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Chemical access to the vomeronasal organs of garter snakes
Physiology & Behavior, Vol. 24, pp. 367-371. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.
Chemical Access to the Vomeronasal Organs of Garter Snakes MIMI HALPERN
Department of Anatomy and Cell Biology, Downstate Medical Center, Brooklyn, N Y 11203 and JOHN L. KUBIE
Department of Physiology, Downstate Medical Center, Brooklyn, N Y 11203 R e c e i v e d 9 A u g u s t 1979 HALPERN, M. AND J. L. KUBIE. Chemical access to the vomeronasal organ o f garter snakes. PHYSIOL. BEHAV.
24(2) 367-371, 1980.--Garter snakes tongue-flicked cotton swabs soaked in a mixture of earthworm extract and 3H-proline. In snakes with intact tongues and patent vomeronasai ducts, large accumulations of radioactive material, as revealed by autoradiography, were observed in the vomeronasal organs. When the vomeronasal ducts were sutured closed, no radioactivity was discerned in the vomeronasal organs. The accumulations of radioactive material in the organs were reduced slightly when the tongue tips were removed. In snakes whose entire tongue was removed, but whose lips touched the swabs, a discernable amount of radioactive accumulation still occurred in the vomeronasal organs. Garter snake
Vomeronasal organ
Tongue flicking
T H E tongue flicking behavior of snakes is commonly thought to be a mechanism for odorant delivery to the vomeronasal (Jacobson's) organs [2]. The evidence for this idea, however, is largely circumstantial. The tongue tips as they reenter the mouth, passes along the fenestra vomeronasalis into which the ducts of Jacobson's organ open (Fig. 1). The vomeronasal organs of snakes are exceptionally well developed [19] and tongue flicking appears to be an important species-specific behavior. Rapid tongue-flicking occurs during prey trailing in a variety of snakes [1, 10, 12, 13, 14, 18, 20, 21] during exploration [8] and during courtship [16]. Increases in tongue flick rate are also observed when relevant prey stimuli [6, 9, 22] or predator odors [5] are presented to snakes. Finally, increased neural activity in the accessory olfactory bulb of snakes has been observed following tongue withdrawal from a stimulating substance [17]. Blocking tongue access to the vomeronasal organ by tongue removal [7] or vomeronasal duct suture [15] causes deficits in behaviors that depend on a functional vomeronasal system, such as prey trailing [15], male courtship [16] and prey attack [7,11]. Furthermore, snakes are unable to follow trails if their tongues are prevented from touching the trail substance [14]. To date, the only direct evidence that the snake's tongue is capable of delivering particles to the vomeronasal organ is the demonstration by Kahmann [12] that a snake sacrificed shortly after tongue flicking on a piece of carbon had a con-
Odorant access
Autoradiography
centration of soot in the lumen of the vomeronasal organ. The present study replicates and extends Kahmann's observation by asking, (1) whether the tongue is necessary for the delivery of particles to the vomeronasal organ, (2) whether substances found in the vomeronasal organ following tongue-flicking are also found in the nasal sacs, (3) whether substances picked up on the tongue can be prevented from entering the vomeronasal organ by closing the vomeronasal ducts, and (4) whether the tongue tips themselves are necessary for particle access to the vomeronasal organ. The general plan of the experiment was to present snakes with cotton swabs soaked with a mixture of earthworm extract and 3H-proline. The snakes tongue-flicked the swabs and the presence of radioactivity in the vomeronasal organs and nasal cavities was determined by autoradiography. METHOD
Subjects The subjects were 18 male and female adult garter snakes of the genus Thamnophis purchased from various laboratory animal suppliers. The " n o r m a l " group (n=4) consisted of snakes that were not subjected to any surgical procedure prior to testing. Four experimental groups were comprised as follows: duct suture (n=3), tongue removal (n=3), tongue removal and duct suture (n=2), tongue tip removal, n = 5 (unilateral)+ 1 (bilateral).
1This study was supported by National Institute of Health Grant NS11713. We thank Hansjurgen Distel for his collaboration in pilot work associated with this study, Ruu-Tong Wang for advice in technical matters, Alice Vagvolgyi and Natalie Frumin for histological processing of the tissue, Jack lllari for the photography, Richard Doering for the art work and Rose Kraus for typing the manuscript.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/020367-05502.00/0
~68
HALPERN
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permitted to touch it. For snakes whose tongues had been removed, the experimenter tapped the snout of the animal with the wet swab 10 times. Six hours following the tongue-flick or snout-touching procedure, the snakes were anesthetized with Sodium Brevital 0.015 mg/kg of body weight injected SC and perfused with 0.09% saline followed by Bodian's fixative [3.Jl.
