Name: Single-unit In vivo Recordings from the Optic Chiasm of Rat (Video) | JoVE Journal
Uploaded: 2010-04-02T15:15:00.000+00:00
Duration: 11 min
Description: Learn single-unit in vivo recordings from the optic chiasm of rat using tungsten-stained glass electrodes. This technique enables high-quality action potential recordings from retinal ganglion cells for visual neuroscience research.

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5 minutes

We thank Dr Dan Green for providing technical input during the development of these experimental techniques. This work was supported by NIH Grant R01-EY016849A and the Smith Family New Investigator Award.

Part I: Fabrication of Tungsten-in-Glass Electrodes
<ol>
 <li>A graphite ring is placed in a beaker filled with a concentrated solution of sodium nitrate (0.71g/mL) and potassium hydroxide (0.34g/mL) dissolved in water1, and the beaker is placed under the sewing arm of a modified sewing machine. </li>
 <li>Tungsten wire (0.5mm diameter, 6cm length) is taped to the sewing arm so that it is partially immersed (1-2cm) in solution. </li>
 <li>2V are applied across the tungsten wire by attaching a positive lead to the sewing arm and a negative lead to the graphite ring. </li>
 <li>The sewing machine is turned on and the tungsten wire is dipped repetitively into solution at a rate of ~4Hz for 2 min, which electrolytically etches the metal to a sharp point. </li>
 <li>The etched wire is placed on a manual micromanipulator and the point aligned under microscopic guidance (40x magnification) with the shaft of a glass pipette (0.25mm OD, 0.175mm ID) pulled to a 1mm tip with a glass puller. To prevent movement during wire insertion, the glass is temporarily secured to the microscope stage with putty.</li>
 <li>The wire is carefully advanced into the unpulled end of the glass, down the shaft, and through the 1&mu;m opening A small piece of glass is seen to break off the tip in the video, indicating a tight fit of the tungsten in the glass. The wire should extend around 5-10mm beyond the tip for rat optic fiber recordings.</li>
 <li>Cyanoacrylate is applied to the unpulled end of the glass and after hardening the electrical impedance is measured, where 1M&Omega; electrodes generally work best.</li>
</ol>
Part II: Stereotaxic Setup and Spike Train Recordings
<ol>
 <li>The stimulus display system is illustrated in the video, along with the stereotaxic apparatus and microdrive for positioning and lowering the electrode into the brain.</li>
 <li>To perform retinal spike train recordings the rat is anesthetized and later paralyzed. Standard procedures (not illustrated) are used for surgical insertion of a femoral vein cannula for drug delivery and a tracheal (Y-tube) cannula for mechanical ventilation during paralysis. The experiments are terminal for the animal. </li>
 <li>After preparatory procedures, the animal is placed on the heating blanket and the head is fixed into stereotaxic position, as defined by rat brain atlas2, with ear and tooth bars. </li>
 <li>The rectal probe is inserted, which controls the heating blanket via a homeothermic control unit that maintains body temperature at 37&deg;C.</li>
 <li>Metal needle are used as leads to measure the electrocardiogram and monitor heart rate. </li>
 <li>The skin is opened on top of the skull and the intersection of skull plates (bregma) is exposed. A custom-designed crossbar frame is set in place, a circle is marked on the skull around begma through a hole in the crossbar, and the crossbar is temporarily removed.</li>
 <li>The stereotaxic system is calibrated by positioning the guide needle over bregma. A 5-mm hole is drilled into the skull at the marked location and the bone is removed, exposing the brain. A thin ring of putty is placed around the opening in the skull to form a seal around the hole in the crossbar, which is reset into place. </li>
 <li>If the animal has been at a stable plane of anesthesia for awhile, it is injected with paralytic in order to prevent eye movements during data collection. After breathing stops the animal is mechanically ventilated. A custom program tracks ventilation and physiological parameters throughout the experiment and informs the experimenter if compensatory action is needed should any parameters go outside normal levels.</li>
