This project was funded by the MINECO grants BFU201343608-P and PSI2013-49348-EXP, and the JCYL grant SA343U14 to MSM and MRC core funding to ARP. YAA held a CONACyT (216106) and a SEP fellowship.

All experimental procedures were carried out at the University of Salamanca with the approval of, and using methods conforming to the standards of, the University of Salamanca Animal Care Committee as well as the standards of the European Union (Directive 2010/63/EU) for the use of animals in neuroscience research.&#160;
1. Tungsten Electrodes
Note: The manufacture of tungsten electrodes is based on the original technique described in Merrill and Ainsworth27 and Ainsworth et al.28 and performed using the workstation setup described in Bullock et al.17.
<ol>
	<li>To record single-unit extracellular activity of IC neurons, use electrodes with a tip impedance of 1.5 - 2.5 M&#937;. Here, the steps involved in the tungsten electrode manufacture are described only briefly, since a detailed description is found in the above-mentioned references.</li>
	<li>Place tungsten wires in a custom-built alignment tool (Figure 1A) to assist cutting them to the desired length. The alignment tool consists of 30 needles (25 G) glued in parallel at 2 mm intervals, with a stop at one end to limit the length of the wires to ~40 mm. Carefully attach the loose ends of the wires with adhesive tape, and cut the rest of the wire with strong scissors.</li>
	<li>Pull gently from the tape in order to remove the tungsten wires from the alignment tool, making sure that all the wires remain attached to the tape. Roll the tape onto a polished brass spindle (Figure 1B,C), holding the wires firmly against the spindle so there is a good electrical connection.</li>
	<li>Connect the spindle to a 5 rpm motor in the workstation, and immerse the protruding wire tips into an etching solution (KNO2, 90 g per 80 ml). Angle the spindle axis 45&#176; relative to the surface of the etching solution. Immerse the wire tips so that ~1/3 of the wires contact the solution.</li>
	<li>Immerse a carbon rod electrode into the etching bath to close the circuit. Use a spring copper braid brush to apply the etching supply to the spindle shaft while the spindle is rotating. Adjust the current that is passed through the electrodes and the etching solution to 250 mA. Stop the tip etching when the current drops to 200 mA. The wire tips will be etched into very fine points.</li>
	<li>Pass one sharpened wire through a flame to remove any grease and adhesive residue. Place the wire (blunt end first) into a borosilicate glass capillary (1.5 mm O.D., 0.86 mm I.D) with its lower end blocked with modelling clay. Clamp the pipette vertically at the top and bottom. Pull the pipette using a heating coil (diameter 5 mm, depth 5 mm) and a suspended plunger. As the wire is pulled down by the weight of the plunger, the molten glass forms a very fine coat around it.</li>
	<li>Remove the glass covering the tip at the sharp end of the electrode with the help of a molten bead of sodium tetraborate. To form the bead, mix the sodium tetraborate (~0.25 g) with a small amount of water (~0.5 ml) and slowly place it on a heating element, until it melts into a bead ~2 mm in diameter. The temperature of the heating element is controlled by a potentiometer. Place the bead and heating element on a manipulator arm fixed to the table, and move it until the bead is focused under the microscope at low magnification.</li>
	<li>Secure the glass-covered wire to a slide and place it under the microscope. Move the slide with the stage knobs until the tip and tetraborate bead are in focus. Heat the bead until it melts slightly, and insert the wire tip ~10 - 15 &#181;m. Turn the heating potentiometer off. As the bead cools down, it contracts and will remove the glass from the tip of the electrode, exposing the underlying tungsten. The tungsten electrode is ready to use.</li>
</ol>
2. Multibarrel Glass Pipette Manufacturing
<ol>
	<li>Protect the ends of the multibarrel glass capillaries (five barrels in H-configuration) with heat-shrink tubing, to allow a better grip to the puller clamps and prevent breakage, and place one in the vertical puller.</li>
	<li>To obtain tip lengths of ~15&#160;mm, set the puller to the following parameters: Heat: 84, Sub magnet force: 49, Main magnet force: 38. Fine-tune these parameters according to the particular setup, as well as after changing the heating element.</li>
	<li>Pull the capillary to obtain two pipettes with the tips occluded. Mount a pipette on a slide using modelling clay and break the pipette tip under the microscope until the outer diameter is ~20 - 30 &#181;m, using fine scissors or forceps, or the flat side of a scalpel. If the broken edge is too rough, refine it using the tetraborate bead technique described in the step 1.7.</li>
	<li>Place a tungsten electrode (from section 1) in a holder made with a 20 G needle at the end of a 3-axis miniature micropositioner mounted on the microscope stage. Using forceps, bend the end of the electrode 5 - 10&#176;, ~30 mm from the tip.</li>
	<li>Under the microscope, carefully align the tungsten electrode over the multibarrel tip. Once aligned, lower the electrode until it contacts the multibarrel, fitting in the groove between the two upper barrels. Slide the tip of the electrode until it protrudes ~15 - 20 &#181;m away from the tip of the multibarrel. Make the angle between the electrode and the multibarrel as small as possible to obtain a better bonding and a thinner ensemble.</li>