Tisslce Preparation Vomeronasal - N ~ W
ium
The heads were fixed in Bodian's fixative, decalcified (see 16 for details) and embedded in Paraplast. Sections were cut at 10 tz, mounted on gelatinized slides, deparaffinized and dipped in Kodak NTB 2 emulsion. Sections were exposed for 3-8 weeks at 10°C, developed in Kodak D-19 and counterstained with cresyl violet. RESULTS
--
- - Mushroom body
Tongue and Sheath
.
.
.
~ct of Vomeronasal organ
.
.
.
Lateral ridge of Fenest m Vomeronasalis
FIG. 1. Schematic drawing of side view of garter snake head illustrating relative position of the vomeronasal organ, its duct and the roof of the mouth. Enlargement illustrates the position of tongue tip cut and path of tongue in relation to the lateral ridge of the fenestra vomeronasalis.
Surgery A paracervical injection of 0.1 cc Xylocaine (2% Xylocaine, Astra Pharmaceutical Products Inc., Worcester, MA) on each side produced a teml0orary local anesthesia of the head which permitted surgery on the animals. Duct suture was performed with the animals restrained on their backs. A wooden rod was placed in the mouth to keep it open. Using fine suture material, (9-0(35 tz) Ethilon; Ethicon, Inc. Somerville, NJ) and fine forceps we inserted four or five individual sutures tieing together the two lateral ridges of the fenestra vomeronasalis (See Fig. 5, reference [15]). Tongue cuts were made by gently pulling the tongue out of its sheath till it was fully extended. The tongue was cut at its root and a cautery (Accu-temp Cautery; Concept, Inc., Clearwater, FL) applied to the cut end to stop bleeding. Tongue tips were cut at the bifurcation of the tongue (Fig. 1). Little bleeding resulted and therefore no cautery was applied to the cut stub.
The vomeronasal organs of normal snakes that tongueflicked the radioactive earthworm extract mixture contained a dense band of reduced silver grains over the apical portion of the vomeronasal epithelium (Fig. 2A,B,C). This is the region occupied by the cell bodies of supporting cells and dendrites of bipolar neurons (receptor cells). Scattered silver grains were present in the more basal regions as well. In these animals no significant accumulation of silver grains was observed over the olfactory epithelium (Fig. 7). In snakes whose vomeronasat ducts were sutured closed, no significant accumulation of silver grains was observed over the vomeronasal (Fig. 3A,B,C) or olfactory epithelia although the epithelium lining the roof of the mouth was densely covered with silver grains (Fig. 8). A low but consistent level of labeling was observed in the vomeronasal epithelium of snakes whose tongues were removed (Fig. 4A,B,C). Apparently touching the lips with the radioactive swabs is sufficient for some substances to reach the vomeronasal organ. When the tips of the tongue were removed unilaterally, reduced silver grains were still observed in the vomeronasal organs bilaterally. In two animals there appeared to be a higher concentration of silver grains in the organ on the side of the intact tongue tip (Fig. 5), however, in most of the animals concentration differences between the two sides were less obvious (Fig. 6). In general the level of labeling was considerably reduced in the animals with tongue tip removed (compare Fig. 2 with Figs. 5 and 6) suggesting that the snakes may have experienced mechanical difficulties in passing their tongues back through the fenestra vomeronasalis. No accumulations of silver grains were observed in the vomeronasal organ, olfactory epithelium or roof of the mouth of the snake that did not contact the proline soaked cotton swab with tongue or lips. DISCUSSION
Procedure One day following surgery, snakes were removed from their cages, held by the experimenter, and presented with cotton swabs dipped in an earthworm extract--aH-proline mixture (approximately 210 t~ Ci concentrated 3H-proline mixed with 0.5 cc earthworm extract prepared by the method of Burghardt [6], 6 g earthworm to 20 cc dH20). The snakes with intact tongues or tongues with only tips removed tongue-flicked the swabs I0 to 50 times. One normal snake was presented with the swab 1 cm from the snout, but not
The results of the present study appear to be straightforward. The tongue of garter snakes is capable of bringing substances into the lumen of the vomeronasal organ as evidenced by the accumulation of silver grains over the epithelium of intact snakes. However, the tongue is not a necessary vehicle for access to the organ since, in the absence of a tongue, substances accumulate, albeit in very low quantities, in the organ. Bilaterally intact tongue tips appear to facilitate odorant delivery to the vomeronasal organ since lower than normal concentrations of silver grains are found
