Stereotactic Surgery and Long-Term Maintenance of Cranial Implants in Research Animals THOMAS W. GARDINER, PHD,1 AND LINDA A. TOTH, DVM, PHD2 Abstract ͉ Most neuroscience research is performed by using anesthetized animals or tissue samples obtained from animals that have been euthanatized. However, study of many important issues requires the use of animals that are alert and capable of engag- ing in behavior. Various methods have been used to humanely perform neuroscience experiments that involved unanesthetized animals. These techniques often involve surgical implantation of an apparatus that permits direct manipulation of brain tissue or measurement of neurochemicals or neuronal activity in conscious animals. We describe here common surgical techniques used to prepare animals for long-term neuroscience studies, discuss several issues related to short- and long-term postoperative care of animals with implants, and offer suggestions that veterinary and research personnel can use to prevent or mitigate some common problems that may develop when preparing and maintaining animals for these studies. Most neuroscience research is performed by using anesthe- tized animals or tissue samples obtained from animals that have been euthanatized. However, the study of many important is- sues sometimes requires the use of animals that are alert and capable of engaging in behavior. Studies that address how the brain controls processes such as learning, sleep, or movement may be meaningful only if brain function is evaluated while the animal performs the behavior of interest. In addition, anesthe- sia can profoundly influence the brain’s responses to some experimental treatments. For example, amphetamine and other drugs can affect the nervous system differently in anesthetized animals as compared to unanesthetized animals (1). Learning how various drugs modulate brain function may require collect- ing data from unanesthetized animals. Various methods have been used to humanely perform neu- roscience experiments on unanesthetized animals. These techniques often involve surgical implantation of an apparatus that permits direct manipulation of brain tissue or measure- ment of neurochemicals or neuronal activity in conscious FIG. 1. Photograph of an unrestrained rat with an implant for animals (Figure 1). To perform these types of studies, an ani- electrophysiologic recording. The electrical connector in the headpiece mal is anesthetized, and a headpiece apparatus is permanently permits the attachment of wires that conduct electrical signals to ampli- attached to its skull. The animal then is allowed a period of fiers and data collection equipment. The headpiece was surgically attached to the skull of the rat while it was anesthetized, and experimen- postsurgical recovery before collection of data actually begins. tal recordings can later be obtained without causing pain or discomfort Design of a headpiece apparatus depends on the type of study to the conscious rat. that will be performed. For example, a headpiece might con- tain electrodes that record electrical activity from single neurons in specific brain regions, electrodes that measure insertion of electrodes or other probes into the brain. In fact, electroencephalographic activity or chemical changes in the they sometimes are so relaxed that they fall asleep during the brain, or cannula tubes that enable collection of cerebrospinal surgical procedure (2). Such observations confirm that physi- fluid or administration of drugs and other substances directly cal penetration of brain tissue is not painful and that despite into specific brain regions. the strange or unnatural appearance of headpieces, long-term Permanently implanted headpieces allow experimental mea- experimental manipulations of the central nervous system can surements to be taken or manipulations to be performed be performed without causing stress or discomfort to the ani- without causing pain or distress to conscious animals. In fact, mals. To accomplish this, however, surgical implantation must in many instances, such as during measurement of neuronal be performed correctly, and the headpiece must be maintained activity, animals seem to be unaware of the experimental ma- properly after surgery. nipulation. Verbal reports from human patients undergoing We describe here common surgical techniques used to pre- neurosurgery reinforce this impression. Surgical treatment of pare animals for long-term experimental study of the brain. We some disorders requires that patients communicate with the also address several issues related to short- and long-term post- neurosurgeon during surgery so that their sensory responses operative care of animals with implants, including procedures and motor or cognitive states can be evaluated as the surgical that can be used to keep the brain surface healthy and protected procedures are performed. Anesthesia for these patients con- in animals implanted with headpieces that incorporate remov- sists only of locally acting agents applied to the scalp, yet these able electrodes. Although our examples reported here focus on patients report a complete lack of sensation associated with implants designed for electrophysiologic recording from indi- vidual neurons, the surgical procedures and considerations for Department of Anatomy and Neurobiology1, University of Tennessee, Memphis, postoperative care apply equally well to the implantation of other TN 38163; Department of Infectious Diseases2, St. Jude Children’s Research types of devices, such as microdialysis probes, cannulas used for Hospital, Memphis, TN 38105 drug infusions, and electrodes used for stimulating specific ar- 56 CONTEMPORARY TOPICS © 1999 by the American Association for Laboratory Animal Science Volume 38, No. 1 / January 1999 FIG. 2. Diagram of an implant for use in unanesthetized rats. The electrophysiologic recording electrode is attached to the skull. Two small mounting screws are anchored in the skull, and acrylic cement is used to bond the headpiece apparatus to the screws and directly to the cranium. The specific design of the headpiece depends on the nature of the ex- periment. In this example, the recording electrode is fixed only to the upper platform of the headpiece apparatus. Large screws positioned in the threaded posts in the base of the headpiece permit the upper plat- form to be moved in small increments, resulting in gradual insertion of the electrode into the brain so that neurons located at various depths can be monitored during subsequent experimental sessions. eas of the brain. We have not attempted to review all available modifications of these techniques, and we do not believe that any specific method described here is innately superior to alter- native procedures used by other researchers. Rather, our FIG. 3. A rodent stereotactic apparatus. The micromanipulator attached intention is to provide personnel who evaluate and manage ani- to the stereotactic frame can be moved in 3 dimensions by turning the mals used for long-term brain studies with an overview of these calibrated thumbscrews on each axis and, thus, can be used to accu- commonly used surgical techniques and to supply suggestions rately position an electrode or other device within a target structure in for managing or avoiding various problems. the brain during stereotactic surgery. Principles of Stereotactic Surgery Techniques for recording the activity of individual neurons involve inserting fine-tipped microelectrodes into the brain and positioning their active recording tips in close proximity to single neurons so that ongoing changes in electrical activity can be measured. By preparing animals with appropriate cra- nial implants, researchers can use this approach to measure neuronal activity in conscious, active animals. One type of head- piece (Figure 2) is designed to permit permanent implantation of an array of electrophysiologic recording electrodes in the brain of a rat, and illustrates the basic features of most perma- nently attached headpieces. The surgical procedure first involves exposing the surface of the skull by incising and re- tracting the overlying skin and fascia. Next, small screws are placed in the skull to provide a supporting base for the head- piece assembly. A hole is then drilled through the skull over FIG. 4. Anesthetized rat positioned in a stereotactic apparatus. The skull the target location in the brain, and the dura is incised. The is anchored between the ear bars, and the incisor bar is used to adjust electrode or other device that is to be implanted is attached to the forward angle of the snout and to hold the skull firmly in position. a micromanipulator and slowly inserted to a depth consistent with the location of the intended target structure in the brain. sitioning, animals are placed in a stereotactic frame during sur- Acrylic cement is used to secure the device in the correct posi- gery. The probe is then inserted into position using a finely tion. Some electrode assemblies, including the one illustrated, calibrated micromanipulator. Some stereotactic frames are in- allow the electrode to be gradually advanced through the brain tended only for use with a specific species (Figure 3), whereas to permit evaluation of various neurons during sequential re- other designs include adaptors that accommodate animals rang- cording sessions. Other assemblies secure the electrode tip in a ing in size from rodents to primates. Two components of the permanent position at the time of the initial surgery, permit- stereotactic