Olfactory pedunculotomy induced anosmia in the wolf (Canis lupus)

Physiology&Behavior,Vol. 27, pp. 543-546. PergamonPress and Brain Research Publ., 1981.Printed in the U.S.A.

Olfactory Pedunculotomy Induced Anosmia in the Wolf (Canis lupus) E R I C K. P E T E R S O N , * M A R C A. L E T E L L I E R , t J O N A T H A N A. P A R S O N S , * E D W A R D D. P L O T K A , * L. D A V I D M E C H § A N D U L Y S S E S S. S E A L ¶

*Department of Anatomy, University of Minnesota, 4-135 Jackson Hall, 321 Church Street SE Minneapolis, MN 55455 tTheda Clark Medical Center, Neenah, WI 54956 *Marshfield Medical Foundation, Inc., Marshfield, WI 54449 §U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, MD 20811 and SResearch Service, V.A. Hospital, Minneapolis, MN 55417 and the Departments of Biochemistry and Entomology, Fisheries and Wildlife, University of Minnesota St. Paul, MN 55108 Received 15 May 1981 PETERSON, E. K., M. A. LETELLIER, J. A. PARSONS, E. D. PLOTKA, L. D. MECH AND U. S. SEAL. Olfactory pedunculotomy inducedanosmia in the wolf(Canislupus). PHYSIOL. BEHAV. 27(3)543-546, 1981.--A procedure for the surgical induction of anosmia in the wolf is described. Six wolves (4 altered and 2 sham) and one mongrel dog were operated; four wolves and the dog were bilaterally pedunculotomized, and two wolves were sham operated. Behavioral tests with the wolves confirmed that they were anosmic, and anatomical verification in the dog showed that the olfactory peduncle (corresponds to the human olfactory tract) was completely sectioned. Use of this procedure will make possible tests of the role of olfaction in these animals. Olfactory pedunculotomy

Anosmia

Wolf

THE wolf uses olfactory cues for marking of trails and territory, pair-bonding, breeding, and seeking food [6, 7, 8, 9]. In some subordinate individuals reproduction is deferred during the annual breeding season as the alpha male and female tend to be the only breeders in packs containing multiple mature members [7]. This selective suppression of reproduction in subordinate animals may be induced by behavioral cues, endocrine changes, olfactory information, or a combination of these [7]. A procedure for inducing total anosmia in the timber wolf is described as an approach for determining the role of olfaction in deferred reproduction. METHOD One mongrel dog, age unknown, and six timber wolves, approximately 25 kg, at age 2 years 6 months were anesthetized with 300-500 mg ketamine hydrochloride (Ketaset ®, Bristol), 100 mg promazine (Sparine ®, Wyeth) and 10 mg diazepam (Valium ®, Roche) intramuscularly (IM). Additional doses were given intravenously (IV) as required. Four wolves and the dog were pedunculotomized and two wolves were sham operated. Six wolves served as intact, unoperated controls. All procedures employed standard sterile surgical techniques. The animals were shaved, and Betadine ® (Purdue Frederick Co.) was applied over the skull from the nasion to the inion and laterally to the zygomatic arches. The skin incision extended about 6-8 cm from a point just posterior to the attachment of the orbital ligament with the zygomatic

arch to a point midway between the bregma and the inion, thus exposing the frontalis and interscutularis muscles. The incision was continued through these muscles exposing the dense external fascia of the temporalis muscle. The fascia and muscle, which is approximately three times thicker in the wolf than in the dog, was incised parallel to the muscle fibers through the periosteum to the underlying parietal and temporal bones. Bleeding subsequent to muscle incision was controlled by bipolar electrocautery. Caution was exercised in all procedures near the infratemporal region to avoid the orbital contents. A periosteal elevator was used to retract the periosteum and muscle from the underlying bone on each side of the incision followed by placement of a self-retaining retractor. A hole (approximately 0.5 cm) was drilled (Hudson Burr and Ball drill, diameter 13 mm) through the skull at the junction of the sphenoid with the anterior-inferior parietal (area corresponds to the human pterion). The burr hole had to be inferior to the zygomatic process of the frontal bone to ensure proper exposure of the superior aspect of the olfactory peduncle and to avoid exposure of the frontal sinuses. The base of the anterior cranial fossa was identified by placing a small elevator between the dura and the bone, and the burr hole was enlarged subsequently down to that level by use of Leksell cranial rongeur forceps (angular) and Kerrison punch forceps (2 mm) to allow entrance of the bipolar coagulation forceps (Scoville-Greenwood). All bone chips removed were stored in sterile saline for later replacement. Bleeding of the dipole was controlled, and

