Table of Contents

Table of Contents--------------------------------------------------------------------------------2

Description of Benchmark Stereotaxic Instrument------------------------------------------3

Manipulator Movements and Offsets ---------------------------------------------------------4

Alignment and Calibration ---------------------------------------------------------------------5

Installing Animal and Operating --------------------------------------------------------------6

Care and Cleaning-------------------------------------------------------------------------------9

Specifications ------------------------------------------------------------------------------------10

Useful References -------------------------------------------------------------------------------11

Warranty------------------------------------------------------------------------------------------11

Appendix A Vernier Scales -------------------------------------------------------------------12

Appendix B Initial Assembly of Digital -----------------------------------------------------13

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Description of Benchmark Stereotaxic Instruments

A stereotaxic instrument is a device for placing a probe in a precise anatomical location in the brain or spinal cord of an animal. It consists of two major sections that must be in alignment with each other, 1) a head or spinal holder for an animal that locks the target in a defined position and orientation, and 2) a manipulator to enable precise movements of a probe relative to the head or body.

Manipulator Movements and Offsets Benchmark Stereotaxic Instrument manipulators all have 3 linear movements and 3 rotary movements, as well as 2 settable offsets. These are labeled in Figure 1.

The linear movements are referred to relative to the animal:

• (A/P) Anterior/posterior, movements along a line parallel to the midline of the brain

• (M/L) Medial/Lateral, movements along a horizontal line perpendicular to the midline

• (D/V) Dorsal/Ventral, generally vertical, the last movement to advance into brain

The rotary movements are referred to by the effect on manipulator orientation:

• Rotation, changes the direction of the tilt

• Tilt, tilts the manipulator and angle of probe approach in either the A/P or M/L direction

• Swing-Out, swivels the probe out of the way, and then restores it precisely to its original position

The V clamp probe offset adaptor shifts the range of a movement to reach a different area

• The probe offset adaptor set screw can be loosened to vertically move the probe in the D/V direction, and then relocked

• The probe holder offset clamp can be flipped to offset in the anterior or posterior direction, and has two holes for the thumb screw giving the user two choices for the degree of offset, four possible positions, two rostral, and two caudal, to the manipulator

Other optional offsets are available for special needs. If you have a special request, please contact us.

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Figure 1. Movements and Offsets of Stereotaxic Instrument Swing Out is a tapered square block that allows the M/L drive to point in any of 4 directions. It is used to move the probe holder away from the skull, e.g. in order to drill a hole, and then return to exactly the position it was in, defined by the block face. Using the Rotation to achieve this purpose will give substantially less accuracy of return. To operate, push up and around after loosening locking screw. Tilt is a pivot movement that allows tilting the probe holder to approach brain at an angle. In Figure 1, tilt is oriented to allow tilt in the A/P plane. It may be made to tilt in the M/L plane by turning the Rotation movement 90˚ either way, then turn the Swing Out 90˚ back to its M/L orientation. Tilt would then be possible in the M/L plane. Obviously, complex angles of tilt can be achieved by manipulating the rotation less than 90˚. This capability is rarely used with stereotaxic instruments less sophisticated than the Benchmark Angle One™ or Angle Two™, as it then becomes very difficult to calculate where the movements will take the probe tip. Rotation has no stops or tapered faces, so is free to rotate any number of degrees. The distance from the pivot point to the graduations for this movement is much less than the distance from the pivot to the probe holder. Therefore, very small errors of graduated mark alignment produce multifold larger errors of probe tip position. Any time the Rotation is used, alignment should be rechecked. (See the procedure in Alignment and Calibration.) The clamp that attaches the shaft of the probe holder to the manipulator includes two offset mechanisms. Vertical Offset is achieved by loosening the set screw with a 5/64 inch Allen hex wrench, sliding the shaft up or down, and retightening the set screw. At this time, the shaft can also be rotated. A/P Offset is achieved in two ways. The user may select from two holes through the clamp that will accommodate the thumb screw to set the degree of offset. The user may also remove the clamp from the

