Methods in Neurosciences: Electrophysiology and Microinjection

preparation)

Recording Methodologies

Outline

Single-Unit Recording in Conscious Sheep

Single-Unit Recording from Pontomedullary Neuraxis in Awake, Freely Behaving Animals

Measurement of Whole-Cell Calcium Current in Cardiac Myocytes

Measurement of Noninactivating Calcium Current in Smooth Muscle Cells

Two-Suction Electrode Voltage-Clamp Recording

1

Single-Unit Recording in Conscious Sheep

K.M. Kendrick and B.A. Baldwin

Publisher Summary

This chapter discusses single-unit recording in conscious sheep. The use of sheep in neuroscience offers considerable economic and laboratory management advantages over primates. Electrophysiological work over the past few years in the fields of central control of food recognition and intake, and visual recognition of individuals has shown that this species can be used in this context and has many similarities with primates. The large size of the sheep and the fact that it readily adjusts to the restraint system that such recordings normally require make it an ideal candidate for use as a conscious experimental animal for electrophysiology. Although the sheep is not as easy to train to perform operant tasks as some other laboratory animals, it is possible to do this and it can therefore be used in experiments concerned with neuronal correlates of learning and memory. The chapter also discusses techniques developed for neurochemical sampling, using microdialysis, recording electroencephalogram and evoked potentials, and electrical or thermal stimulation of localized regions of the brain.

Introduction

Although the sheep is an established experimental animal in the field of neuroendocrinology, it has not been used extensively by neuroscientists outside of this field. The use of sheep in neuroscience offers considerable economic and laboratory management advantages over primates. Our electrophysiological work over the past few years in the fields of central control of food recognition and intake (1–4) and visual recognition of individuals (5–7) has shown that this species can be used in this context and has many similarities with primates. We have developed techniques for neurochemical sampling, using microdialysis (8, 9), recording electroencephalogram (EEG) and evoked potentials (10, 11), and electrical (11, 12) or thermal (13) stimulation of localized regions of the brain. In this chapter we will describe the technique for the extracellular recording of single-unit activity from the brain of the conscious sheep and present examples from our work in the context of central control of food recognition and intake, and visual recognition of animals and humans. The method used is an adaptation of one that has been described previously for use in sheep and goats (14).

Methods

Animals

For single-unit recording we currently use a horned breed of sheep (Dalesbred). The presence of horns allows for an easy method of keeping the head still (see below) although, where this is not essential, it is also possible to use nonhorned breeds. We have been able to make successful recordings from Clun Forest ewes, for example. Whatever breed is used, however, it is essential to habituate the animals to the presence of humans and to the experimental procedures used. Prior to and during recordings animals are housed in individual pens, although with both visual and auditory contact with other sheep, and fed twice a day on a diet of concentrates and chopped hay with ad libitum water and access to a mineral lick.

Surgical Preparation

Surgery is performed under general anesthesia [induced by a 10 mg iv injection of sodium methohexitone (Brietal, Elanco Products, Ltd., London, England) followed by closed circuit halothane] and with full sterile precautions. The animals are placed in a canvas sling (see Fig. 1), a hole drilled through each horn, and stainless steel bolts (8 cm × 8 mm) inserted into the horns. The bolts are then fixed in position using a nut tightened onto a short length of nylon tubing shaped to fit the angle of the horn. The skin over the skull is reflected, the periosteum removed, and a 2.0-cm-diameter hole trephined in the skull over the brain region of interest, leaving the dura intact. To aid localization dorsoventral and/or lateral X rays are taken and bony landmarks such as the optic recess used for reference. For more accurate localization a hole can be drilled in the skull 1 cm lateral and 1 cm anterior to bregma and a needle (24 gauge) inserted into the lateral ventricle. A radioopaque agent is then injected into the ventricle (1 ml of Ultravist 300; Schering, Berlin, FRG) and dorsoventral and lateral X rays taken starting 30 sec after the end of the injection. The brain ventricular system is then clearly indicated on the X rays and the radioopaque agent clears away in <15 min. The hole drilled for the ventricular injection is sealed with bone wax and a stainless steel well (see Fig. 2) is placed over the hole in the skull and fixed in position using stainless steel self-tapping screws and dental acrylic. The well is filled with Aureomycin (chlortetracycline) mastitis suspension, which contains a steroid (Cyanamid, Ltd., Gosport, Hants, England) to prevent infection and to inhibit granulation of the dura, and closed with a removable nylon cap. To protect the well a 1-cm-high stainless steel ring is then cemented in position surrounding the well. The skin is sutured so that it seals against the protective ring. A topical antibiotic is applied to the skin around the implant (Aureomycin powder) and the animals given a 10-ml im injection of long-lasting antibiotic (Propen; Glaxovet, Uxbridge, England). Animals are allowed to recover for 2–4 weeks after surgery before the start of single-unit recording.

