Brain Research, 575 (1992) 132-137 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
132 BRES 17547
Area postrema mediation of physiological and behavioral effects of lithium chloride in the rat Ilene L. Bernstein, Mark Chavez, Donald Allen and Eugene M. Taylor Department of Psychology, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted 5 November 1991)
Key words: Gastric emptying; Hypothermia; Emetic mechanism; Learned taste aversion
The area postrema (AP), a chemoreceptor trigger zone for nausea and vomiting, has been implicated in taste aversion conditioning with LiCI. In addition to taste aversion acquisition, the present studies indicate that a number of other responses to LiC1 administration are eliminated by lesions of the AP. These include a behavioral response, 'lying-on-belly' as well as two physiological responses, delayed stomach emptying and hypothermia. These findings suggest that the area postrema is critically involved in the detection of LiCI and in a wide range of responses to this toxin. They also provide strong evidence that the failure to acquire conditioned taste aversions to LiCl-paired flavors after AP lesions can be attributed to the absence of a significant 'illness' response in lesioned animals. INTRODUCTION The area p o s t r e m a ( A P ) , a circumventricular organ in the brainstem, has been identified as the site of a c h e m o r e c e p t o r trigger zone for nausea and emesis 3'4. In rats, lesions of this region eliminate or attenuate taste aversion learning in response to some, but not all, unconditioned stimuli 3A8. Lithium chloride (LiCI) is widely used as the unconditioned stimulus in studies of taste aversion learning 17. Several studies have d e m o n s t r a t e d that lesions of the area p o s t r e m a interfere with the acquisition of learned taste aversions when LiC1 is employed as the unconditioned stimulus 5'7'8'16. Thus, the A P appears to play a critical role in lithium-induced taste aversions. This role could involve the detection of the conditioned stimulus (taste); detection of the unconditioned stimulus (drug) and/or the integration of taste and visceral cues to establish a learned association. It is generally believed that the A P is important in detecting the presence of the unconditioned stimulus and/or triggering illness or nausea 8'18. If the role played by the A P in conditioned responses to drugs like LiC1 primarily involves detection of the unconditioned stimulus, additional questions remain. In particular, when an AP-lesioned rat fails to acquire a conditioned taste aversion after the administration of LiCI it is unclear whether the animal fails to respond to the LiC1 at all or whether a partial response to the LiC1 occurs which does not support taste aversion acquisition. Some investigators have addressed the question of
whether animals with lesions of the A P show responses to the administration of LiC1, responses, that is, other than the d e v e l o p m e n t of conditioned taste aversions. A P - l e s i o n e d rats fail to show the anti-aggressive effect of LiC1 which is generally observed in intact rats 1°. Lesioned animals also have been r e p o r t e d to fail to show behavioral depression after lithium s. In contrast to the latter findings are reports of elevations in oxytocin 5 and 'spastic movements and a b d o m e n dragging '7 after LiCI treatments in A P - l e s i o n e d rats. Because all these responses can be viewed as indices of illness or malaise, there would a p p e a r to be discrepant findings with regard to whether LiC1 induces illness in A P - l e s i o n e d animals. The present studies examined a n u m b e r of responses known to be triggered by the administration of LiC1 to determine whether any of these responses occur in animals with complete destruction of the area postrema. Since the rat is a non-emetic species, responses other than retching and vomiting need to be e m p l o y e d as indices of a physiological response to lithium. The behavioral response to lithium administration has been characterized as a general suppression of activity as well as a specific response described as 'lying-on-belly' ( L O B ) TM 14. Physiological responses to lithium include dramatic delays in gastric emptying 9 and a m a r k e d decline in body t e m p e r a t u r e 1'21. These responses were evaluated in A P lesioned rats along with their ability to display a conditioned taste aversion to a saccharin solution paired with lithium.
Correspondence: I.L. Bernstein, Department of Psychology, University of Washington, Seattle, WA 98195, U.S.A. Fax: (1) (206) 685-3157.
133 MATERIALS AND METHODS Subjects were male Long-Evans and Wistar rats weighing between 280 and 350 g at the start of the study. The rats were maintained in individual cages in a room with a 12:12 h light:dark cycle. Food and water were continually available except as noted. A single group of Long-Evans male rats was used in the taste aversion, behavioral assessment and body temperature phases of this project. A separate group of Wistar male rats of comparable body weights was used to assess gastric emptying.
Area postrema lesions Rats were matched for body weight and received either lesions of the area postrema (APX) or sham (SHAM) operations 2. The area postrema was exposed by slight enlargement of the foramen magnum. Under direct visualization using a dissecting microscope, AP tissue was removed in the manner of a 'brush' biopsy, using the tightly twisted filaments teased from the tip of a cotton applicator. Residual AP tissue was destroyed by application of an ophthalmic cautery. In sham-operated animals the region was exposed, but no lesion was made. At least 6 weeks elapsed to allow for recovery from the acute effects of the lesion and for stabilization of body weight. At this stage lesioned animals weighed 60-100 g less than sham-operated controls. Weight differences, which were statistically significant, reflect the weight loss known to be associated with AP lesions TM.