 <li>The guide needle is backloaded with the tungsten electrode and lowered into the brain. After brain penetration the crossbar hole is filled with agar (not shown), and the microdrive assembly is clamped to the crossbar for mechanical stability. The tungsten electrode is then slowly advanced (2&mu;m/sec) out the guide needle until the tip is about 8-9mm below the skull, where the optic chiasm is located.</li>
 <li>Spike trains belonging to retinal ganglion cells are identified by their response to visual input. Upon isolation of a single optic fiber the receptive field of the recorded cell is mapped in visual space and its response properties are characterized with drifting gratings or other visual patterns of interest. The stability of the system allows for single-unit recordings of several hours3.</li>
</ol>
Materials
Adult Brown-Norway rats (250-400g) were purchased from a commercial vendor and housed under a regulated (12/12) light/dark cycle. Anesthesia was induced with an intraperitoneal injection of ketamine hydrochloride and xylazine (70 and 2 mg/kg, respectively, Henry Schein Inc) and maintained for the duration of the experiment with an intravenous infusion of ketamine and xylazine (30 and 1 mg/kg/hr) mixed with dextrose, saline, and gallamine triethiodide (40 mg/kg/hr, Fischer Inc) through a catheter (0.13mm OD, Small Parts Inc) in the right femoral vein. Gallamine is included in the mixture to paralyze eye movements. The infusion rate of the pump (WPI Inc) was adjusted as needed to maintain a stable plane of anesthesia as assessed by heart rate and blood pressure variability. After paralysis, the animal was mechanically ventilated (Harvard Apparatus, Model 683) through a tracheal cannula at 60-80 breaths/min (2cc volume), with the rate adjusted as necessary to maintain end-tidal CO2 measured with an in-line capnometer (Novametrix Inc, Model 710Sp) at 30%. Body temperature was regulated via a homeothermic blanket control system (Harvard Apparatus Inc). Body temperature, end-tidal CO2, heart rate, and blood pressure were continuously monitored throughout the experiment by a LABVIEW program. Visual stimuli were presented on a Sony Multiscan 17e CRT monitor (mean luminance of 30 cd/m2) running at 100 Hz with a resolution of 800x600 pixels. Data acquisition and monitor output were controlled with custom software written in Matlab and LabView in conjunction with a video image processor (Cambridge Research Systems Inc, Bits++) and the Psychophysics Toolbox4. The animal viewed the stimulus display (40.4 x 30.2 cm) at a distance of 16.5cm through contact lenses (Ocular Instruments Inc), and ophthalmic solution was applied periodically during the experiment to keep the eyes moist. The visual field accessible to stimulation was maximized by reverse mounting the animal in a stereotaxic apparatus (Stoelting Co) elevated 14cm above the surface of a floating table (TMC Inc) and setting the monitor on a custom-designed sled that moved on the table along a &plusmn;100 deg arc about the nose. This provided a repositionable display field that extended 60-deg above and 35-deg below eye level and &plusmn;42-deg laterally from the center of monitor. Tungsten electodes were fabricated with wire obtained from Small Parts Inc and custom borosilicate glass obtained from Friedrich and Dimmock Inc. A standard micropippete puller (Sutter Instruments, Model P-97) was used to shape the glass tip. The electrode impedance tester was purchased from Bak-Electronics. Electrodes were advanced with a motorized microdrive system (Newport Inc, StepperMike). Electrocardiogram and optic nerve signals were amplified and filtered with a high input impedance multielectrode amplifier (FHC Inc, X-Cell 3x4). Heart beats were detected with a window discriminator (WPI Inc, Model 121). Nerve action potentials were detected and time stamped with 0.1ms resolution by a digital spike discriminator (FHC Inc, APM). The spike discriminator was gated by a trigger signal from the video image processor so that spike times were locked to the stimulus.