Fig. 1 Sheep suspended in a canvas hammock for surgery or for making single-unit recordings. The apertures for the legs are padded with foam to prevent chafing.

Fig. 2 Left: A stainless steel well for attachment to the animal’s skull which is closed with a nylon cap. Middle: The well with a Trent Wells X/Y table mounted onto it. Right: The Trent Wells hydraulic microdrive, with 20-gauge guide tube and microelectrode drawn up inside it, ready to be inserted into the brain through the central hole in the X/Y table.

Microelectrode Construction

Although (for ease of manufacture and reproducibility) most electrophysiologists prefer to use glass micropipet electrodes filled with an electrolyte, these are not ideal when they are required to penetrate through a large amount of tissue. In the sheep, for example, it is approximately 4 cm from the dura to the base of the brain. For this reason, most experimenters use microelectrodes constructed from metal and the material most commonly used for conscious single-unit recording is tungsten. We use a modification of the method described by Merrill and Ainsworth (15), in which tungsten microelectrodes are constructed with glass insulation. Briefly, this involves initially etching one end of a 15-cm length of cleaned, straightened tungsten wire (0.13-mm diameter; Phillips Element Co., Lewiston, ME) in Levick’s solution and inserting it into a 10-cm length of glass capillary tube (0.3-mm o.d. × 0.2-mm i.d.; supplied by Plowden and Thompson, Ltd., Dial Glassworks, Stourbridge, West Midlands, England). The etched tip of the metal is pulled up approximately 2 cm into the glass tubing and fixed in position using molten wax applied to the other end. The glass is pulled over the tip of the tungsten wire using a microelectrode puller, and a small amount of the glass insulation is finally pulled off the tip of the tungsten wire by inserting it into a molten glass bead. With practice it is possible to remove a sufficiently small amount of glass from the microelectrode with the molten glass bead so that it is available for immediate use. If not, the tungsten can be etched for a second time using a very low current. In the original method Merrill and Ainsworth subsequently coated the tip of the tungsten wire with platinum black to lower the resistance; however, we have not found this to be necessary for our application. A similar modification has also been used by Rolls et al. .

Preparation for Single-Unit Recording

Animals are taken from their pens and lifted into the canvas hammock shown in Fig. 1. Head restraint is achieved by inserting the bolts in their horns into Tufnol blocks attached to horizontal bars fixed to the frame of the sling. To prevent the bolts from slipping out of their blocks additional nuts are used to fix them in position. A horizontal bar is placed on the front of the frame so that the animals can comfortably rest their front legs on it. The animals are habituated to this form of restraint on a number of occasions before actual recordings are attempted. The sheep can be restrained in this way for up to 6 hr and both behavioral (rumination occurs and they will consume either food or liquid offered to them) and physiological indicators (only slight increases in plasma cortisol concentrations and respiration rate) suggest that the animals do not find their circumstances very stressful. This form of restraint does not prevent the animals from making body movements and if the initial placement in the hammock is uncomfortable for them they rapidly indicate this by moving their bodies until they find a more comfortable position. It should be emphasized that successful recordings from conscious animals can be achieved only if they are comfortable and relaxed. During recording sessions the animals should not be left alone and we usually give them frequent food rewards. Recordings are normally made only once or twice a week.

Once the sheep has been placed in the sling the nylon cap is removed from the well on its head and the Aureomycin mastitis compound removed from it using sterile swabs. A sterile solution of local anesthetic [3–4 ml of 5% (w/v) tetracaine hydrochloride; Sigma, St. Louis] is injected into the well and left for 5–10 min to anesthetize the dura. The local anesthetic is removed with sterile swabs and X/Y micropositioner (Trent Wells, South Gate, CA) placed and locked in position on the top of the well (see Fig. 2). A 20-gauge blunt stainless steel needle is gently lowered down through the central hole in the X/Y table until the tip touches the dura. A depth measurement is then taken and a length of the 20-gauge tubing cut so that it will penetrate 5 mm into the brain when mounted into the microdrive (Trent Wells) which fits onto the top of the X/Y table (see Fig. 2). This same length of guide tube is sterilized and then used on all subsequent recording occasions. The dura is pierced using a pointed 20-gauge needle and the microdrive assembly, with a microelectrode drawn up 1–2 mm inside the guide tube, is then placed on the X/Y table and locked in position. The microelectrode is advanced to the point when it should be level with the end of the guide tube and a depth reading taken. This depth reading is then equivalent to 5 mm below the surface of the dura. The microelectrode is then lowered down to whatever depth is required for the structure where recordings are to be made.