Taste aversion conditioning Long-Evans rats were placed on a 19-h, water deprivation schedule for 3 days, with water available for 5 h a day during the light phase of the lighting cycle. Within each lesion condition animals were assigned to either an experimental or control group (n = 8 per group). On the conditioning day rats received a novel 0.15% saccharin solution in place of water for 30 min. Immediately after the drinking period experimental animals received an i.p. injection of 0.15 M LiCl in a volume of 20 ml/kg of body weight (APX/LiC1; SHAM/LiC1) while control animals received a comparable volume of isotonic saline (0.15 M NaC1; APX/NaC1; SHAM/NaCl). Three days after conditioning animals were tested for saccharin preference in a two-bottle test. Two calibrated drinking tubes, containing tap water and 0.15% saccharin solution were presented for 30 min. Sampling of each solution was insured by briefly introducing each drinking tube alone until the animals had sampled both the saccharin and the water. At the end of the test period intake of water and saccharin were recorded. Preference scores for saccharin were calculated for individual animals by dividing the volume of saccharin solution consumed by total fluid intake during the test (saccharin + water).
recorded the behaviors in which the subjects engaged; thus, for every minute each subject was observed for 10 s and its behavior was recorded for a total of at least 30 scores. Interrater reliability for 'lying on belly' was 0.89.
Body temperature responses Biotelemetry devices for detecting body temperature (MiniMitter Co., Sunriver, OR) were implanted intraperitoneally under halothane anesthesia into 4 APX and 4 SHAM animals. These devices send out AM-band signals proportional to the temperature within the peritoneal cavity and allow for remote monitoring of body temperature with an accuracy of about 0.1°C. Three days after implantation of thermistors animals were placed in recording chambers for 20 min and baseline temperatures were recorded every 5 min. Animals were then given an i.p. injection of 0.15 M LiC1 (20 ml/kg) and returned to the recording chamber for an additional 80 min. Temperatures were recorded every 5 min.
Gastric emptying This response was measured in a separate group of AP-lesioned and control Wistar rats. Rats were accustomed to receiving an afternoon meal of moist chow (approximately 1 part Wayne Lab Blox to 1.5 parts tap water). For testing they were deprived of food for 24 h (to allow for emptying of stomach contents) and then offered a meal of moist chow for 30 min. Intake was measured by weighing the food jars before and after the meal. Rats then received i.p. injections of LiC1 (APX/LiC1, n = 7; SHAM/LiCI, n = 5) or NaCI (APX/NaCI, n = 6; SHAM/NaC1, n = 4) (0.15 M; 20 ml/kg). Three hours after injection animals were sacrificed with an overdose of Equithesin. Both ends of the stomach were ligated and the stomach was removed and weighed. Stomach emptying was estimated for individual animals by calculating a retention ratio, the weight of the full stomach divided by the weight of the mash meal.
Histology Rats were sacrificed by an overdose of Equithesin (Nembutal/ chloral hydrate) and were perfused with saline and then phosphatebuffered formalin. Brains were removed, fixed in 30% glucose-formalin, and then frozen and cut into serial sections of 30 /~m thickness. Serial sections were mounted on slides and stained with thionin for microscopic examination.
RESULTS
Taste aversion conditioning S a c c h a r i n p r e f e r e n c e s c o r e s , d e p i c t e d in Fig. 1, indi-
Behavioral responses to LiCl administration
cate that strong, significant aversions developed to the
Behavioral responses to LiCI administration were assessed in the same animals at the time of conditioning. Cylindrical Plexiglas cages (20 cm in diameter; 30 cm in height) served as the experimental chambers. The floors of the chambers were made of stainless steel rods positioned 2.5 cm apart. Six chambers were placed side-by-side on a table that was situated along one wall of the conditioning room. Animals were habituated to the chambers prior to testing. After injection with LiCl or NaC1 animals were placed in the chambers and behaviors were recorded by two observers, blind to group membership, who scored a set of 9 different behaviorsa (adapted from Parkerl3). Observers sat in the conditioning room with a clear view of the chambers. At 10-s intervals the observers
l i t h i u m - p a i r e d s a c c h a r i n s o l u t i o n in s h a m - o p e r a t e d anim a l s (t14 = 4.45; P < 0.001) w h i l e n o s a c c h a r i n a v e r s i o n s w e r e e v i d e n t in a n i m a l s w i t h A P l e s i o n s . T h e s e results a r e c o n s i s t e n t w i t h a n u m b e r o f o t h e r s t u d i e s w h i c h indicate that, after destruction of the AP, animals do not a c q u i r e c o n d i t i o n e d t a s t e a v e r s i o n s w h e n LiCI is t h e u n conditioned stimulus.
Behavioral responses to LiCl administration A s c a n b e s e e n in Fig. 2, ' l y i n g - o n - b e l l y ' ( L O B ) is t h e
a Behavioral categories included: 'lying on belly', defined as lying prostrate on belly with no discernable body movement, loss of muscle tone, head and nose slumping to the floor of the cage; 'doggy scratch', limb flicking, grooming, chin rubbing, 'wet dog shake', freezing, rearing and 'other'.