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probe holder, and flip it 180˚ to change the direction of A/P offset. All probe holders available from MyNeuroLab use the same size shaft, and the same offset clamp. The clamp comes with each probe holder (in most cases), and can be offset as described. The three linear movements are obvious in function. All three axis should be perpendicular to each other, unless deliberately set at other angles (See Alignment and Calibration). For instruction on reading Vernier scales, refer to Appendix A. If the stereotaxic instrument includes digital scales, these may be zeroed by touching the zero buttons when the probe is at Bregma (or earbar zero if an instrument reference is to be used as zero.) Alignment and Calibration Accuracy of measurement depends on the assumption that all 3 axes are perpendicular to each other (or, if tilt or rotation is used, at a known angle relative to each other.) The Rotation movement in particular can easily be slightly out of alignment and impair accuracy. For the Benchmark Basic, Digital and Deluxe, good alignment may be achieved by a visual gap method (below). Please note that the Angle One™ and Angle Two™ include an alignment tool kit and corresponding manual. This kit may be purchased separately for use with other stereotaxic instruments. With any stereotaxic instrument, check alignment and probe straightness before starting any projects and after probe changes. The human eye can see very small differences, tens of microns, in the size of an air gap between two objects. This can be used to check alignment of the manipulator axes with the head holder, and the straightness of the probe before use. To check alignment, install a probe, either known straight (a drill bit or glass pipette blank), or the actual probe to be used. The standard probe holder has 10 grooves in the plastic block on each of 3 faces that run parallel to the probe holder shaft (the fourth such face is flat and ungrooved. Lay the probe in one of the grooves, and tighten the clamp to secure it in place. Push both earbars in to where they touch in the middle. Both earbars vernier zero should align with zero on the scales. Tighten the locking levers. If they do not read zero, the scales were misaligned at the factory. The scales can be adjusted, but should be adjusted when the earbars meet in the center between the U frame arms. If you do not have the equipment to measure this, you might want to send the instrument to the factory for alignment. Use the 3 linear movement drives to move the probe until it is next to an earbar, just separated from it by a barely visible air gap. Move the M/L drive from one side to the other, while closely watching the air gap. If the gap gets larger or smaller, the probe is not moving parallel to the earbars, and Rotation is out of alignment. Loosen the locking screw on the Rotation mechanism (Figure 1) and rotate slightly to restore alignment. Relock. Try again. By trial and error, you will achieve alignment. Unlock and rotate an ear bar 180°, so the blunt end is on the inside of the stereotaxic U frame. Relock. Move the probe until it is a barely visible air gap away from the blunt end. Operate the D/V drive while observing the air gap. If the air gap changes in size as the probe moves vertically, the probe is not straight in an M/L plane. The probe may be bent or not installed straight in a groove of the probe holder, or the tilt mechanism may be slightly tilted. Adjust the tilt, or straighten the probe. Repeat with the probe a barely visible air gap away from one side of the ear bar. If the air gap changes in size, the