	<li>Apply a small drop of light-curable adhesive ~ 5 mm away from the tips. Be careful the glue does not reach the tip to avoid blocking the multibarrel channels.</li>
	<li>Cure the glue using a blue light LED lamp. Repeat the glue application/curing if needed, for better bonding.</li>
	<li>Move the microscope stage gently until the end of the tungsten electrode is released from its holder. Remove the piggy-back ensemble from the slide.</li>
	<li>Wrap the middle section of the piggy-back electrode in a short length of heat-shrink tube and apply rapid curing epoxy appropriate for glass to further secure the tungsten electrode to the multibarrel.</li>
</ol>
3. Drugs for Microiontophoresis
Note: The drugs used for microiontophoresis must have an electrical charge when dissolved in water. Check the literature to see if the drug of interest is appropriate for this procedure. Here, the procedure for gabazine, an antagonist of the GABAA receptor, is described.
<ol>
	<li>Filter distilled water using a 0.2 &#181;m syringe filter to sterilize the water and secure containing no solutes. Prepare ~1,000 &#181;l&#160;of 20 mM gabazine using this distilled water.</li>
	<li>Adjust the pH of the dilution to 4 with 0.2 &#181;m filtered NaOH using a fine-tip pH electrode.</li>
	<li>Store 100 - 200 &#181;l&#160;aliquots at -20 &#186;C. Defrost on the day of the recording. That day, before the animal is restrained, fill the barrels (3 - 5 &#181;l) of the multibarrel electrode with the drugs, using a 100 &#181;l&#160;microsyringe fitted with a flexible plastic needle.</li>
</ol>
4. Surgery and Headpost Implantation
<ol>
	<li>Perform experiments in two male CBA/J mice (Mus musculus) with weights of 27 and 30 g. Follow surgical and recording procedures of Bryant and colleagues29, Portfors and collaborators30-32, and Duque and Malmierca26.</li>
	<li>In the days prior to the surgery, handle the animals and habituate them to the recording chamber by allowing them to explore freely. Perform 3 sessions per day, each one lasting at least 30 min. This way the animals will be calmer and comfortable during the recordings.</li>
	<li>Anesthetize the mouse using a mix of ketamine (50 mg/kg) and xylazine (10 mg/kg) injected intramuscularly. With this dose the animal is deeply anesthetized for around 1 hr. Give supplementary doses of one third of the initial dose as needed.</li>
	<li>Place the animal on a heating blanket set to maintain a temperature of 38 &#177; 1 &#186;C and stabilize the animal&#39;s head in a stereotaxic frame by using two ear bars and a bite bar. Be careful not to pierce the tympanic membrane with the ear bars.</li>
	<li>Protect the eyes by applying a drop of ophthalmic gel.</li>
	<li>Shave the scalp using scissors and apply povidone-iodine to disinfect the skin.</li>
	<li>Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the parietal and the more rostral part of the occipital bones.</li>
	<li>Glue a lightweight duralumin headpost (weight ~0.65 g, length 30 mm, Figure 2A) with a round and flat base (5 mm diameter) on the skull. Glue a silver wire to the middle of the headpost leaving its ends free. The silver wire will be used as the reference electrode for the electrophysiological recording.</li>
	<li>Apply a thin layer of light-curable adhesive on the skull and the base of the headpost. Wait 10 sec and place the headpost over the rostral area of the parietal bones, along the midline.</li>
	<li>For an effective bonding strength, light cure the adhesive using a blue light source for 20 sec.</li>
	<li>Drill three holes around and behind the base of the headpost using an electric drill and a small drill bit.</li>
	<li>Place two watch screws (~2 mm length) in the drilled holes to serve as additional contact points between the skull and the headstage. The screws require 1&#189; turns to achieve a snug fit. Test the screws with forceps to make sure they are not loose.</li>
	<li>Insert the tip of the silver ground wire into the third hole making sure it contacts the dura. Apply a small drop of water resistant cyanoacrylate glue to bind the wire to the skull.</li>
	<li>Apply light-curing composite to reinforce the bond of the headpost with the skull. Cover the base of the headpost, the low part of the silver wire, and the screws, being careful not to extend behind lambda, to allow access during recording. Cure using a blue light source.</li>
	<li>Retract the muscle at the back of the neck near its insertion on the skull. Using a small trephine (2.35 mm diameter), drill a round window just below lambda suture and lateral to the midline, to expose the inferior colliculus (IC). When the borders are loose, pull up the covering bone with fine tweezers. If bleeding occurs, rinse with cold sterile saline to stop it.</li>
	<li>Make a small (~1 mm wide and ~1 mm high) wall around the window with composite and light-cure it.</li>
	<li>Cover the exposed skull and muscle with antibiotic ointment. Then, wet the exposed surface of the IC with sterile saline and cover it with petroleum jelly, filling the well generated after making the wall around the recording window.</li>
	<li>Inject buprenorphine subcutaneously&#160;(0.03 mg/kg, diluted 1:10 in sterile saline) as an analgesic.</li>