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544 closure of any exposed sinuses was accomplished by impacting with bone wax. The exposed dura was opened with an inverted T-shaped incision. The resultant triangular flaps were retracted over the exposed bone. The frontal lobe was lightly retracted medially using a strip of small cottonoid (Codman surgical patties). This exposed the olfactory peduncle (which corresponds to the human olfactory tract). The peduncle was identifiable by its color (white as compared to the grey-pinkish of the frontal lobe). The peduncle was sectioned using bipolar coagulation at a setting of four on the power supply (Malis Bipolar Coagulator) with concomitant saline irriation and suction. In the subject animals, the peduncle was ribbonlike, approximately 5 mm high and 1 cm wide. Thus it was imperative to identify the arachnoid surrounding the peduncle inferiorly to ensure complete division of the peduncle. Upon completion of sectioning, the cottonoid was removed, gelfoam was placed between the sectioned portions and on the exposed brain, the dural flaps were replaced without suturing, and the bone chips placed within the skull opening. During this procedure it was important to avoid traumatizing the optic nerve which lay adjacent to the surgical field. The temporalis was sutured in two layers (internal muscle mass and external muscle mass) with Neurolon ® (0, Ethicon, Inc.). The external fascia and peripheral muscles were closed with Neurolon and the skin with Surgilon ® (4-0, American Cyanamid Co.) using individually placed mattress sutures. This operation was repeated on the opposing side, and then both incisions were coated with a topical antibiotic. Bilateral completion of this surgery required approximately 2 hours per animal. Two sham operated controls were treated exactly as the four ablated animals except for sectioning of the olfactory peduncles. The animals were given 3 x l0 s units of Flo-cillin® (Bristol) IM prior to surgery and oral antibiotics (tetracycline, amphicilin) for 5 days postoperatively.

Behavioral Tests

To determine the effectiveness of the ablative surgery, behavioral tests were developed to evaluate the presence or absence of anosmia in wolves 2 months after pedunculotomy. Twelve wolves were isolated in kennels with access to a 4 × 4 m enclosure in which they had been born, raised, and fed (Fig. 1). They were fasted for 24 hr prior to the tests. Behavioral test no. 1 (familiar feeding situation). Deer meat, a favorite food of the wolf, was placed on the cement floor of the enclosure. The wolves were released into the enclosure singly, starting with the surgically altered animals, and they were permitted to explore the area for 10 min. Observations on each animal were recorded from an isolated viewing room. Responses were recorded in two categories: either the animal ate the meat (positive response) or it did not (negative response) within a time span of 10 min. Behavioral test no. 2 (novel feeding situation). Five black 475 ml paper cups were placed around the enclosure. Approximately 150 g of deer meat were placed in one cup while the remaining cups were filled with the same volume of corn cobs. The animals were again tested for 10 min while their behavior was observed. The same responses were recorded as in test no. 1.

PETERSON ET AL.

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Statistics

Where applicable a one-way analysis of variance was conducted. Differences were considered significant if a p<0.01 was obtained. RESULTS

All surgically operated animals recovered without complications, and within 24 hr post operatively they were able to eat their pre-conditioned diet of dog food. The one exception to this was the dog, which had to be force fed for 3 days before it would eat on its own. The sham operated and intact control animals devoured the meat within 30 sec of their entrance into the enclosure; the surgically altered animals ignored the meat (Fig. 2). In behavioral test no. 2 all animals investigated the cups with glances or cursory sniffs, but only the sham and control animals ate the deer meat from the appropriate cup (Fig. 3). Since the solves were involved in behavioral studies anatomical verification of the completeness of the lesion was accomplished only with the dog, which was studied 2 weeks post operatively. Grossly, the olfactory peduncles were completely separated about 5 mm behind the olfactory bulb. The gelfoam within the sectioned region of the peduncle was adherent to the underlying dura. The skull opening was difficult to locate as the gelfoam packing and the dura formed a strong continuous seal over the opening, resembling the surrounding dura mater. Histologically, the peduncles were severed at the point of section. DISCUSSION

Olfaction has been implicated in the control of behavior, including reproductive behavior, in many species [2, 10, 11]. Endocrine changes inducing responses in target tissues (i.e. testosterone from the testes affecting secretions from the anal glands, and estrogens affecting vaginal secretions) are detected by the olfactory apparatus of other members of the species [4]. To test olfactory effects on behavior and endocrine changes in the wolf it was necessary to develop a technique to induce total anosmia in the animals. Previous studies have utilized diverse modes of producing anosmia in rats, mice,