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probe is not properly aligned in an A/P plane. Position the probe just above an earbar, with a small air gap down to the earbar. Move horizontally along the earbar, and back and forth in the anterior/posterior direction across the earbar. If the air gap to the tip changes in size, the instrument is moving in the vertical dimension when the other drives are being operated. If you have performed all three linear movement tests, and cannot align your instrument, call MyNeuroLab for assistance. Both the vernier and digital scales are permanently engraved by a laser guided by a computer. Just as the lines on a ruler can not move once made, the instrument can not become un-calibrated in this sense. Installing the Animal and Operating. The animal’s head must be securely and symmetrically affixed in the stereotaxic instrument, reproducibly aligned with the movement axes of the manipulator section. Different head holder adaptors are designed for each species. For most species, bars into the ear canal on each side and a bar behind the incisors provide a stable, reproducible and defined hold. Traditional 18˚ ear bar tapers break the tympanic membrane. Tapers of 45˚ are almost as stable, and do not break the tympanic membrane, and are thus better if post-surgical testing is planned. An excellent book, photographically revealing each detailed step for stereotaxic surgery in the rat, is available from MyNeuroLab. MyNeuroLab recommends, “Stereotaxic Surgery in the Rat, a Photographic Series” By R. J. Cooley and C.H. Vanderwolf., even it you intend to work with a different species. Installing the earbars is the most difficult skill required for stereotaxic surgery. If a novice, practice with an injection anesthetized animal or a sacrificed animal. Learn to feel the correct placement before beginning on an experimental animal. Note that the earbar locks on the Benchmark are unique locking levers. Other companies use thumb screws, which require at least two fingers. You will notice the ease of one-finger locking of the last earbar when installing an animal. Anesthetize the animal and shave its head. If using gas anesthesia, go back to the induction chamber and re-anesthetize. Install the earbars. Set the left earbar tip about 1 cm left of the center position, and lock it in place. Remove the right earbar, and leave the lock ready to drop the earbar back into place. Loosen the thumb screw that holds the incisor bar in place, and slide the incisor bar plate all the way back away from the animal. Holding the animal’s body with the left hand, and its head with the right hand, move the animal to insert the left earbar fully into the ear canal. Pointing the nose slightly to the right, allowing the earbar to enter from a slightly dorsal-caudal direction, will ease insertion. When the earbar slips into the canal, hold the head in place with the left hand, and pick up the right earbar. Moving the right earbar freely, insert it into the right ear. While holding the earbar snug in the ear, lower the body of the earbar into the right slot and lock. Check centering. If both earbars do not have identical readings, loosen both earbar locks while holding the bars snug from the outside with one finger, and push back and forth until both earbar scales line up on the same number. Lock the earbars. Pushing down with one finger on either earbar lever will lock it.

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More pressure can be applied with rat bars than with mouse bars. The mouse skull is extremely thin and flexible, and the breathing passages run directly between the ears. The mouse is easily strangled by ear bars. The rat earbars will not fit into mouse ear canals, and the rat tooth bar is too rounded for the mouse. For stereotaxic mouse surgery, an optional adapter is needed, specific to the mouse. Hamsters and gerbils fit into the rat head holder equipment. Check that the earbars were correctly installed. Gently try to move the nose from side to side. If you can, the earbars are not correctly placed in the ear canals or they are not snug enough. Push the nose up and then down. If it stays right where you left it, the earbars are correctly placed. If it springs back a bit, the earbars are probably not correctly placed. If there is movement, remove the earbars and repeat the last paragraph. Once the earbars are securely installed, slide the incisor bar into the mouth, hook the incisor teeth over the bar and push the nose down. Slide the bar back until it is snug behind the incisors and lock down the thumb screw. If using an injectable anesthetic, snugly tighten the nose clamp down. If using a gas anesthetic, slide the nose cone forward around the animal’s nose, and unclamp the gas flow through it. Next, place two fingers on the animals scalp, straddling the midline, where you want the incision to begin. Pull the skin tight by pressing caudally. Set the blade of the scalpel down with some pressure between the two fingers, sharp side down and rostral. Holding the scalpel steady, pull the skin rostrally to cut. For a better incision, move the skin against the blade rather then the blade against the skin. Retract the skin with locking forceps. Scrape the scalp membranes away from the midline with the handle of the scalpel or another metal scraping edge, in order to fully expose the bone. The midline, bregma and lambda should be visible on the skull (Figure 3). Swab off any blood with a cotton swab, wetted with saline if needed.