	<li>Keep the animal on the heating blanket until it awakes and return it to its housing cage to recover from surgery for three days prior to recording sessions. House animals in individual rat&#180;s housing cage to prevent cage mates cleaning out the jelly and the headpost touch the metal-grid cover. Place some enrichment in the cage when the animal is single housed. Also, change daily the sawdust to prevent infection and carefully check that the animal recovers properly and does not show any signs of discomfort. Also check if petroleum jelly is over the recording window protecting the exposed brain.</li>
</ol>
5. Electrophysiological Recording and Microiontophoresis
<ol>
	<li>After recovery, give the animal time to acclimate to the recording environment and having its head restrained. Acclimate the animal by fixing the headpost to the holder while the mouse is sitting on a custom-made cushioned foam restrainer carved to its body&#160;size (Figure 2B). This will limit the movements of the mouse and will contribute to its calmness.</li>
	<li>Start with 10 min sessions and progressively increase the duration up to 120 min. During the session, reward the animals with sweet liquids (condensed milk, diluted 30:70 in water) at given intervals33. Later, during the recording session, give the same reward at the start and at the end of the session or while no neuron is isolated.</li>
	<li>If animal is very excited prior to the recording, inject the mild sedative acepromazine (2 mg/kg, intraperitoneal). According to the neural system of interest, the sedative can affect your results.</li>
	<li>Let the animal freely explore the recording chamber for 5 - 10 min.</li>
	<li>Place the animal&#39;s body into the foam custom-made cushioned restrainer (Figure 2B).</li>
	<li>Secure the headpost to a holder attached to the stereotactic frame (Figure 2C). This is a good time to give the animal a liquid reward.</li>
	<li>Cover the animal&#39;s body with a cotton blanket and loosely fasten it using a plastic hemicylinder and adhesive tape.</li>
	<li>Remove the petroleum jelly from the recording window and rinse with warm sterile saline.</li>
	<li>Record neural activity and iontophoretic application of gabazine.
	<ol>
		<li>Place the multibarrel electrode on its holder, controlled by a microdrive and attached to a micromanipulator.</li>
		<li>Connect the wires for recording, using small crocodile clips. Connect the positive terminal to the end of the tungsten electrode, and the ground terminal to the silver wire that contacts the dura.</li>
		<li>Place an uncoated silver wire inside each barrel, so one end contacts the solution and the other end is accessible. Connect those ends to the microiontophoresis device, following the instructions of the manufacturer.</li>
		<li>Move the multibarrel electrode until the tip contacts the surface of the brain. Apply warm sterile saline to prevent the desiccation of the brain tissue. At that point apply a retention current (-10 nA for gabazine, check the literature for the particular drug) to avoid leakage of the drug from the barrel tip while looking for single-unit activity.</li>
		<li>Use standard techniques for isolating single neuron extracellular activity. Obtain the frequency response area (FRA) of the neuron, i.e., the combination of frequencies and intensities capable of evoking a response because the neuron is sensitive to these sounds34-36.</li>
		<li>Choose two frequencies (f1 and f2) that evoke a similar firing rate and pattern. Present each of those frequencies as rare and repetitive stimulus under the oddball paradigm. 
		Note: Briefly, in the oddball paradigm, one frequency (f1) is presented as the repetitive stimulus with a high probability of occurrence, while the second (f2) is the rarely occurring deviant stimulus, interspersed randomly among the repetitive ones. In a second oddball sequence, the relative probabilities of the two stimuli are reversed. Details of the stimulation paradigm have been described previously22,37.</li>
		<li>Release the gabazine by shifting the current to positive values (10 - 20 nA). Obtain again the FRA and repeat the oddball paradigm under the gabazine application. Maintain the ejection until a visible effect in the firing rate is observed; it usually takes 5 - 20 min, depending on the particular drug, its concentration, and the magnitude of the ejection current. Repeat the recording protocol to obtain the values under the effect of the drug.</li>
		<li>Stop the injection by returning the current to retention values. Wait for the effect of the drug to wash away by monitoring that the FRA or response to the oddball paradigm returns to control values. 
		Note: The recording sessions can last 2 - 3 hr&#160;per day, and should be ended earlier if the animal shows any sign of discomfort or struggling. Give reward liquid and cover the recording chamber with petroleum jelly.</li>
	</ol>
	</li>
</ol>
6. Data Analysis
<ol>
	<li>Measure the FRA before and during the gabazine injection as well as the level of SSA.</li>
	<li>Quantify the strength of SSA response by the common-SSA index (CSI) and the frequency-specific SSA index (SI) as described&#160;previously19,22,23,38,39. The CSI and SI reflect the normalized difference between the neuronal response to the rare stimulus and the response to the repetitive one. Positive CSI and SI values indicate the neural response (spikes per stimulus) was higher to rare than to repetitive tones.</li>
	<li>Compare data from the control and drug ejection condition.</li>
</ol>