C H E M I C A L ACCESS-VOMERONASAL ORGANS
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FIG, 2. Horizontal section through the vomeronasal organ of a normal snake that tongue flicked a mixture of ~H-proline and earthworm extract. Rectangles in A and B represent areas magnified in B and C respectively. Autoradiograph counterstained with Cresyl violet. Bar= 100 /x (A), 50/x (B and C). FtG. 3. Horizontal section through the vomeronasal organ of a snake whose vomeronasal ducts were sutured closed. Otherwise same as Fig. 1. Abbreviations: Bp= Bipolar cell layer; Bv = Blood vessel; Cap = Capsule of organ; Lu = Lumen of organ; MB = Mushroom Body; S = Septum; SC=Supporting Cell Layer; Sep=Sensory epithelium; Ud=Undifferentiated cell layer. FIG. 4. Horizontal section through the vomeronasal organ of a snake whose entire tongue was removed. A cotton swab soaked with a mixture of '~H-prolineand earthworm extract was tapped against the animal's snout 10 times. Otherwise same as Fig. 2.
in the vomeronasal organs of animals whose tongue tips are removed unilaterally or bilaterally. Finally, stimulus access to the vomeronasal organ can be completely blocked by suturing closed the vomeronasal ducts. At no time was silver grain accumulation observed in the olfactory epithelium. These results fully confirm the work of Kahmann [12] and extend them by ruling out access to the olfactory epithelium by either tongue flicking or snout touching, Furthermore, the present results clarify some results reported previously by Wilde [22]. He reported that garter snakes with their tongues severed failed to attack earthworm extract swabs presented at a distance, but did attack if the swab touched the snout. Our results confirm Wilde's suggestion that although the tongue may normally act to bring substances to the vomeronasal organ, the tongue is not necessary for such access. In addition, our results demonstrate that non-volatiles are
capable of gaining access to the vomeronasal organ but do not gain access to the olfactory epithelium via a tongueflicking mechanism. Wilde [21] and Burghardt and Pruitt [7] found diminished responding to earthworm extracts when the tips of the tongue were severed. Our results suggest that less of the stimulating substance is gaining access to the vomeronasal organ when the tips are severed. Thus one might expect less consistent behavior from animals in which stimulus levels are hovering around the threshold level. Our demonstration that suturing closed the ducts to the vomeronasal organ effectively prevents stimulus access to the organ suggests that this procedure is an ideal means of reversibly deafferenting the vomeronasal system. We have used this technique in a study in which snakes trailed earthworm extract [15] and in a study of male courtship be-
370
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.i~ FIG. 5. Horizontal section through vomeronasal organ of a snake with one tongue tip removed. The left side of the figure is the side of the tongue tip removal. Otherwise same as Fig. 2. Bar 100/z (A), 50 (B-EL havior (Halpern and Kubie, unpublished observations). In both cases the behavior, which we had independently determined was d e p e n d e n t on a functional v o m e r o n a s a l system, ceased to be elicitable from the snakes as long as the duct sutures were in place. As soon as the sutures were r e m o v e d , the b e h a v i o r s returned to presuture levels.
.....
FIG. 6. Same as Fig. 5.
Our results further suggest that caution should be used in interpreting the results o f tongue cutting procedures. Frequently the removal of the tongue is thought to be equivalent to r e m o v i n g v o m e r o n a s a l stimulation. H o w e v e r , if the tongueless snake can touch its snout to the stimulating substance the v o m e r o n a s a l organ may be stimulated.
¸
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'-7A FIG. 7. Horizontal section through olfactory epithelium of a normal snake that tongue flicked the 3H-proline-earthworm extract mixture. Bar=50/z; BG=Bowman's Gland; Lu=Lumen of nasal cavity; SET,-Sensory eipthelium.
CHEMICAL
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FIG. 8. Horizontal section through roof of the mouth epithelium of a snake with tongue removed. Lips were tapped with radioactive swab. A: close to front of mouth. B: adjacent to duct of the vomeronasal organ.