134
1.0 o w e=
m
F.-
38 37
o
36
&
[] saline c
[]
E
"
APX
LiCl
o.
35 APX
SHAM
Fig. 1. Mean saccharin preference scores (+ S.E.M.) in AP-lesioned (APX) and sham-operated (SHAM) animals after conditioning with LiCI (striped bars) or saline (white bars). Differences between LiC1 and saline groups is significant (P < 0.001) in SHAM but not APX animals.
dominant behavior displayed by intact rats after LiC1 injections; with L O B scores significantly higher after LiCI injections than after saline (t14 = 12.8; P < 0.001). In contrast, L O B scores were low in all A P - l e s i o n e d rats, and did not differ as a function of whether animals had received LiC1 or saline. Thus, L O B , a postural index of malaise, did not occur after LiC1 injections in animals with lesions of the AP.
Body temperature responses Baseline body t e m p e r a t u r e (mean of the 4 measurements obtained before the injection of LiCI) was the same in the A P - l e s i o n e d and control animals, averaging 37.9°C (see Fig. 3). Mean body t e m p e r a t u r e of control animals declined steadily during the 80-min period following injection of LiC1, reaching a mean of 35.3°C. A P lesioned animals displayed a non-significant decline in
2'0
4'0 6~0 8~0 TIME (minutes)
SHAM I 00
Fig. 3. Mean body temperature (°C) in APX and SHAM animals during the baseline period and for 80 rain after treatment with LiCI. LiCI was administered at 20 rain.
body t e m p e r a t u r e of less than I°C. A r e p e a t e d measures A N O V A indicated a significant effect of time on body t e m p e r a t u r e (F15.9o = 7.43; P < 0.001) and a significant interaction between lesion condition and time (F15,90 = 7.27; P < 0.001).
Gastric emptying Mash meals consumed prior to drug treatments were larger for sham-lesioned animals (16.5 g + 2.0) than for animals with A P lesions (9.0 g _+ 1.4). To examine gastric emptying, stomach contents were expressed as retention ratios relative to the size of individual test meals. The p r o p o r t i o n of the test meal which r e m a i n e d in the stomach 3 h after LiCI injections are presented in Fig. 4. In s h a m - o p e r a t e d animals a dramatic effect of LiC1 is evident; it appears that virtually none of the meal consumed before lithium t r e a t m e n t had e m p t i e d from the stomach, whereas in saline-treated animals around half of the ingested meal had already e m p t i e d (t7 --- 5.64; P
1.2-
c =
I-Isaline ~LiCI
m co 0
~.0 0.8
8
0.6
[] saline T
m
o
E (n
OA
T
[] ucl
1
O2
0.0
APX
SHAM
Fig. 2. Mean LOB ('lying-on-belly') scores (+ S.E.M.) in AP-lesioned (APX) and sham-operated (SHAM) animals after treatment with LiCI (striped bars) or saline (white bars). Differences between LiCI and saline groups is significant (P < 0.001) in SHAM but not APX animals.
APX
SH-AM
Fig. 4. Mean stomach contents (+ S.E.M.) expressed as a ratio to weight of mash meal in APX and SHAM animals after treatment with LiC1 (striped bars) or saline (white bars). Differences between LiC1 and saline groups is significant (P < 0.001) in SHAM but not APX animals.
135
Fig. 5. Schematics (adapted from ref. 15) display coronal sections through the brainstem at the level of the lesion in an intact and lesioned brain (lesioned region is blackened). AP, area postrema; CUL, lateral cuneate nucleus; CUM, medial cuneate nucleus; FC, fasiculus cuneatus; FG, fasiculus gracilis; FLM, medial longitudinal fasiculus; GR, nucleus gracilis; NTST, nucleus of the spinal tract of V; PC1, inferior cerebellar peduncle; SOL, nucleus solitarius; TCS, corticospinal tract; TSC, spinocerebellar tract; TSL, lateral nucleus of tractus solitarius; TST, spinal tract of V; TSTH, spinothalamic tract; X, nucleus of vagus; XII, nucleus of hypoglossal.
< 0.001). In contrast, LiC1 had no apparent effect on stomach emptying after destruction of the AP; in lesioned groups there was no difference between lithiumand saline-treated animals in the proportion of the mash meal which remained in the stomach. A comparison between the retention ratios in A P X NaCI and SHAM-NaC1 groups suggests that stomach emptying may have been accelerated in AP-lesioned animals relative to controls, although this effect was not statistically reliable.