Single-Unit Recording and Data Analysis

Once the animal has been prepared for single-unit recording it is placed inside a Faraday cage to reduce 50-Hz interference. The microelectrode is connected to a preamplifier (Digitimer, NL100; Welwyn Garden City, Herts, England) mounted on the hydraulic microdrive assembly (thereby keeping the connection between the preamplifier and the electrode as short as possible). This in turn is connected, using low-noise shielded cable, to an ac amplifier (Digitimer, NL103) and low- and high-pass filters (Digitimer, NL125). Signals are fed into a window discriminator with audio output (Digitimer, D130) and displayed on an oscilloscope. The shape of each single unit recorded is also displayed using a digital storage scope since this is the best way to ensure that the same single unit is being recorded from over long periods of time.

For on-line analysis of single-unit activity the TTL output from the window discriminator is fed into an interface (Microlink interface; Biodata, Manchester, England) and statistical analysis of changes in firing rate and poststimulus histograms carried out using a microcomputer (Hewlett-Packard model 86 or IBM). Hard copy of single-unit activity is made using an electrostatic recorder (Gould ES1000, Longjumeau, France) and single-unit records can be also stored on magnetic tape (Teac R-71, Tokyo, Japan).

Visual Stimulation

Much of our work is concerned with the response of single units to complex visual stimuli such as food, faces, or body shapes. While it is possible in some cases to use real stimuli, particularly food, in other cases, where more controlled visual presentation is required, images are either displayed on a screen, using a slide projector system, or on a television screen, using a computerized image capture and display system. The simplest method involves the back projection of images from slides (Digitimer, pattern reversal projector). An electronic shutter (Prontor, Stadtteil Calmbach, FRG) is positioned between the projector and the screen to control image presentation, and this in turn triggers data collection so that response latencies can be calculated. Using the above projector system images can also be automatically moved about on the screen. For computer-controlled capture and presentation of images a Pluto 2i, 24-bit system is used (I. O. Research, Ltd., Barnet, London, England). This is a high-resolution (768 × 576 pixels and 16.7 million colors) image-capture system which can be used to display real, or artificial, images on a 27-in. screen television (Sony, Trinitron, KV27XRU with SCART interface). This system can hold between two and four pictures in memory at any time and other images can be rapidly called up from hard disk. Response times can also be calculated by triggering data collection via the serial port on the computer. The main advantage of this system is the facility that the editing software gives for systematic changes to images (i.e., mirror images, changing features, colors, and distorting the image).

Operant Responses

Sheep can easily be trained to perform a visual discrimination task for a food reward (18). Initially the animals are tethered in a wooden stand facing a large black panel fitted with a hinged transparent response panel (4 × 5 cm) which the sheep can press with their muzzle. A projector, mounted behind the Perspex panel, is used to display different geometric symbols. During recording, a metal screen (65 × 35 cm) with a Perspex screen mounted on it is placed in front of the animal so that it just touches its muzzle. The visual stimuli used in the original visual discrimination task can then be displayed. The sheep is able to move its muzzle forward the necessary few millimeters to activate a microswitch which in turn sets off a buzzer. Correct responses can then be rewarded by the experimenter by hand feeding.

