needle with the fixed glass pipette with a solution of 0.5% Chicago Sky Blue (CSB) and 300 &#181;M &#947;-aminobutyric acid (GABA) or a solution of 2% lidocaine with 0.5% CSB 9. Dilute all solutions with saline.
	<ol>
		<li>In the case of an abundant substance, fill using a regular syringe and use the usual precautionary techniques to avoid the formation of air bubbles.</li>
		<li>In the case of more expensive substances, use mineral oil to fill the injectrode and the chemical agent can then be introduced by aspiration. As the density difference between mineral oil and water is relatively high, this substance is a good candidate for the injection of aqueous solutions. 
		NOTE: A dye can be added to confirm the separation between both liquids.</li>
	</ol>
	</li>
	<li>To fill the injection pipette, fill a 1 ml syringe with the solution by aspiration and then slowly inject the solution into the injection pipette.</li>
	<li>Pay careful attention for leaks in regions indicated in Figure 1 by swabbing these areas clean and observing leakages by further injecting the solution slowly with the syringe.</li>
	<li>Remove the 1 ml syringe. When doing so, be sure to keep light pressure on the plunger so that the vacuum does not remove the solution from the injection pipette.</li>
	<li>Fill the microsyringe with mineral oil by aspiration and attach it firmly to the filled injection pipette, then carefully wipe away any excess solution from the assembled injectrode with gauze.</li>
	<li>Verify that the tip is not blocked by injecting a very small volume, enough to see a small drop forming at the tip of the glass pipette.</li>
	<li>Mount the injectrode on the micropump system and ensure that it is well fixed.</li>
	<li>Carefully position the injectrode tip at the target coordinates and lower the tip to the surface of the cortex.</li>
	<li>Slowly lower the injectrode using the stereotaxic apparatus to the target structure (superior colliculus in this case) using the appropriate anatomical coordinates.</li>
	<li>Cover the exposed cortex with warm agar to prevent tissue desiccation.</li>
</ol>
4. Injection and Reversible Inactivation
<ol>
	<li>Set the microinjection pump to inject 400 to 800 nl at 40 nl/min and press Run to start the injection. Note that spike rate will show reduction during the injection. 
	NOTE: In the experimental setup, neural activity recovered within an hour after the end of GABA delivery. Any calibrated mechanical device can be used to apply pressure on the microinjection syringe in order to conduct the injection.</li>
	<li>After acquisition of the electrophysiological data, euthanize using a method approved by the local Animal Ethics Community.</li>
</ol>

<ol>
	<li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inactivation+in+the+analysis+of+the+central+control+of+movement.">Pharmacological inactivation in the analysis of the central control of movement.</a> Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).</li><li>Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+indirect+pathways+to+visual+response+properties+in+macaque+middle+temporal+area+MT.">Contributions of indirect pathways to visual response properties in macaque middle temporal area MT.</a> The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).</li><li>Lomber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+limitations+of+permanent+or+reversible+deactivation+techniques+in+the+assessment+of+neural+function.">The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).</li><li>Malpeli, J., Schiller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+reversible+inactivation+of+small+regions+of+brain+tissue.">A method of reversible inactivation of small regions of brain tissue.</a> Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).</li><li>Lomber, S. G., Payne, B. R., Horel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryoloop:+an+adaptable+reversible+cooling+deactivation+method+for+behavioral+or+electrophysiological+assessment+of+neural+function.">The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 179-194 (1999).</li><li>Gonzalez-Perez, O., Guerrero-Cazares, H., Qui&#241;ones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs,+viral+vectors,+or+cell+transplants.">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> Journal Of Visualized Experiments. (46), (2010).</li><li>Hup&#233;, J., Chouvet, G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+parameters+of+cortical+inactivation+by+GABA.">Spatial and temporal parameters of cortical inactivation by GABA.</a> Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).</li><li>Casanova, C., McKinley, P., Molotchnikofff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+of+Reorganized+Primary+Somatosensory+(SI)+Cortex+after+Local+Inactivation+of+Normal+SI+Cortex+in+Chronic+Spinal+Cats.">Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats.</a> Somatosensory &amp; Motor Research. 8 (1), 65-76 (1991).</li><li>Malpeli, 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+subcortical+sites+by+drug+injection.">Reversible inactivation of subcortical sites by drug injection.</a> Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).</li><li>Minville, K., Casanova, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+frequency+processing+in+posteromedial+lateral+suprasylvian+cortex+does+not+depend+on+the+projections+from+the+striate-recipient+zone+of+the+cat&#8217;s+lateral+posterior-pulvinar+complex.">Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat&#8217;s lateral posterior-pulvinar complex.</a> Neuroscience. 84 (3), 699-711 (1998).</li><li>Diao, Y., Wang, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+of+the+binocular+visual+field+in+the+superior+colliculus+of+the+albino+rat.">Representation of the binocular visual field in the superior colliculus of the albino rat.</a> Experimental Brain Research. 52 (1), 67-72 (1983).</li></ol>