Histology Inspection of brain slices taken from lesioned animals revealed that lesions were essentially limited to ablation of the area postrema with slight intrusion into the dorsal edge of the solitary tract. The most extensive lesion, displayed in Fig. 5, involved minimal damage to the border of the dorsal motor nucleus of the vagus and hypo-
glossal nucleus and minimal invasion of dorsal column fibers. None of the lesioned brains showed significant damage to the vagal or hypoglossal nuclei. DISCUSSION The area postrema lacks a blood-brain barrier and contains chemoreceptors sensitive to certain drugs 3'4. This chemosensitive region participates importantly in emetic responses in many mammalian species. Lithium chloride, a toxin with a wide range of peripheral as well as central effects, is known to penetrate the blood-brain barrier 6,~z. Nonetheless, the area postrema has been implicated in the response to administration of LiC1 in the rat. Taste aversion conditioning, with LiCI as the unconditioned stimulus, is attenuated or eliminated in animals with lesions of the A P s'ts. Additionally, the present studies indicate that a number of other responses to LiC1
136 administration are eliminated by lesions of the AP. These include a behavioral response, 'lying-on-belly' as well as two physiological responses, delayed stomach emptying and hypothermia. Since the rat is a non-emetic species, responses like delayed stomach emptying and 'lying-onbelly' have been e m p l o y e d as indirect indices of nausea or malaise, Thus, the present data provide additional support for the suggestion that receptors in the region of the area p o s t r e m a are critically involved in detecting LiCI and in a wide range of responses to this toxin. The present findings are somewhat inconsistent with a report by Carter and Lightman 5. The main focus of that p a p e r was the mediation of cholecystokinin-stimulated oxytocin secretion but groups receiving LiCI were also included. It was found that the oxytocin secretion provoked by LiC1 administration was not attenuated by A P lesions. Those authors concluded that, although the nausea-promoting effects of LiCI may involve the AP, neuroendocrine effects m a y be m e d i a t e d by other regions. This would not be consistent with the idea that, in the rat, oxytocin release is a reliable biological m a r k e r of nausea 22 since elevations in oxytocin in A P - l e s i o n e d animals should indicate that they were experiencing nausea. Since the Carter and Lightman 5 data are the only systematic evidence that A P - l e s i o n e d animals respond to LiC1 administration with illness, they represent an anomalous finding which p r o b a b l y needs confirmation. O u r data provide evidence that the area p o s t r e m a is important in orchestrating physiological responses to
LiC1, including delaying gastric emptying and hypothermia. The A P has strong connections with the nucleus of the solitary tract and the dorsal m o t o r nucleus of the vagus as well as the parabrachial nucleus 2°. Thus, lesions to the A P would be likely to interfere with vagal mechanisms regulating gastric emptying. Involvement of the A P in lithium's hypothermic effect could be based on input to the parabrachial nucleus which projects to the hypothalamus 19. The loss of physiological responses to LiC1 is correlated with the loss of behavioral indications of discomfort (lying on belly) and visceral malaise (taste aversion learning) normally induced by the drug. This pattern of results is consistent with the hypothesis that the failure to acquire a conditioned taste aversion to a lithiumpaired flavor after A P lesions reflects the absence of a significant 'illness' response in lesioned animals. These findings do not rule out a m o r e general role of the A P in integration of sensory information important for formation of learned associations about food. For example Kosten and Contreras 7 recently p r o p o s e d that the lesion interferes with the ability to recognize taste solutions as novel. However, it should be noted that such a role for the A P would not explain the b r o a d range of lithiuminduced responses which are attenuated or eliminated by A P lesions. Acknowledgements. This work was supported in part by NIH
Grant CA26419.
REFERENCES 1 Batson, J.D., Effects of repeated lithium injections on temperature, activity and flavor conditioning in rats, Animal Learn. Behav., 11 (1983) 199-204. 2 Bernstein, I.L., Taylor, E.M. and Bentson, K.L., TNF-induced anorexia and learned food aversions are attenuated by area postrema lesions, Am. J. Physiol., 260 (1991) R906-R910. 3 Borison, H.L., Area postrema: chemoreceptor circumventricular organ of the medulla oblongata, Prog. Neurobiol., 32 (1989) 351-390. 4 Borison, H.L. and Wang, S.C., Physiology and pharmacology of vomiting, Pharmacol. Rev., 5 (1953) 193-230. 5 Carter, D.A. and Lightman, S.L., A role for the area postrema in mediating cholecystokinin-stimulated oxytocin secretion, Brain Research, 435 (1987) 327-330. 6 Davenport, V.D., Distribution of parenterally administered lithium in plasma, brain and muscle of rats, Am. J. Physiol., 163 (1950) 633-641. 7 Kosten, T. and Contreras, R.J., Deficits in conditioned heart rate and taste aversion in area postrema-lesioned rats, Behav. Brain Res., 35 (1989) 9-21. 8 Ladowsky, R.L. and Ossenkoff, K.P., Conditioned taste aversions and changes in motor activity in lithium-treated rats, Neuropharmacology, 25 (1986) 71-77. 9 McCann, M.J., Verbalis, J.G. and Stricker, E.M., LiC1 and CCK inhibit gastric emptying and feeding and stimulate OT secretion in rats, Am. J. Physiol., 256 (1989) R463-R468. 10 McGlone, J.J., Ritter, S. and Kelley, K.W., The antiaggressive
11 12
13 14 15 16
17 18 19