Verification of Microelectrode Placements

Recording sites can be localized using a combination of X rays taken at various times during recording sessions, and at the end of experiments by histology. The tungsten microelectrodes will show up on X rays, as illustrated in Fig. 3, and so some indication of placements can be made by reference to bone landmarks. However, the most accurate localization is through postmortem histology. At the conclusion of a series of recording sessions the animals are anesthetized with an intravenous injection of barbiturate (Sagatal, May and Baker, Dagenham, England). A stainless steel marker electrode, insulated except for the tip, is lowered into the brain at selected coordinates. An anodal dc current (100 μA for 10 sec) is passed through this electrode to deposit ferrous ions into the tissue. Generally two to four such lesions are made to provide a number of different reference points. The animals are then given a lethal dose of anesthetic (Expiral) and the brain perfused via the carotid arteries with 0.9% NaCl followed by 10% formol–saline with a small amount of potassium ferrocyanide and potassium ferricyanide added. This has the effect of staining the deposited ferrous ions Prussian Blue. The brain is then stored in 10% formol–saline until histological sections are cut at 50–100 μm on a freezing microtome, using the plane of a sheep stereotaxic atlas (19), and stained with Neutral Red and Luxol Fast Blue. Neutral Red is preferable to Cresyl Violet for the nuclear stain since it does not mask the Prussian Blue spot at the end of the electrode track.

Fig. 3 Photograph taken from an X-ray plate showing a microelectrode inserted in the brain. The inset shows the wave form of an action potential recorded from a single unit at this location.

Results

We have used the above technique to make conscious single-unit recordings from various brain regions for periods of between 6 months and 1 year from the same animal. The steroid in the Aureomycin mastitis suspension reduces the granulation of the dura, but the dura does progressively harden and thicken over time. The combination of an antibiotic inside the recording well (Aureomycin mastitis compound) and the use of sterile swabs and local anesthetics, when preparing for single-unit recording, also effectively prevents any local infections occurring throughout the recording period.

Quality and Stability of Recordings

In general, we have been able to make successful single-unit recordings using this technique with the ability to pick up selectively the activity of one cell (see Fig. 4 for some examples). Occasionally the output of two or three cells is picked up simultaneously but these can easily be distinguished using the window discriminator. We have frequently been able to record from the same single cell for periods in excess of 4 hr, even when the sheep exhibits a large amount of motor movements such as during ingestion of food. This ability to hold cells for long periods has allowed us to investigate changes in neuronal responses during on-line discrimination learning procedures and to monitor the effects of satiety (2, 4). Figure 5 shows an example of a conditioned neuronal response to the sight of a colored bottle associated with a food reward. It has also been possible to record from cells during visual discrimination tasks requiring operant responses (1). On some occasions chewing movements do cause some artifacts in the recordings, but in general these are small and do not interfere substantially. Figure 6 shows an example of recordings from cells in the zona incerta where specific responses to the sight or ingestion of food can be found (2–4). On a few occasions we have also made recordings from nonhorned animals without head restraint and have found that movement artifacts are minimal under these circumstances.

Fig. 4 Traces showing neuronal responses to specific directions of visual movement, different auditory frequencies, and somatosensory stimuli in the sheep temporal cortex.

Fig. 5 Activity of a single neuron in the sheep zona incerta (ZI), which responds to the sight of food, during a visual discrimination task. The sight of a yellow bottle is paired with a food reward and after 15 trials (given at 1-min intervals) the sight of this colored bottle evokes a neuronal response. Correspondingly, the sight of a black bottle, which is not paired with a food reward, does not produce this effect. T, Thalamus; LH, lateral hypothalamus; HIPP, hippocampus.

Fig. 6 Responses of single neurons in the sheep zona incerta to the sight (S) of food, the visual approach of food (App), and the ingestion of food (Eat). Note the lack of movement artifacts produced by mouth movements which occur during the end of the visual approach phase of the stimulus and throughout the ingestion of food.

Discussion

The methods we have described for making single-unit extracellular recordings from the brain of the conscious sheep are similar to those used in primates [see the review by Lemon (20)]. The large size of the sheep, and the fact that it readily adjusts to the restraint system that such recordings normally require make it an ideal candidate for use as a conscious experimental animal for electrophysiology. Although the sheep is not as easy to train to perform operant tasks as some other laboratory animals, it is possible to do this and it can therefore be used in experiments concerned with neuronal correlates of learning and memory. While the only stereotaxic atlas available (19) is not very detailed, and is not easily applicable to the wide variety of different breeds of sheep which are available, localization is best achieved using X rays and ventriculography rather than by stereotaxic coordinates.

In summary, the sheep is an excellent subject for neuroscience experiments using single-unit recording techniques, and presents a useful alternative to species that are less readily available, such as primates.