OLFACTORY P E D U N C U L O T O M Y I N D U C E D ANOSMIA

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Treatment FIG. 2. Behavioral test no. 1. Response of wolves exposed to familiar feeding situation. *All animals in these groups responded within 30 sec, our minimal detectable time, therefore no variance is shown. **None of the animals in this group responded within 10 min, therefore no variance is shown.

gerbils, rabbits, guinea pigs, cats, and dogs [1, 3, 4, 5], including destroying or inhibiting peripheral olfactory receptors, olfactory bulbectomy [1,5] or lesions of the olfactory tract [3, 4, 5]. The peripheral approach to anosmia was inappropriate for our studies due to the reversibility of its effects and bulbectomy was deemed a too radical surgical approach for the initial studies [1,4]. The advantage of lesioning the olfactory peduncle lies in the fact that the olfactory bulbs are still present and can be removed at a later date to determine if the olfactory bulbs alone have any effects on the parameters being investigated. The preceding technique adequately fulfilled our requirements of inducing anosmia that would be maintained over the course of a year. The behavioral tests to verify the completeness of the lesion and the subsequent development of anosmia consisted of a familiar feeding situation (test no. 1, Fig. 2) where deer meat was in plain view. Since this procedure was similar to the animals' previous experiences when they received deer meat for other purposes (i.e. giving medications, hand feeding), it could be argued that recognizing and eating the deer meat (positive response) was learned behavior based upon visual cues and therefore not dependent on olfaction. However, the surgically ablated animals' failure to recognize the

FIG. 3. Behavioral test no. 2. Response of wolves exposed to a novel feeding situation. Bars indicate mean _+SEM for (n) animals in each group. **None of the animals in this group responded within 10 min, therefore no variance is shown for this group. Pedunculotomy treated animals were significantly different from the other groups at a p<0.01 level. It is important to remember that our animals were not domestic and that they could feed indiscriminately in tests preceded by fasting. During test no. 2 the control animals ingested not only the meat but the paper cups as well. Therefore the use of non-toxic, ingestible material was suggested.

deer meat as food strongly suggests that visual cues were unimportant in this situation and that therefore the surgery had induced anosmia. Further substantiation was provided by the second test involving a novel feeding situation (Fig. 3). In that test, the wolves could only see dark cups not previously associated with feeding. The deer meat and corn cobs occupied the bottom of the cups and were similar in volume. Because the control animals selectively located the deer meat in all cases and the surgically altered animals were totally unsuccessful in this experiment, we conclude that the surgery induced anosmia. ACKNOWLEDGEMENTS The authors gratefully acknowledge the assistance of Larry Peterson for technical advice and handling of the wolves. We appreciate the cooperation of the Minnesota Department of Natural Resources and, in particular, Loyd Knutsen and Roger Johnson. Partial financial support for this study was obtained from the Wilkie Fund of the Bell Museum of Natural History. We also wish to thank Theda Clark Hospital for use of surgical equipment and supplies.

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4. Doty, R. L. and I. Dunbar. Attraction of beagles to conspecific urine, vaginal and anal sac secretion odors. Physiol. Behav. 12: 825-833, 1974. 5. Macrides, F., A. C. Firl, Jr., S. P. Schneider, A. Bartke and D. G. Stein. Effects of one stage or serial transections of the olfactory tract on behavior and plasma testosterone levels in male hamsters. Brain Res. 109: 97-109, 1976.

546 6. Mech, L. D. The Wolf. Garden City, NY: Natural History Press, 1970. 7. Packard, J. M. Deferred reproduction in wolves (Canis lupus). Ph.D. thesis, University of Minnesota, Minneapolis, MN, August, 1980. 8. Peters, R. P. and L. D. Mech. Scent marking in wolves. Am. Scient. 63: 628--638, 1975. 9. Rothman, R. J. and L. D. Mech. Scent marking in lone wolves and newly formed pairs. Anim. Behav. 27: 750-760, 1979.

PETERSON ET AL. 10. Whitten, W. K. and A. K. Champlin. The role of olfaction in mammalian reproduction. In: Handbook o f Physiology, Section 7, Endocrinology, edited by R. O. Greep. Washington, DC: American Physiological Society, 1973, pp. 109--123. 11. Wysocki, C. J. Neurobehavioral evidence for the involvement of the vomeronasal system in mammalian reproduction. Neurosci. Biobehav. Rev. 3: 301-341, 1979.