REFERENCES
1. Baumann, F. Experimente fiber den Geruchsinn der Viper. Revue suisse Zool. 34: 173-184, 1927. 2. Bellairs, A. The Life o f Reptiles. New York, Universe Books, 1970. 3. Bodian, D. A method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat. Rec. 65: 8%97, 1936. 4. Bodian, D. The stain of paraffin sections of nervous tissue with activated protargol: The role of fixatives. Anat. Rec. 69: 153-162, 1937. 5. Bogert, C. M. Sensory cues used by rattlesnakes in their recognition of ophidian enemies. Ann. N.Y. Acad. Sci. 41: 32%343, 1941. 6. Burghardt, G. M. Stimulus control of the prey attack response in naive garter snakes. Psychon. Sci. 4: 37-38, 1%6. 7. Burghardt, G. M. and G. H. Pruitt. The role of the tongue and senses in feeding of naive and experienced garter snakes. Physiol. Behav. 14: 185-194, 1975, 8. Chiszar, D. and T. Carter. Reliability of individual differences between garter snakes (Thamnophis radix) during repeated exposure to an open field. Bull. Psychon. Soc. 5: 507-509, 1975. 9. Chiszar, D., K. Scudder and L. Knight. Rate of tongue flicking of garter snakes (Tharnnophis radix haydeni) and rattlesnakes (Crotalus v. viridis, Sistrurus catenatus tergerninus, and sistrurus catenatus edwards) during prolonged exposure to food odors. Behav. Biol. 18: 273-283, 1976. 10. Gehlbach, R. F., J. F. Watkins and J. C. Kroli. Pheromone trail-following of typhlopid and colubrid snakes. Behavior 40: 282-294, 1971. 11. Halpern, M. and N. Frumin. Roles of the vomeronasal and olfactory systems in prey attack and feeding in adult garter snakes. Physiol. Behav. 22: i183-1189, 1979. 12. Kahmann, H. Sennesphysiologische studien an Reptilien: I. Experimentelle untersuchungen fiber das jakobsonische organ der eidenschsen und schlangen. Zool. Jahr. Abteil. Allgem. Zool Physiol. Tiere 51: 173-238, 1932.
13. Kubie, J. L. and M. Halpern. Laboratory observations of trailing behavior in garter snakes. J. comp. physiol. Psychol. 89: 667-674, 1975. 14. Kubie, J. L. and M. Halpern. Garter snake trailing behavior: Effects of varying prey-extract concentration and mode of prey-extract presentation. J. comp. physiol. Psychol. 92: 362373, 1978. 15. Kubie, J. L. and M. Halpern. The chemical senses involved in garter snake prey trailing. J. comp. physiol. Psychol. 93: 648667, 1979. 16. Kubie, J. L., V. Vagvolgyi and M. Halpern. Roles of the vomeronasal and olfactory systems in courtship behavior of male garter snakes. J. comp. physiol. Psychol. 92: 627-641, 1978. 17. Meredith, M. and G. M. Burghardt. Electrophysiological studies of the tongue and accessory olfactory bulb in garter snakes. Physiol. Behav. 21: 1001-1008, 1978. 18. Naulleau, G. La biologie et le comportement predateur de Vipera aspis au laboratorie et dans la nature. Bull. Biol. Belg. 99: 395--524, 1%5. 19. Parsons, T. S. The nose and Jacobson' organ. In: Biology o f Reptilia Vol. 11 Morphology B, edited by C. Gans and T. S. Parsons. New York: Academic Press, pp. 99-191, 1970. 20. Weideman, E. Zur biologie der Nahrungs-aufnahme eurap/iischer Schlangen. Zool. Jber. Abstr. Syst. 61" 621-636, 1931. 21. Weideman, E. Zur biologie der Hahrungs-aufnahme der Kreuzotter, Vipera berus L. Zool. Anz. 97: 278-286, 1932. 22. Wilde, W. S. The role of Jacobson's organ in the feeding reaction of the common garter snake, Thamnophis sirtalis sirtalis (Linn). J. exp. Zool. 77: 445-465, 1938.
Chemical Access to the Vomeronasal Organs of Garter Snakes MIMI HALPERN
Department of Anatomy and Cell Biology, Downstate Medical Center, Brooklyn, N Y 11203 and JOHN L. KUBIE
Department of Physiology, Downstate Medical Center, Brooklyn, N Y 11203 R e c e i v e d 9 A u g u s t 1979 HALPERN, M. AND J. L. KUBIE. Chemical access to the vomeronasal organ o f garter snakes. PHYSIOL. BEHAV.