References

1. Maddison, S., Baldwin, B. A. Brain Res.. 1983; 278:195.

2. Kendrick, K. M., Baldwin, B. A. Brain Res.. 1986; 375:320.

3. Kendrick, K. M., Baldwin, B. A. Neurosci. Lett.. 1986; 63:237.

4. Kendrick, K. M., Baldwin, B. A. Brain Res.. 1989; 492:211.

5. Kendrick, K. M., Baldwin, B. A. Science. 1987; 236:448.

6. Kendrick, K. M., Baldwin, B. A. Neurosci. Lett.. 1989; 100:193.

7. K. M. Kendrick, J. Anim. Sci. (in press).

8. Kendrick, K. M.Conn, P.M., eds. Methods in Enzymology; 168. Academic Press, San Diego, California, 1989:182.

9. K. M. Kendrick, J. Neurosci. Methods (in press).

10. Hill, M. W., Heavens, R. P., Baldwin, B. A. Brain Res. Bull.. 1985; 15:453.

11. I. S. Ebenezer, K. M. Kendrick, and B. A. Baldwin, this volume [15].

12. Baldwin, B. A., Parrott, R. F. Physiol. Behav.. 1982; 28:77.

13. Baldwin, B. A., Yates, J. O. J. Physiol. (London). 1977; 265:705.

14. Baldwin, B. A., Siegel, J., Yates, J. O. Physiol. Behav.. 1971; 7:935.

15. Merrill, E. G., Ainsworth, A. Med. Biol. Eng.. 1972; 10:662.

16. Rolls, E. T., Burton, M. J., Mora, F. Brain Res.. 1976; 111:53.

17. Rolls, E. T., Judge, S. J., Sanghera, M. K. Brain Res.. 1977; 130:229.

18. Baldwin, B. A. Anim. Behau.. 1981; 29:830.

19. Richard, P.Atlas Stereotaxique du Cerveau de Brebis.. Paris: Inst. Natl. Rech. Agron., 1967.

20. Lemon, R.Methods for Neuronal Recording in Conscious Animals,. Chichester, England: Wiley, 1984. [Int. Brain Res. Organ. Handb. Ser.: Methods Neurosci. 4.].

[2]

Single-Unit Recording from Pontomedullary Neuraxis in Awake, Freely Behaving Animals

Fat-Chun Tony Chang

Publisher Summary

This chapter discusses single-unit recording from pontomedullary neuraxis in awake, freely behaving animals. The single-unit recording technique has played a crucial role in the identification and characterization of various pontomedullary neural substrates and their involvement in the control of life-defining functions such as cardiorespiratory rhythm, states of consciousness, and processing of sensorimotor information. Recording of single-unit activities from pontomedullary neural substrates in unanesthetized, freely behaving animals is an extremely difficult procedure.. The chapter describes a nonintrusive procedure that permits hours of stable unit recording from pontomedullary neurons in awake, freely behaving animals. Electromechanical components of this procedure consist of three principal elements. In the first principal element, single-strand fine wires are used as recording electrodes. The single-strand fine wire offers the advantage of flexibility. A fine-wire electrode introduced into an unstable environment, such as the medulla, floats and moves with each cardiac pulsation, respiratory movement, or motor activity-induced brainstem movement, while a relatively constant distance from the signal source is maintained. In the second element, a miniature, skull-mounted microdrive that permits discrete dorsoventral movement of the fine-wire recording electrode is used to advance the fine-wire electrode and to isolate single-unit activities. In the third element, a head-mounted voltage follower is employed to condition and stabilize the neuronal signals prior to transmission or for further signal processing. The chapter also discusses methods for integrating these three elements into a working electrophysiological recording procedure.

Introduction

The single-unit recording technique has played a crucial role in the identification and characterization of various pontomedullary neural substrates and their involvement in the control of such life-defining functions as cardiorespiratory rhythm, states of consciousness, and processing of sensorimotor information (see, e.g., Refs. 1 and 2). However, in contrast to its use in forebrain areas where single neuronal activities are routinely being studied in freely moving animals, the application of the single-unit recording technique in the pontomedullary neuraxis has been limited, because of a number of formidable technical difficulties, to anesthetized, paralyzed, or conscious but behaviorally restrained preparations. In response to an ever-increasing awareness and concern about the confounding effects of anesthetic and paralytic agents (see, e.g., Refs. 3 and 4), an electrophysiological model system that permits recording of lower brainstem single-unit activities in awake, freely behaving animals was developed.