The authors have nothing to disclose.

The microiontophoresis of neuroactive substances in awake animals is a powerful technique to probe and dissect the role of specific synaptic inputs on the activity of single neurons40,41. More importantly, this procedure allows the determination of the impact of the neurotransmitters and neuromodulators on neural circuits without the potential interference of anesthetics. Here, we demonstrate that the application of gabazine in the IC of awake mice robustly changed the frequency tuning (Figure 4A</stro...

The interplay of neural excitation and inhibition is fundamental for the processing of sensory information1. It is also known that anesthesia has a strong impact on the dynamics of cortical activation and the temporal pattern of synaptic inputs2,3. For example, it has been observed that anesthetics alter the duration of visually-evoked responses in cortical neurons3,4. Moreover, the ratio between excitatory and inhibitory synaptic inputs is different in anesthetized and awake animals4,5, altering both evoked and spontaneous activity rates6,7. By measuring the synaptic conductances, Haider and colleagues4 found that inhibition matched excitation in amplitude under anesthesia whereas during wakefulness, inhibition was stronger than excitation. These findings prompt the development of experimental procedures to study the impact of specific synaptic inputs on sensory processing in awake animals.
The controlled ejection of charged neuroactive substances by applying small current injections (on the order of nA) has been extensively used to study the contribution of synaptic inputs and the role of putative cell receptors in sensory processing8-13. This technique, known as microiontophoresis, allows the application of drugs in the vicinity of the recorded neuron, which contributes to a rapid and confined effect. This procedure is more suitable for studying local effects of neuroactive substances, compared to the widespread effect elicited by other experimental manipulations such as systemic injections, microdialysis or the use of optogenetic techniques. Usually, a piggy-back electrode configuration14,15 is used to simultaneously record the target neuron and deliver the neuroactive substances of interest. It consists of a recording electrode attached to a multibarrel pipette that carries the neuroactive substances. Modifications of the original procedure described by Havey and Caspary14 have been implemented. For example, a tungsten electrode, instead of a glass one,&#160;can be used to record the neural activity16. Previously published methods for the manufacture of tungsten electrodes17,18 involve three general steps: electrolytic etching of tungsten wire tips, glass insulation, and adjustment of the tip exposure to meet recording requirements.
An interesting and emergent field in auditory neuroscience is the study of stimulus-specific adaptation (SSA19). SSA is a specific decrease in the neural response to repetitive sounds that does not generalize to other, rarely presented sounds. The importance of SSA resides in its potential role as a neural mechanism underlying deviance detection in the auditory brain, as well as a possible neuronal correlate for the late mismatch negativity component of the auditory evoked potential20,21. SSA occurs from the IC up to the auditory cortex19,22-24. GABAA-mediated inhibition has been demonstrated to act as a gain control mechanism on SSA7,16,25, which has also been shown to be affected by anesthesia26. Here we present a protocol that combines previously described methods for recording the single-unit activity of IC neurons before and during the application of a selective antagonist of the GABAA-receptors in awake mice. First, we describe the manufacture of piggy-back electrodes and next, the surgical and recording methods. To test for the efficacy of drug release, we compared the receptive field as well as the level of SSA of IC neurons before and during the microiontophoretic ejection of gabazine.