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Figure 3. Exposed Scalp with drilled holes. The atlas you are using, particularly if by Paxinos, will describe how to locate Bregma and lambda. The anterior point where a M/L bone suture crosses the midline suture is called Bregma. The caudal point barely visible at the bottom of the picture, is called lambda. As you can see, the irregularity of the biological lines makes these points somewhat indeterminate. Most atlases recommend using the best approximation of where the intersection would be if the lines were straight and smooth, and crossed the midline at a point. This also creates some examiner variability. You can only be consistent in how you do it, and adjust your coordinates if need be. Use a permanent ink fine point marker to put a dot on the bone where you think Bregma and lambda ought to be. Use Bregma rather than the intra-aural line for the zero point (Paxinos, Watson, Pennisi, & Topple, 1985). Touch a probe down at Bregma and lambda, and take a vertical reading on the D/V at each point. By trial and error, adjust the vertical position of the incisor bar until Bregma and lambda get the same vertical reading. This is the ‘Skull Flat’ reference plane. Position the probe tip at Bregma. If using a vernier model stereotaxic instrument, record the coordinates. If using a digital stereotaxic instrument, zero the scales. Raise the probe slightly along the D/V axis, and then move to the target A/P and M/L coordinates. Place a dot on the skull directly below

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the tip, labeling the area for drilling. Make a contralateral mark if needed. Unlock the Swing Out lock, and pivot the manipulator and probe out of the way. Be careful the probe does not strike anything on the way around. If need be, raise the manipulator further in the D/V direction. The dental drill bits used for drilling into skull have a spherical head. This means that the hole is deeper in the middle than around the edges. If you do not go deep enough, there will be bone ledges around the outside of the hole, hidden under blood, that could break a glass pipette tip. If you go too deep in order to remove the ledges, there may be a small lesion in cortex just under the center of the drilled hole. Care should be taken to avoid either outcome. The smallest possible drill head, moved continuously in a small circle as you are drilling, will help avoid either outcome. Use of a manual drill with settable depth stops, mounted on the stereotaxic manipulator, will give you precise stereotaxic control of placement and depth of hole, highly reproducible. The rest is highly variable, depending on the research project. A wide variety of species adaptors and probe holders are available for special applications. When the surgery is complete, close with Autoclips or Tissumend suture adhesive or suture thread. Be careful to abut cut skin to cut skin for faster healing. The small holes through the skull are insignificant and can be ignored. Care and Cleaning Do not Autoclave! The bushings on the linear movements, and the acrylic block on the probe holder, will not survive repeated autoclaving. Do not lubricate the sliding shafts that go into plastic bushings, or the lead screw, that goes into a plastic nut. Petroleum products will absorb on the plastic, and cause it to swell. This may result in the opposite of the desired effect, the drive may lock up when the plastic swells after greasing or oiling. The dovetail slides and other metal to metal sliding surfaces have a thin layer of grease lubricant. Do not remove it with heat or solvents. Contact us for a supply of this lubricant. Earbars and incisor bar may be sterilized with alcohol or Cidex (a commercial brand of highly dilute glutaraldehyde). Probe holder can be sterilized with Cidex. Wipe blood off the base plate or stainless steel parts with soapy water or alcohol.

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Specifications for Benchmark Digital Deluxe™ Stereotaxic Instrument

Resolution, all scales: Vernier 0.1 mm Digital Displayed 0.01 mm Linear Advance/Revolution (screw pitch) 5 mm/rev. Fine Drive Advance 20:1 reduction, or 0.25 mm/rev. Manipulator Movement Ranges AP 80 mm ML 80 mm DV 62.5 mm Tilt and Rotation ML or AP axes, or combined: ±90° Rotation 360° Toothbar Movement Ranges DV 20 mm AP 44.5 mm Compatibility: Earbars and nose holders interchangeable with other common makes of stereotaxic instruments. Probe Holder: Shaft Diameter: 7.88 mm (0.31 inches) Length: 168 mm Length, Extended Shaft 200 mm Probe Holder Block 11 mm square,