Recording of single-unit activities from pontomedullary neural substrates in unanesthetized, freely behaving animals is an extremely difficult procedure. In addition to the usual technical difficulties associated with chronic unit recording, a far more trying and unique problem is encountered in the lower portion of the brainstem which moves relative to the circumjacent bones with each respiratory cycle and cardiac pulsation, as well as with locomotion and postural modifications. Under these circumstances, it is virtually impossible to perform single-unit recording in awake, behaving animals with conventional rigid metal or glass electrodes. The magnitude of the movement problem may be reduced with the use of head restraints combined with arthrodesis (e.g., artificial ankylosis) of the skull and cervical vertebras (5–7). Approaches such as these, however, are not without problems. With the use of a head restraint, the movement of the animal must be kept to an absolute minimum in order to allow stable unit recording and to preserve the mechanical integrity between the restraining devices and the calvarium. As such, the procedure seriously limits the extent to which behavioral correlates of neurophysiology can be studied. Surgical fusion of cervical vertebras, on the other hand, necessitates not only extremely delicate surgery, but also extensive exposure of neck muscles which significantly increases the risk of infection and the potential for postoperative discomfort and prolonged incapacitation of the animal.

In this chapter, a nonintrusive procedure that permits hours of stable unit recording from pontomedullary neurons in awake, freely behaving animals is described. Electromechanical components of this procedure consist of three principal elements. First, single-strand fine wires are used as recording electrodes. The single-strand fine wire offers the advantage of flexibility. A fine-wire electrode introduced into an unstable environment such as the medulla floats and moves with each cardiac pulsation, respiratory movement, or motor activity-induced brainstem movement, while a relatively constant distance from the signal source is maintained. Second, a miniature, skull-mounted microdrive which permits discrete dorsoventral movement of the fine-wire recording electrode is used to advance the fine-wire electrode as well as to isolate single-unit activities. Third, a head-mounted voltage follower is employed to condition and stabilize the neuronal signals prior to transmission or for further signal processing. Methods follow for integrating these three elements into a working electrophysiological recording procedure.

Technical Descriptions

Recording Electrode

Recording electrodes are constructed from stress-relieved Formvar- or poly-amide-insulated platinum (90%)–iridium (10%) fine wires (California Fine-wire Company, Grover City, CA). Platinum and iridium are both chemically and biologically inert. The added iridium serves to stiffen the alloy and thus increases the ease with which the wire penetrates the neural tissues. Other less expensive metals or alloys may be used. It should be noted, however, that some metals possess hyperallergenic properties that could promote gliomatosis at, and insulation of, the fine-wire electrode tip and ultimately result in loss of neuronal signals within hours after implantation. The diameter of the fine wire is selected on the basis of desired degree of flexibility. Used singly, a 37.5-μm (0.0015-in.) diameter platinum–iridium fine wire is sufficiently stiff to penetrate the brain tissue and is flexible enough for most chronic unit recording applications. Should added flexibility be required, a 25-μm (0.0010-in.) diameter fine wire, braced to a strut (e.g., a length of 37.5-μm fine wire) in a staggered fashion, may be used (see Fig. 1a). Generally, a smaller diameter fine wire is usually more difficult to manipulate and it may not significantly improve the quality of unit recording.

Fig. 1 in. long); a stainless steel compression spring; an outer cannula (22-gauge hypodermic tubing, 15 mm in length) cemented to the rectangular block; an inner guide tube (26-gauge hypodermic tubing, 22 mm in length) affixed to the carrier plate; a female gold-plated connector (Amphenol part #220−SO2) seated in a connector strip (Amphenol part #221–2653); and a capped outer protective housing made of square brass tubing.

Single-strand fine-wire recording electrodes are prepared as follows. Straight lengths of insulated microwires approximately 8 cm long are cut with a pair of scissors. Approximately 2 cm of insulation on one end is burnt off with either a small flame or a microforge; care should be taken not to ignite the metal. After the burnt residue has been removed with emery cloth, the stripped portion is folded and compressed into a 2- to 3-mm-long pile and crimped to a gold-plated miniature connector (Amphenol part #220-PO2) as shown in Fig. 1b. The opposite end, which is to be used as the recording tip, is cut afresh immediately before surgical implantation. The tip can be cut at various oblique angles to broaden the scope of signal detection. This strategy works well under most circumstances, and may be adapted to accommodate to such factors as the population density of neural substrate, the orientation and the soma dendritic morphometry of target neurons, and the profile and gradient of extracellular current source density.