effect of lithium is abolished by area postrema lesion, Physiol. Behav., 24 (1980) 1095-1100. Meachum, C.L. and Bernstein, I.L., Conditioned responses to a taste CS paired with LiC1 administration, Behav. Neurosci., 104 (1990) 711-715. Morrison, J.M., Pritchard, H.D., Brande, M.L. and Aguanns, W.D., Plasma and brain lithium levels after lithium carbonate and lithium chloride administration by different routes in rats, Proc. Soc. Exp. Biol. (N.Y.), 137 (1971) 889-892. Parker, L.A., Nonconsummatory and consummatory behavioral CRs elicited by lithium- and amphetamine-paired flavors, Learn. Motiv., 13 (1982) 281-303. Parker, L.A., Hills, K. and Jensen, K., Behavioral CRs elicited by a lithium- or an amphetamine-paired contextual test chamber, Learn. Motiv., 12 (1984) 307-315. Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn. Plenum, New York, 1979. Rabin, B.M., Hunt, W.A. and Lee, J., Attenuation of radiation- and drug-induced conditioned taste aversions following area postrema lesions in the rat, Radiat. Res., 93 (1983) 388394. Riley, A.L. and Tuck, D.L., Conditioned taste aversions: a behavioral index of toxicity, Ann. N Y Acad. Sci., 443 (1985) 272292. Ritter, S., McGlone, J.J. and Kelley, K.W., Absence of lithium-induced taste aversion after area postrema lesion, Brain Research, 201 (1980) 501-506. Saper, C.B. and Loewy, A.D., Efferent connections of the
137 parabrachial nucleus in the rat, Brain Research, 197 (1980) 291317. 20 Shapiro, R.E. and Miselis, R.R., The central nervous connections of the area postrema of the rat, J. Comp. Neurol., 234 (1985) 344-364. 21 Taukulis, H.K., Attenuation of pentobarbital-elicited hypother-
mia in rats with a history of pentobarbital-LiC1 pairings, Pharmacol. Biochem. Behav., 17 (1982) 695-697. 22 Verhalis, J.G., McHale, C.M., Gardiner, T.W. and Stricker, E.M., Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats, Behav. Neurosci., 100 (1986) 466-475.
132 BRES 17547
Area postrema mediation of physiological and behavioral effects of lithium chloride in the rat Ilene L. Bernstein, Mark Chavez, Donald Allen and Eugene M. Taylor Department of Psychology, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted 5 November 1991)
Key words: Gastric emptying; Hypothermia; Emetic mechanism; Learned taste aversion
The area postrema (AP), a chemoreceptor trigger zone for nausea and vomiting, has been implicated in taste aversion conditioning with LiCI. In addition to taste aversion acquisition, the present studies indicate that a number of other responses to LiC1 administration are eliminated by lesions of the AP. These include a behavioral response, 'lying-on-belly' as well as two physiological responses, delayed stomach emptying and hypothermia. These findings suggest that the area postrema is critically involved in the detection of LiCI and in a wide range of responses to this toxin. They also provide strong evidence that the failure to acquire conditioned taste aversions to LiCl-paired flavors after AP lesions can be attributed to the absence of a significant 'illness' response in lesioned animals. INTRODUCTION The area p o s t r e m a ( A P ) , a circumventricular organ in the brainstem, has been identified as the site of a c h e m o r e c e p t o r trigger zone for nausea and emesis 3'4. In rats, lesions of this region eliminate or attenuate taste aversion learning in response to some, but not all, unconditioned stimuli 3A8. Lithium chloride (LiCI) is widely used as the unconditioned stimulus in studies of taste aversion learning 17. Several studies have d e m o n s t r a t e d that lesions of the area p o s t r e m a interfere with the acquisition of learned taste aversions when LiC1 is employed as the unconditioned stimulus 5'7'8'16. Thus, the A P appears to play a critical role in lithium-induced taste aversions. This role could involve the detection of the conditioned stimulus (taste); detection of the unconditioned stimulus (drug) and/or the integration of taste and visceral cues to establish a learned association. It is generally believed that the A P is important in detecting the presence of the unconditioned stimulus and/or triggering illness or nausea 8'18. If the role played by the A P in conditioned responses to drugs like LiC1 primarily involves detection of the unconditioned stimulus, additional questions remain. In particular, when an AP-lesioned rat fails to acquire a conditioned taste aversion after the administration of LiCI it is unclear whether the animal fails to respond to the LiC1 at all or whether a partial response to the LiC1 occurs which does not support taste aversion acquisition. Some investigators have addressed the question of
whether animals with lesions of the A P show responses to the administration of LiC1, responses, that is, other than the d e v e l o p m e n t of conditioned taste aversions. A P - l e s i o n e d rats fail to show the anti-aggressive effect of LiC1 which is generally observed in intact rats 1°. Lesioned animals also have been r e p o r t e d to fail to show behavioral depression after lithium s. In contrast to the latter findings are reports of elevations in oxytocin 5 and 'spastic movements and a b d o m e n dragging '7 after LiCI treatments in A P - l e s i o n e d rats. Because all these responses can be viewed as indices of illness or malaise, there would a p p e a r to be discrepant findings with regard to whether LiC1 induces illness in A P - l e s i o n e d animals. The present studies examined a n u m b e r of responses known to be triggered by the administration of LiC1 to determine whether any of these responses occur in animals with complete destruction of the area postrema. Since the rat is a non-emetic species, responses other than retching and vomiting need to be e m p l o y e d as indices of a physiological response to lithium. The behavioral response to lithium administration has been characterized as a general suppression of activity as well as a specific response described as 'lying-on-belly' ( L O B ) TM 14. Physiological responses to lithium include dramatic delays in gastric emptying 9 and a m a r k e d decline in body t e m p e r a t u r e 1'21. These responses were evaluated in A P lesioned rats along with their ability to display a conditioned taste aversion to a saccharin solution paired with lithium.