24(2) 367-371, 1980.--Garter snakes tongue-flicked cotton swabs soaked in a mixture of earthworm extract and 3H-proline. In snakes with intact tongues and patent vomeronasai ducts, large accumulations of radioactive material, as revealed by autoradiography, were observed in the vomeronasal organs. When the vomeronasal ducts were sutured closed, no radioactivity was discerned in the vomeronasal organs. The accumulations of radioactive material in the organs were reduced slightly when the tongue tips were removed. In snakes whose entire tongue was removed, but whose lips touched the swabs, a discernable amount of radioactive accumulation still occurred in the vomeronasal organs. Garter snake
Vomeronasal organ
Tongue flicking
T H E tongue flicking behavior of snakes is commonly thought to be a mechanism for odorant delivery to the vomeronasal (Jacobson's) organs [2]. The evidence for this idea, however, is largely circumstantial. The tongue tips as they reenter the mouth, passes along the fenestra vomeronasalis into which the ducts of Jacobson's organ open (Fig. 1). The vomeronasal organs of snakes are exceptionally well developed [19] and tongue flicking appears to be an important species-specific behavior. Rapid tongue-flicking occurs during prey trailing in a variety of snakes [1, 10, 12, 13, 14, 18, 20, 21] during exploration [8] and during courtship [16]. Increases in tongue flick rate are also observed when relevant prey stimuli [6, 9, 22] or predator odors [5] are presented to snakes. Finally, increased neural activity in the accessory olfactory bulb of snakes has been observed following tongue withdrawal from a stimulating substance [17]. Blocking tongue access to the vomeronasal organ by tongue removal [7] or vomeronasal duct suture [15] causes deficits in behaviors that depend on a functional vomeronasal system, such as prey trailing [15], male courtship [16] and prey attack [7,11]. Furthermore, snakes are unable to follow trails if their tongues are prevented from touching the trail substance [14]. To date, the only direct evidence that the snake's tongue is capable of delivering particles to the vomeronasal organ is the demonstration by Kahmann [12] that a snake sacrificed shortly after tongue flicking on a piece of carbon had a con-
Odorant access
Autoradiography
centration of soot in the lumen of the vomeronasal organ. The present study replicates and extends Kahmann's observation by asking, (1) whether the tongue is necessary for the delivery of particles to the vomeronasal organ, (2) whether substances found in the vomeronasal organ following tongue-flicking are also found in the nasal sacs, (3) whether substances picked up on the tongue can be prevented from entering the vomeronasal organ by closing the vomeronasal ducts, and (4) whether the tongue tips themselves are necessary for particle access to the vomeronasal organ. The general plan of the experiment was to present snakes with cotton swabs soaked with a mixture of earthworm extract and 3H-proline. The snakes tongue-flicked the swabs and the presence of radioactivity in the vomeronasal organs and nasal cavities was determined by autoradiography. METHOD
Subjects The subjects were 18 male and female adult garter snakes of the genus Thamnophis purchased from various laboratory animal suppliers. The " n o r m a l " group (n=4) consisted of snakes that were not subjected to any surgical procedure prior to testing. Four experimental groups were comprised as follows: duct suture (n=3), tongue removal (n=3), tongue removal and duct suture (n=2), tongue tip removal, n = 5 (unilateral)+ 1 (bilateral).
1This study was supported by National Institute of Health Grant NS11713. We thank Hansjurgen Distel for his collaboration in pilot work associated with this study, Ruu-Tong Wang for advice in technical matters, Alice Vagvolgyi and Natalie Frumin for histological processing of the tissue, Jack lllari for the photography, Richard Doering for the art work and Rose Kraus for typing the manuscript.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/020367-05502.00/0
~68
HALPERN
,\ N t ) K L 13 II:,
permitted to touch it. For snakes whose tongues had been removed, the experimenter tapped the snout of the animal with the wet swab 10 times. Six hours following the tongue-flick or snout-touching procedure, the snakes were anesthetized with Sodium Brevital 0.015 mg/kg of body weight injected SC and perfused with 0.09% saline followed by Bodian's fixative [3.Jl.