Miniature Microdrive Assembly

A chronic microdrive design originally introduced by Harper and McGinty (8) was modified for use in the present procedure. Figure 1c is a schematic diagram of the skull-mountable miniature microdrive assembly. The forward–reverse drive mechanism is provided by clockwise or counterclockwise rotations of the 0–80 machine screw with a jeweler’s screwdriver. The motion is transferred, via the carrier plate, to produce a dorsoventral movement of the sliding inner guide tube through which a fine-wire electrode is affixed. The resolution of the miniature microdrive made with components described in Fig. 1c is approximately 320 μm/revolution of the 0–80 screw. The magnitude of resolution and associated mechanical properties offered by this miniature microdrive are adequate to meet the maneuverability and stability requirements of most chronic single-unit recording applications. An 0–90 machine screw may be used should a higher resolution drive ratio be required.

At the time of surgical implantation, the tip of the inner guide tube is stereotaxically positioned above the target area with the use of a microdrive holder. Figure 2 is a schematic representation of the microdrive holder attached to a standard stereotaxic electrode carrier arm. The holder is not a permanent part of the chronic instrumentation but is attached to the miniature microdrive only for the duration of surgical implantation.

Fig. 2 Schematic representation of the microdrive holder attached to a standard stereotaxic electrode carrier arm. The body of the miniature microdrive is secured in the holder by the set screw (SS) only for the duration of the initial phase of the implantation procedure.

One of the unique features offered by this miniature microdrive is the provision for replacement of fine-wire electrodes in the event that multiple penetrations are desired or the tip of the fine-wire electrode is insulated as a result of gliomatosis. In view of the compactness and the delicate nature of the miniature microdrive components, the animal should be anesthetized with a short-acting anesthetic agent, such as ketamine, and its head secured in a stereotaxic frame prior to replacement of the fine-wire electrode. The procedure for replacement is as follows. The fine wire is first unplugged from the SO2 receptacle and removed, together with the inner guide tube and the carrier plate, by turning the 0–80 screw counterclockwise until the threads are completely out of the tapped hole. A fresh inner guide tube is affixed to the underside of the carrier plate with a small amount of cyanoacrylate adhesive (Krazy Glue). Care should be taken not to block the hole of the inner guide tube. The 0–80 screw, compression spring, and inner guide tube are assembled and reinserted onto the miniature microdrive block. Finally, a new fine-wire electrode is introduced into the target area as described in the section, Chronic Instrumentation.

Head-Mounted Voltage Follower

Movement-induced electrical artifact is undoubtedly the most formidable and irritating technical problem faced by those desiring to record single-unit activities in freely behaving animals. Moreover, the single-unit signal is also subject to distortion by (1) capacitative modulation throughout the signal path, (2) static interference caused by the movement of the cable and other related artifacts during behavior, and (3) electrical noise such as 60 Hz and the harmonics thereof. The most ideal solution is to condition and stabilize the signal as close to the signal source (target neuron) as possible. In this section of the chapter the use of a compact voltage follower circuit that can be plugged directly onto the dental acrylic head mound at the time of experimentation is described.

The circuit diagram of the head-mounted voltage follower is shown in Fig. 3a. The principal element of this circuit is a wide dynamic range N-channel 2N-5247 junctional field effect transistor (JFET). The 2N-5247 JFET, having a frequency response up to 400 MHz, is designed for a variety of common-source or common-gate VHF/UHF amplifier, mixer, and oscillator applications. Other single-stage, N-channel JFETs (e.g., 2N-5196 through 2N-5199 series) designed for analog switch applications requiring low ON resistance and moderate capacitance may also be used. The 2N-5247 JFET is typically used in a self-biased, single-stage mode, thus circumventing the need for extensive biasing networks. This feature is desirable from the standpoint of chronic electrophysiological applications: the voltage follower can be fabricated with only a few electronic components and is light and compact, as shown in Fig. 3c. Discrete JFETs are also well known for their low noise performance characteristics, and are virtually free from problems of current noise, popcorn noise, and limited bandwidth that plague most bipolar transistors and operational amplifiers (op amps). A significant drawback of JFETs is the limited voltage gain. However, monolithic op amps such as μA-741 and LF-356 are inexpensive, flexible in gain (unity to high), and easy to use, though they suffer from poor noise performance. Combining a JFET (Fig. 3a) with an op amp (Fig. 3b) results in a signal follower/amplifier that has the best features of both: low noise, gain flexibility, and wide dynamic range. Table I is a list of values and specifications of all electronic components for the voltage follower