Correspondence: I.L. Bernstein, Department of Psychology, University of Washington, Seattle, WA 98195, U.S.A. Fax: (1) (206) 685-3157.
133 MATERIALS AND METHODS Subjects were male Long-Evans and Wistar rats weighing between 280 and 350 g at the start of the study. The rats were maintained in individual cages in a room with a 12:12 h light:dark cycle. Food and water were continually available except as noted. A single group of Long-Evans male rats was used in the taste aversion, behavioral assessment and body temperature phases of this project. A separate group of Wistar male rats of comparable body weights was used to assess gastric emptying.
Area postrema lesions Rats were matched for body weight and received either lesions of the area postrema (APX) or sham (SHAM) operations 2. The area postrema was exposed by slight enlargement of the foramen magnum. Under direct visualization using a dissecting microscope, AP tissue was removed in the manner of a 'brush' biopsy, using the tightly twisted filaments teased from the tip of a cotton applicator. Residual AP tissue was destroyed by application of an ophthalmic cautery. In sham-operated animals the region was exposed, but no lesion was made. At least 6 weeks elapsed to allow for recovery from the acute effects of the lesion and for stabilization of body weight. At this stage lesioned animals weighed 60-100 g less than sham-operated controls. Weight differences, which were statistically significant, reflect the weight loss known to be associated with AP lesions TM.
Taste aversion conditioning Long-Evans rats were placed on a 19-h, water deprivation schedule for 3 days, with water available for 5 h a day during the light phase of the lighting cycle. Within each lesion condition animals were assigned to either an experimental or control group (n = 8 per group). On the conditioning day rats received a novel 0.15% saccharin solution in place of water for 30 min. Immediately after the drinking period experimental animals received an i.p. injection of 0.15 M LiCl in a volume of 20 ml/kg of body weight (APX/LiC1; SHAM/LiC1) while control animals received a comparable volume of isotonic saline (0.15 M NaC1; APX/NaC1; SHAM/NaCl). Three days after conditioning animals were tested for saccharin preference in a two-bottle test. Two calibrated drinking tubes, containing tap water and 0.15% saccharin solution were presented for 30 min. Sampling of each solution was insured by briefly introducing each drinking tube alone until the animals had sampled both the saccharin and the water. At the end of the test period intake of water and saccharin were recorded. Preference scores for saccharin were calculated for individual animals by dividing the volume of saccharin solution consumed by total fluid intake during the test (saccharin + water).
recorded the behaviors in which the subjects engaged; thus, for every minute each subject was observed for 10 s and its behavior was recorded for a total of at least 30 scores. Interrater reliability for 'lying on belly' was 0.89.
Body temperature responses Biotelemetry devices for detecting body temperature (MiniMitter Co., Sunriver, OR) were implanted intraperitoneally under halothane anesthesia into 4 APX and 4 SHAM animals. These devices send out AM-band signals proportional to the temperature within the peritoneal cavity and allow for remote monitoring of body temperature with an accuracy of about 0.1°C. Three days after implantation of thermistors animals were placed in recording chambers for 20 min and baseline temperatures were recorded every 5 min. Animals were then given an i.p. injection of 0.15 M LiC1 (20 ml/kg) and returned to the recording chamber for an additional 80 min. Temperatures were recorded every 5 min.
Gastric emptying This response was measured in a separate group of AP-lesioned and control Wistar rats. Rats were accustomed to receiving an afternoon meal of moist chow (approximately 1 part Wayne Lab Blox to 1.5 parts tap water). For testing they were deprived of food for 24 h (to allow for emptying of stomach contents) and then offered a meal of moist chow for 30 min. Intake was measured by weighing the food jars before and after the meal. Rats then received i.p. injections of LiC1 (APX/LiC1, n = 7; SHAM/LiCI, n = 5) or NaCI (APX/NaCI, n = 6; SHAM/NaC1, n = 4) (0.15 M; 20 ml/kg). Three hours after injection animals were sacrificed with an overdose of Equithesin. Both ends of the stomach were ligated and the stomach was removed and weighed. Stomach emptying was estimated for individual animals by calculating a retention ratio, the weight of the full stomach divided by the weight of the mash meal.
Histology Rats were sacrificed by an overdose of Equithesin (Nembutal/ chloral hydrate) and were perfused with saline and then phosphatebuffered formalin. Brains were removed, fixed in 30% glucose-formalin, and then frozen and cut into serial sections of 30 /~m thickness. Serial sections were mounted on slides and stained with thionin for microscopic examination.