10 grooves spaced at 1 mm on 3 sides Standard Clamp Holds 0.2 to 2 mm shaft Side Clamp Holds 0.2 to 4.5 mm shaft “C” Clamp Holds 0.2 to 4.5 mm shaft. Set Screw on Shaft holder: 3/32” Second manipulator may be added on right (facing open end of “U”) side. Power: Digital Display – 1.5 Volt Digital Linear Sensors – 5 Volts DC power supply (integrated circuit)

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Useful References Paxinos G, Watson C, Pennisi M, Topple A., Bregma, lambda and the interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. Journal of Neuroscience Methods 1: 39-43, 1985 Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordinates, Fourth Edition. Academic Press, New York, 1998 Cooley, R. J, & C. H. Vanderwolf, Stereotaxic Surgery in the Rat, a Photographic Series. A.J Kirby, 1990    

MyNeuroLab Limited Warranty   MyNeuroLab warrants this instrument to be free from defects in material and workmanship for a period of one year from the date of original purchase. This warranty covers parts and labor. We may repair or replace defective instruments at our option. There is no warranty on consumables supplied with the instrument. The warranty does not cover damage from abuse, neglect or use for purposes other than those intended and described in the manual. MYNEUROLAB/CORETECH HOLDINGS DISCLAIMS ALL RESPONSIBILITY FOR CONSEQUENTIAL DAMAGES OR INCIDENTAL LOSSES CAUSED BY USE OF THIS INSTRUMENT, except where such disclaimer is not allowed by law. To claim, visit our website at URL http://www.myneurolab.com and find the latest contact information, or call 314 522 0300. Do not ship the instrument back without first contacting the company and obtaining a routing number. This warranty gives you specific legal rights, which may vary from state to state or province to province.

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Appendix A: How to read a Vernier Scale Your Benchmark Stereotaxic, whether digital or not, still includes the traditional Vernier scales to measure the movement. Of course, these are redundant and little used on the digital units. The Vernier scale allows more precise measurements that could otherwise be done with a simple ruler. The idea was invented by Pierre Vernier A(1580-1637). A Vernier measurement requires two facing scales. One is the main scale of any length, and graduated in standard units like a ruler. The other, called the vernier, is short, graduated from one to ten, and graduated in units 9/10ths of the standard unit.

Figure 2. Main Scale and facing Vernier reading 1.23. Because of this, one and only one of the lines on the vernier scale can be aligned with a graduation on the main scale. This gives the last decimal place in the final reading. In this case it is the 3 on the vernier. Note that the zero mark on the vernier is just after 1.2 on the main scale. And the 3 on the vernier lines up with a mark on the main scale. The reading is 1.23.

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Appendix B: Initial assembly of Digital Instrument Your Benchmark Digital Stereotaxic Instrument (Angle One and Two have different instructions) comes with a single or dual display panel, appropriate to either a single or dual stereotaxic instrument. This display panel has a power inlet near the switch, for which a power cord with a transformer in the middle and a plug for the instrument are provided. On the opposite side, there are 3 labeled inputs for linear encoders, using plastic telephone-like connectors. Each is labeled, and for each there is a labeled plug connector coming from the Stereotaxic instrument. Connect these as shown. Incorrect placement will not hurt the instrument; just that, for example, the A/P number may change when you operate the D/V scale if you have switched those plugs. Once everything is connected, power is connected, and the switch is on, the displays will light up and show a number. Moving any of the stereotaxic movements will change the digital display, showing mm’s from the zero point to 0.01 (10 microns). At any position, you may press the zeroing button, and restore the display to zero. There is a second button on the counter, it is not assigned, has no function. In use, with an animal installed in the stereotaxic instrument, position the probe at the zero point (usually Bregma) and press the zero button to zero each scale. Some researchers zero the D/V scale last when the probe touches the surface of brain. Once zero’ed, move each axis until the desired target coordinate is displayed, and operate the DV to move down into brain. You never need write down the zero point, do any arithmetic, or read a vernier scale.

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