<ol><li>Levick, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Another+tungsten+microelectrode%5BTitle%5D)+AND+%22Med+Biol+Eng%22%5BJournal%5D)">Another tungsten microelectrode.</a> Med Biol Eng. 10 (4), 510-515 (1972).</li>
<li>Paxinos, G., Watson, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+rat+brain+in+stereotaxic+coordinates%22">The rat brain in stereotaxic coordinates</a>. , Fourth Edition, Academic Press. London, UK. (1998).</li>
<li>Freeman, D. K., Heine, W. F., Passaglia, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maintained+discharge+of+rat+retinal+ganglion+cells%5BTitle%5D)+AND+%22Vis+Neurosci%22%5BJournal%5D)">The maintained discharge of rat retinal ganglion cells.</a> Vis Neurosci. 25 (4), 535-548 (2008).</li>
<li>Brainard, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+psychophysics+toolbox%5BTitle%5D)+AND+%22Spat+Vis%22%5BJournal%5D)">The psychophysics toolbox.</a> Spat Vis. 10, 433-436 (1997).</li>
</ol>

All experimental procedures were approved by the Institutional Animal Care and Use Committee at Boston University.

Optic fiber recordings are an attractive approach for addressing experimental questions about retinal information encoding and transmission that require an intact eye. Moreover, the signaling properties of both eyes can be studied in virtually the same physiological state if the electrode is positioned in the optic chiasm or tract where the activity of crossed and uncrossed optic nerve fibers can be recorded with a single electrode penetration. Optic fiber recordings are common in cat but not in other popular animals mod...

single-unit in vivo recordings from the optic chiasm of rat

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

We'll be demonstrating today a recording system we developed to record in vivo from the optic chiasm of rat. These types of recordings have been very rare in rat, in part because the retinal ganglion cell axons that make up the optic nerve are very small, making it difficult to record from individual cells, and since rat is a common animal model for retinal degeneration, these types of recordings have the potential to produce a lot of useful information. The key to achieving these recordings was in the tung stain glass electrodes that we designed that have a long sharp tip that allow action potentials to be recorded from thin diameter axons with a high signal to noise ratio.We'll walk you through the fabrication process of these electrodes and we will show you how to, how we set up the animal on the stereotactic apparatus, and then we will show you an example of recording spike trains from a single ganglion cell. Three main pieces of equipment are needed to fabricate electrodes, a standard glassblower to make the micro pipettes. A sewing machine is used to sharpen the tungsten and a standard microscope with 40 x objective.In addition, a graphite ring is needed with attached pole and it will be placed in the concentrated solution of potassium hydroxide and sodium nitrate. This is placed under the sewing arm, and then a thin piece of tungsten wire is then taped to the sewing arm, partially immersed in solution. Leads are then applied to the graphite pole on one end into the sewing arm.On the other two volts will be applied across the leads and the sewing machine is turned on. This dips the dunston into solution and electromagnetically sharpens it for two minutes. Once the tungsten is sharpened, it is carefully removed from the sewing arm, Then dipped into water to remove excess solution, and then placed under the microscope under 40 x objective.The glass micro pipette has a small diameter opening about one to two microns in diameter. The sharpened tungsten is then carefully inserted into the back end of the micro pipette and pushed with a mechanical micro manipulator. The tungsten is pushed through the end of the glass very carefully.It is important that there is a tight fit between the tungsten and the glass, and it is common to push enough so that a small amount of glass will actually break off. A small amount of crazy glue can be applied to the back end of the micro pipet to fasten the tung to the glass. Now that the electrode fabrication is complete, its impedance is measured using a metal electrode Impedance testing.Generally we look for impedances near one mega ohm. The electrode is then put away in a case for safekeeping, as it is very fragile. Visual Stimuli are presented on a monitor that is placed on a movable platform.Heating blanket is used to control the body temperature of the animal, and the stereotactic system allows gross movement of the micro drive, which is shown here. The metal case in the hypodermic guide needle shield, the recording electrode in act to reduce noise levels. The guide needle is inserted into the brain and a micro drive is used to slowly lower the electrode through the guide needle.This is illustrated here where the metal case is lowered onto a crossbar that will rest on the animal's skull. The guide needle will stick out below the crossbar as shown, and the electrical contact between the metal case and the crossbar acts to reduce noise levels. The animal is then placed on the heating blanket.The ear bars are carefully inserted. It is important at this point to make sure the head remains level. A tooth bar is then inserted to make sure the head is in all directions.A rectal Probe is inserted using Vaseline. This will measure body temperature, which is then used to control the temperature of the heating blanket. The body temperature should remain between 36 and 37 degrees.A single EKG lead is inserted into the skin to monitor heart rate, which is generally around 300 feet per minute. Heart rate is a good indicator of anesthetic depth. The vital signs of the animal are continuously monitored with custom software.These include blood pressure entitled CO2 body temperature and heart rate. Skin is then removed from the top of the skull and the skull is exposed. It is important to thoroughly clean the skull in order to see the proper skull markers that are used to find the optic chiasm.The crossbar assembly is then put into place. This is used to trace out a five millimeter circle on the skull that will then be drilled and removed. The hole will be drilled around the bgma skull Marker illustrated here.The exposed skull is shown with a circular pencil mark around bgma, which will be drilled before the skull is drilled. The stereotaxic system must be calibrated torema, and this is done by lining up the guide needle precisely on bgma and documenting the position of the stereo. After drilling, the skull has been removed and the brain is exposed.After the skull is drilled, the animal is paralyzed and ventilated in order to prevent eye movements. This is done with an intravenous injection of gamine. The breathing is carefully monitored as it is, and when the breathing stops, the ventilation tubing is inserted into the tracheal connector, at which point the animal is artificially respir.The guide needle is then lowered into the brain. The recording electrode will be lowered through the gyal to about eight to 10 millimeters below the skull where action potentials from visual cells in the optic chiasm can be recorded. The electrode is slowly load into the brain.With The micro drive, a visual cell can be heard spiking spontaneously listen to the cell, respond to the presence of a handheld wand in its receptive field. Drifting grading is then presented, and at this point, any visual stimuli of interest can be used to study the response properties of retinal ganglion cells using optic chiasm.Recording.

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Research

JoVE Journal

Neuroscience

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Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

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Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

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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.

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Learn single-unit in vivo recordings from the optic chiasm of rat using tungsten-stained glass electrodes. This technique enables high-quality action potential recordings from retinal ganglion cells for visual neuroscience research.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

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