RESULTS
Taste aversion conditioning S a c c h a r i n p r e f e r e n c e s c o r e s , d e p i c t e d in Fig. 1, indi-
Behavioral responses to LiCl administration
cate that strong, significant aversions developed to the
Behavioral responses to LiCI administration were assessed in the same animals at the time of conditioning. Cylindrical Plexiglas cages (20 cm in diameter; 30 cm in height) served as the experimental chambers. The floors of the chambers were made of stainless steel rods positioned 2.5 cm apart. Six chambers were placed side-by-side on a table that was situated along one wall of the conditioning room. Animals were habituated to the chambers prior to testing. After injection with LiCl or NaC1 animals were placed in the chambers and behaviors were recorded by two observers, blind to group membership, who scored a set of 9 different behaviorsa (adapted from Parkerl3). Observers sat in the conditioning room with a clear view of the chambers. At 10-s intervals the observers
l i t h i u m - p a i r e d s a c c h a r i n s o l u t i o n in s h a m - o p e r a t e d anim a l s (t14 = 4.45; P < 0.001) w h i l e n o s a c c h a r i n a v e r s i o n s w e r e e v i d e n t in a n i m a l s w i t h A P l e s i o n s . T h e s e results a r e c o n s i s t e n t w i t h a n u m b e r o f o t h e r s t u d i e s w h i c h indicate that, after destruction of the AP, animals do not a c q u i r e c o n d i t i o n e d t a s t e a v e r s i o n s w h e n LiCI is t h e u n conditioned stimulus.
Behavioral responses to LiCl administration A s c a n b e s e e n in Fig. 2, ' l y i n g - o n - b e l l y ' ( L O B ) is t h e
a Behavioral categories included: 'lying on belly', defined as lying prostrate on belly with no discernable body movement, loss of muscle tone, head and nose slumping to the floor of the cage; 'doggy scratch', limb flicking, grooming, chin rubbing, 'wet dog shake', freezing, rearing and 'other'.
134
1.0 o w e=
m
F.-
38 37
o
36
&
[] saline c
[]
E
"
APX
LiCl
o.
35 APX
SHAM
Fig. 1. Mean saccharin preference scores (+ S.E.M.) in AP-lesioned (APX) and sham-operated (SHAM) animals after conditioning with LiCI (striped bars) or saline (white bars). Differences between LiC1 and saline groups is significant (P < 0.001) in SHAM but not APX animals.
dominant behavior displayed by intact rats after LiC1 injections; with L O B scores significantly higher after LiCI injections than after saline (t14 = 12.8; P < 0.001). In contrast, L O B scores were low in all A P - l e s i o n e d rats, and did not differ as a function of whether animals had received LiC1 or saline. Thus, L O B , a postural index of malaise, did not occur after LiC1 injections in animals with lesions of the AP.
Body temperature responses Baseline body t e m p e r a t u r e (mean of the 4 measurements obtained before the injection of LiCI) was the same in the A P - l e s i o n e d and control animals, averaging 37.9°C (see Fig. 3). Mean body t e m p e r a t u r e of control animals declined steadily during the 80-min period following injection of LiC1, reaching a mean of 35.3°C. A P lesioned animals displayed a non-significant decline in
2'0
4'0 6~0 8~0 TIME (minutes)
SHAM I 00
Fig. 3. Mean body temperature (°C) in APX and SHAM animals during the baseline period and for 80 rain after treatment with LiCI. LiCI was administered at 20 rain.
body t e m p e r a t u r e of less than I°C. A r e p e a t e d measures A N O V A indicated a significant effect of time on body t e m p e r a t u r e (F15.9o = 7.43; P < 0.001) and a significant interaction between lesion condition and time (F15,90 = 7.27; P < 0.001).
Gastric emptying Mash meals consumed prior to drug treatments were larger for sham-lesioned animals (16.5 g + 2.0) than for animals with A P lesions (9.0 g _+ 1.4). To examine gastric emptying, stomach contents were expressed as retention ratios relative to the size of individual test meals. The p r o p o r t i o n of the test meal which r e m a i n e d in the stomach 3 h after LiCI injections are presented in Fig. 4. In s h a m - o p e r a t e d animals a dramatic effect of LiC1 is evident; it appears that virtually none of the meal consumed before lithium t r e a t m e n t had e m p t i e d from the stomach, whereas in saline-treated animals around half of the ingested meal had already e m p t i e d (t7 --- 5.64; P
1.2-
c =
I-Isaline ~LiCI
m co 0
~.0 0.8
8
0.6
[] saline T
m
o
E (n
OA
T
[] ucl
1
O2
0.0
APX
SHAM
Fig. 2. Mean LOB ('lying-on-belly') scores (+ S.E.M.) in AP-lesioned (APX) and sham-operated (SHAM) animals after treatment with LiCI (striped bars) or saline (white bars). Differences between LiCI and saline groups is significant (P < 0.001) in SHAM but not APX animals.