<table><tbody><tr><td>Tungsten wire</td><td>Harvard Apparatus LTD</td><td>33-0099</td><td>0.005 inches x 3 inches</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus LTD</td><td>30-0053</td><td>Borosilicate standard wall without filament, 1.5 mm OD, 0.86 mm ID, 100 mm long</td></tr><tr><td>Multibarrel glass capillaries&amp;nbsp;</td><td>World Precision Instruments</td><td>5B120F-4&amp;nbsp;</td><td>5-barrel capillary, 4 inches long, 1.2 mm OD, with filament</td></tr><tr><td>Diaplus</td><td>DiaDent</td><td>2001-2101</td><td>Light-curing adhesive, used to attach the tungsten electrode to the glas multibarrel pipette</td></tr><tr><td>G-Bond</td><td>GC Corporation</td><td>2277</td><td>Light-curing adhesive, used to attach the headpost to the animal&#039;s skull</td></tr><tr><td>Charisma</td><td>Heraeus Kulzer</td><td>66000087</td><td>Light-curing composite, used to reinforce the bond of the headpost with the skull</td></tr><tr><td>Araldit Cristal</td><td>Ceys</td><td></td><td>2-component expoxy, used to further secure the attachment of the tungsten electrode to the glass multibarrel pipette</td></tr><tr><td>Heating blanket</td><td>Cibertec</td><td>RTC1</td><td></td></tr><tr><td>Stereotactic frame</td><td>Narishige</td><td>SR-6N</td><td>Modified for mice</td></tr><tr><td>Microiontophoretic device</td><td>Harvard Apparatus LTD</td><td>Neurophore BH-2</td><td>Including IP-2 iontophoresis pumps (one for each drug delivery channel) and a balance module</td></tr><tr><td>Multibarrel glass pipette puller</td><td>Narishige</td><td>Model PE-21</td><td></td></tr><tr><td>LED lamp</td><td>Technoflux</td><td>CV-215</td><td>5 W, 430-485 nm</td></tr><tr><td>MicroFil</td><td>World Precision Instruments</td><td>MF34G-5</td><td>Flexible plastic needle, 34 AWG</td></tr><tr><td>Imalgene</td><td>Merial</td><td></td><td>Ketamine, 100 mg/mL</td></tr><tr><td>Rompun</td><td>Bayer</td><td></td><td>Xylazine, 20 mg/mL</td></tr><tr><td>Gabazine / SR-95531</td><td>Sigma</td><td>S106</td><td>Prepare ~ 1000&amp;micro;l of 20 mM gabazine in distilled water and adjust the pH to 4</td></tr></tbody></table>

extracellular recording of neuronal activity combined with microiontophoretic application of neuroactive substances in awake mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We recorded the single-unit activity of 4 well-isolated neurons of the IC. Typical signal-to-noise ratios obtained during extracellular recordings in awake mice are shown in Figure 3B. Figure 4A shows the frequency response area (FRA) of each neuron before and during the blockade of the GABAA-receptors with gabazine. An increment in the response strength (spikes/stimulus) as well as a broadening of the spectral tuning was observed. The incremen...

Watch this Scientific Journal Video about Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice at JoVE.com

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

extracellular-recording-neuronal-activity-combined-with

Research

JoVE Journal

Neuroscience

10.0K Views.  University of Salamanca. We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Video: Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Behavior

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

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

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

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

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

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.

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 ...

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Angled Silicon Multielectrode Recordings for Deep Brainstem Structures in Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

Author Spotlight: Investigating Anesthesia-Induced Sleep Pathways and Neuronal Excitability in Mice

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Samenvatting

Meer video's verkennen