We have nothing to disclose.

The proposed protocol was designed to solve the challenges arising from current reversible inactivation methods. Specifically, this project aimed at refining the methods used for chemical microinjections of substances modulating neural activity, particularly in deep brain structures. A technical challenge emerging from this type of setup is the need for both probes to be colocalized in the same restricted space in vivo in order to derive precise recordings at the injection site. This issue can be overcome by usi...

The inactivation of cortical areas and sub-cortical nuclei is important in the study of functional relations between various brain structures2-4. Recent literature has employed loss-of-function chemical or cryogenic techniques to study the role of brain structures2,5. In regard to pharmacological microinjections, small volumes of drugs can be administered into a brain region at a controlled rate while minimizing the collateral damage to the surrounding tissue6,7. This technique can be used to deliver specific agonists, inverse agonists or antagonists to study the effect of different pharmacological targets on neuronal activity. Such effects can also be studied by measuring changes in neuronal responses from distant locations, allowing researchers to study the relationships between different cortical and subcortical structures.
Here, we demonstrate the assembly of a device, the injectrode, capable of both recording electrophysiological signals and delivering small amounts of drugs at the target location. We demonstrate the capabilities of this system by injecting GABA, a common inhibitor of neuronal activity, in the rat superior colliculus. This region is sensitive to visual stimulation, which allowed us to use visually evoked multiunit activity&#160;to confirm injectrode localization. The reversibility of the inactivation was assessed by the recovery of normal neuronal activity following the end of GABA injection.
The ability to monitor multi-unit activity from the injection site allows for the fine tuning of the injection rates and volumes needed to achieve the desired pharmacodynamic response. Therefore, an advantage of this technique is the potential limiting of tissue damage caused by microperfusion, since the smallest effective volumes are injected. The proposed protocol provides a cost efficient method for generating the disposable hardware necessary for conducting experiments where drug delivery and local neuronal activity recording is desired.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Name of the reagent</td>
			<td style="width:209px;">Company</td>
			<td style="width:184px;">Catalogue number</td>
			<td style="width:308px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection pump (UltraMicroPump III)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#UMP3</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection console (Micro4 Controller)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#SYS-MICRO4</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Hamilton syringe</td>
			<td style="width:209px;">Hamliton</td>
			<td style="width:184px;">(80301) 701LT 10 &#181;L SYR</td>
			<td style="width:308px;">Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Gel cyanoacrylate adhesive</td>
			<td style="width:209px;">Krazy Glue</td>
			<td style="width:184px;">KG86648R</td>
			<td style="width:308px;">The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Glass pipettes</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#TW100F-4</td>
			<td style="width:308px;">Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">720 Needle Pipette Puller</td>
			<td style="width:209px;">Kopf</td>
			<td style="width:184px;">720</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Silver wire</td>
			<td style="width:209px;">A-M Systems, Inc.</td>
			<td style="width:184px;">782500</td>
			<td style="width:308px;">Bare 0.010&#8221;</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

The construction of the injectrode is illustrated in Figure 1. A silver wire (C) is fed into a glass pipette (D) with a portion of the wire bent and protruding out from the opening. A 30 G needle (B) is attached and sealed to the opening of the glass pipette with glue. After the pipette has been filled with the injection substance, a glass micro syringe (A) is attached to the needle. It is important that there is a good seal where the micro syringe connects with the needle (E) and where the silver wire p...

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain at JoVE.com

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

simultaneous-electrophysiological-recording-micro-injections

Research

JoVE Journal

Neuroscience

8.0K Views.  Université de Montréal. Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Video: Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

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

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 ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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

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

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

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

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...