APX
SH-AM
Fig. 4. Mean stomach contents (+ S.E.M.) expressed as a ratio to weight of mash meal in APX and SHAM animals after treatment with LiC1 (striped bars) or saline (white bars). Differences between LiC1 and saline groups is significant (P < 0.001) in SHAM but not APX animals.
135
Fig. 5. Schematics (adapted from ref. 15) display coronal sections through the brainstem at the level of the lesion in an intact and lesioned brain (lesioned region is blackened). AP, area postrema; CUL, lateral cuneate nucleus; CUM, medial cuneate nucleus; FC, fasiculus cuneatus; FG, fasiculus gracilis; FLM, medial longitudinal fasiculus; GR, nucleus gracilis; NTST, nucleus of the spinal tract of V; PC1, inferior cerebellar peduncle; SOL, nucleus solitarius; TCS, corticospinal tract; TSC, spinocerebellar tract; TSL, lateral nucleus of tractus solitarius; TST, spinal tract of V; TSTH, spinothalamic tract; X, nucleus of vagus; XII, nucleus of hypoglossal.
< 0.001). In contrast, LiC1 had no apparent effect on stomach emptying after destruction of the AP; in lesioned groups there was no difference between lithiumand saline-treated animals in the proportion of the mash meal which remained in the stomach. A comparison between the retention ratios in A P X NaCI and SHAM-NaC1 groups suggests that stomach emptying may have been accelerated in AP-lesioned animals relative to controls, although this effect was not statistically reliable.
Histology Inspection of brain slices taken from lesioned animals revealed that lesions were essentially limited to ablation of the area postrema with slight intrusion into the dorsal edge of the solitary tract. The most extensive lesion, displayed in Fig. 5, involved minimal damage to the border of the dorsal motor nucleus of the vagus and hypo-
glossal nucleus and minimal invasion of dorsal column fibers. None of the lesioned brains showed significant damage to the vagal or hypoglossal nuclei. DISCUSSION The area postrema lacks a blood-brain barrier and contains chemoreceptors sensitive to certain drugs 3'4. This chemosensitive region participates importantly in emetic responses in many mammalian species. Lithium chloride, a toxin with a wide range of peripheral as well as central effects, is known to penetrate the blood-brain barrier 6,~z. Nonetheless, the area postrema has been implicated in the response to administration of LiC1 in the rat. Taste aversion conditioning, with LiCI as the unconditioned stimulus, is attenuated or eliminated in animals with lesions of the A P s'ts. Additionally, the present studies indicate that a number of other responses to LiC1
136 administration are eliminated by lesions of the AP. These include a behavioral response, 'lying-on-belly' as well as two physiological responses, delayed stomach emptying and hypothermia. Since the rat is a non-emetic species, responses like delayed stomach emptying and 'lying-onbelly' have been e m p l o y e d as indirect indices of nausea or malaise, Thus, the present data provide additional support for the suggestion that receptors in the region of the area p o s t r e m a are critically involved in detecting LiCI and in a wide range of responses to this toxin. The present findings are somewhat inconsistent with a report by Carter and Lightman 5. The main focus of that p a p e r was the mediation of cholecystokinin-stimulated oxytocin secretion but groups receiving LiCI were also included. It was found that the oxytocin secretion provoked by LiC1 administration was not attenuated by A P lesions. Those authors concluded that, although the nausea-promoting effects of LiCI may involve the AP, neuroendocrine effects m a y be m e d i a t e d by other regions. This would not be consistent with the idea that, in the rat, oxytocin release is a reliable biological m a r k e r of nausea 22 since elevations in oxytocin in A P - l e s i o n e d animals should indicate that they were experiencing nausea. Since the Carter and Lightman 5 data are the only systematic evidence that A P - l e s i o n e d animals respond to LiC1 administration with illness, they represent an anomalous finding which p r o b a b l y needs confirmation. O u r data provide evidence that the area p o s t r e m a is important in orchestrating physiological responses to
LiC1, including delaying gastric emptying and hypothermia. The A P has strong connections with the nucleus of the solitary tract and the dorsal m o t o r nucleus of the vagus as well as the parabrachial nucleus 2°. Thus, lesions to the A P would be likely to interfere with vagal mechanisms regulating gastric emptying. Involvement of the A P in lithium's hypothermic effect could be based on input to the parabrachial nucleus which projects to the hypothalamus 19. The loss of physiological responses to LiC1 is correlated with the loss of behavioral indications of discomfort (lying on belly) and visceral malaise (taste aversion learning) normally induced by the drug. This pattern of results is consistent with the hypothesis that the failure to acquire a conditioned taste aversion to a lithiumpaired flavor after A P lesions reflects the absence of a significant 'illness' response in lesioned animals. These findings do not rule out a m o r e general role of the A P in integration of sensory information important for formation of learned associations about food. For example Kosten and Contreras 7 recently p r o p o s e d that the lesion interferes with the ability to recognize taste solutions as novel. However, it should be noted that such a role for the A P would not explain the b r o a d range of lithiuminduced responses which are attenuated or eliminated by A P lesions. Acknowledgements. This work was supported in part by NIH
Grant CA26419.
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