Tisslce Preparation Vomeronasal - N ~ W
ium
The heads were fixed in Bodian's fixative, decalcified (see 16 for details) and embedded in Paraplast. Sections were cut at 10 tz, mounted on gelatinized slides, deparaffinized and dipped in Kodak NTB 2 emulsion. Sections were exposed for 3-8 weeks at 10°C, developed in Kodak D-19 and counterstained with cresyl violet. RESULTS
--
- - Mushroom body
Tongue and Sheath
.
.
.
~ct of Vomeronasal organ
.
.
.
Lateral ridge of Fenest m Vomeronasalis
FIG. 1. Schematic drawing of side view of garter snake head illustrating relative position of the vomeronasal organ, its duct and the roof of the mouth. Enlargement illustrates the position of tongue tip cut and path of tongue in relation to the lateral ridge of the fenestra vomeronasalis.
Surgery A paracervical injection of 0.1 cc Xylocaine (2% Xylocaine, Astra Pharmaceutical Products Inc., Worcester, MA) on each side produced a teml0orary local anesthesia of the head which permitted surgery on the animals. Duct suture was performed with the animals restrained on their backs. A wooden rod was placed in the mouth to keep it open. Using fine suture material, (9-0(35 tz) Ethilon; Ethicon, Inc. Somerville, NJ) and fine forceps we inserted four or five individual sutures tieing together the two lateral ridges of the fenestra vomeronasalis (See Fig. 5, reference [15]). Tongue cuts were made by gently pulling the tongue out of its sheath till it was fully extended. The tongue was cut at its root and a cautery (Accu-temp Cautery; Concept, Inc., Clearwater, FL) applied to the cut end to stop bleeding. Tongue tips were cut at the bifurcation of the tongue (Fig. 1). Little bleeding resulted and therefore no cautery was applied to the cut stub.
The vomeronasal organs of normal snakes that tongueflicked the radioactive earthworm extract mixture contained a dense band of reduced silver grains over the apical portion of the vomeronasal epithelium (Fig. 2A,B,C). This is the region occupied by the cell bodies of supporting cells and dendrites of bipolar neurons (receptor cells). Scattered silver grains were present in the more basal regions as well. In these animals no significant accumulation of silver grains was observed over the olfactory epithelium (Fig. 7). In snakes whose vomeronasat ducts were sutured closed, no significant accumulation of silver grains was observed over the vomeronasal (Fig. 3A,B,C) or olfactory epithelia although the epithelium lining the roof of the mouth was densely covered with silver grains (Fig. 8). A low but consistent level of labeling was observed in the vomeronasal epithelium of snakes whose tongues were removed (Fig. 4A,B,C). Apparently touching the lips with the radioactive swabs is sufficient for some substances to reach the vomeronasal organ. When the tips of the tongue were removed unilaterally, reduced silver grains were still observed in the vomeronasal organs bilaterally. In two animals there appeared to be a higher concentration of silver grains in the organ on the side of the intact tongue tip (Fig. 5), however, in most of the animals concentration differences between the two sides were less obvious (Fig. 6). In general the level of labeling was considerably reduced in the animals with tongue tip removed (compare Fig. 2 with Figs. 5 and 6) suggesting that the snakes may have experienced mechanical difficulties in passing their tongues back through the fenestra vomeronasalis. No accumulations of silver grains were observed in the vomeronasal organ, olfactory epithelium or roof of the mouth of the snake that did not contact the proline soaked cotton swab with tongue or lips. DISCUSSION
Procedure One day following surgery, snakes were removed from their cages, held by the experimenter, and presented with cotton swabs dipped in an earthworm extract--aH-proline mixture (approximately 210 t~ Ci concentrated 3H-proline mixed with 0.5 cc earthworm extract prepared by the method of Burghardt [6], 6 g earthworm to 20 cc dH20). The snakes with intact tongues or tongues with only tips removed tongue-flicked the swabs I0 to 50 times. One normal snake was presented with the swab 1 cm from the snout, but not
The results of the present study appear to be straightforward. The tongue of garter snakes is capable of bringing substances into the lumen of the vomeronasal organ as evidenced by the accumulation of silver grains over the epithelium of intact snakes. However, the tongue is not a necessary vehicle for access to the organ since, in the absence of a tongue, substances accumulate, albeit in very low quantities, in the organ. Bilaterally intact tongue tips appear to facilitate odorant delivery to the vomeronasal organ since lower than normal concentrations of silver grains are found
C H E M I C A L ACCESS-VOMERONASAL ORGANS
369
-':
Ig
r
•
t, I
CAa $C
. tap
• I
,\
,=..ai!'
4B,.~ . - .
+.
- - 4 ¢
FIG, 2. Horizontal section through the vomeronasal organ of a normal snake that tongue flicked a mixture of ~H-proline and earthworm extract. Rectangles in A and B represent areas magnified in B and C respectively. Autoradiograph counterstained with Cresyl violet. Bar= 100 /x (A), 50/x (B and C). FtG. 3. Horizontal section through the vomeronasal organ of a snake whose vomeronasal ducts were sutured closed. Otherwise same as Fig. 1. Abbreviations: Bp= Bipolar cell layer; Bv = Blood vessel; Cap = Capsule of organ; Lu = Lumen of organ; MB = Mushroom Body; S = Septum; SC=Supporting Cell Layer; Sep=Sensory epithelium; Ud=Undifferentiated cell layer. FIG. 4. Horizontal section through the vomeronasal organ of a snake whose entire tongue was removed. A cotton swab soaked with a mixture of '~H-prolineand earthworm extract was tapped against the animal's snout 10 times. Otherwise same as Fig. 2.
in the vomeronasal organs of animals whose tongue tips are removed unilaterally or bilaterally. Finally, stimulus access to the vomeronasal organ can be completely blocked by suturing closed the vomeronasal ducts. At no time was silver grain accumulation observed in the olfactory epithelium. These results fully confirm the work of Kahmann [12] and extend them by ruling out access to the olfactory epithelium by either tongue flicking or snout touching, Furthermore, the present results clarify some results reported previously by Wilde [22]. He reported that garter snakes with their tongues severed failed to attack earthworm extract swabs presented at a distance, but did attack if the swab touched the snout. Our results confirm Wilde's suggestion that although the tongue may normally act to bring substances to the vomeronasal organ, the tongue is not necessary for such access. In addition, our results demonstrate that non-volatiles are
capable of gaining access to the vomeronasal organ but do not gain access to the olfactory epithelium via a tongueflicking mechanism. Wilde [21] and Burghardt and Pruitt [7] found diminished responding to earthworm extracts when the tips of the tongue were severed. Our results suggest that less of the stimulating substance is gaining access to the vomeronasal organ when the tips are severed. Thus one might expect less consistent behavior from animals in which stimulus levels are hovering around the threshold level. Our demonstration that suturing closed the ducts to the vomeronasal organ effectively prevents stimulus access to the organ suggests that this procedure is an ideal means of reversibly deafferenting the vomeronasal system. We have used this technique in a study in which snakes trailed earthworm extract [15] and in a study of male courtship be-
370
H A L P E R N A N D KLiBIE
P
[,
t~
F
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.i~ FIG. 5. Horizontal section through vomeronasal organ of a snake with one tongue tip removed. The left side of the figure is the side of the tongue tip removal. Otherwise same as Fig. 2. Bar 100/z (A), 50 (B-EL havior (Halpern and Kubie, unpublished observations). In both cases the behavior, which we had independently determined was d e p e n d e n t on a functional v o m e r o n a s a l system, ceased to be elicitable from the snakes as long as the duct sutures were in place. As soon as the sutures were r e m o v e d , the b e h a v i o r s returned to presuture levels.
.....
FIG. 6. Same as Fig. 5.
Our results further suggest that caution should be used in interpreting the results o f tongue cutting procedures. Frequently the removal of the tongue is thought to be equivalent to r e m o v i n g v o m e r o n a s a l stimulation. H o w e v e r , if the tongueless snake can touch its snout to the stimulating substance the v o m e r o n a s a l organ may be stimulated.
¸
Lu
713
'-7A FIG. 7. Horizontal section through olfactory epithelium of a normal snake that tongue flicked the 3H-proline-earthworm extract mixture. Bar=50/z; BG=Bowman's Gland; Lu=Lumen of nasal cavity; SET,-Sensory eipthelium.
CHEMICAL
ACCESS-VOMERONASAL
ORGANS
371
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~*
8 B"
FIG. 8. Horizontal section through roof of the mouth epithelium of a snake with tongue removed. Lips were tapped with radioactive swab. A: close to front of mouth. B: adjacent to duct of the vomeronasal organ.
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