Physiology& Behavior, Vol. 51, pp. 207-210. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/92 $5.00 + .00
BRIEF COMMUNICATION
Lithium Chloride and Immunomodulation in Taste Aversion Conditioning M. G A U C I , * D. F. B U L L , t M. S C H E D L O W S K I , t I A. J. H U S B A N D .2 A N D M. G. K I N G t
*Faculty of Medicine and ~Department of Psychology, The University of Newcastle, NSW, 2308, Australia R e c e i v e d 27 M a r c h 1991 GAUCI, M., D. F. BULL, M. SCHEDLOWSKI, A. J. HUSBAND AND M. G. KING. Lithium chloride and immunomodulation in taste aversion conditioning. PHYSIOL BEHAV 51(1) 207-210, 1992.--Lithium chloride has been used in many studies of conditioning to induce taste aversion behaviour, and in some experiments investigating conditioning effects on immunity it has been used on the assumption that it is immunologically neutral. The studies reported here, however, indicate that LiC1 is not immunologically neutral and when used to endow a UCS with noxious properties to enhance the behavioural response in taste aversion conditioned irnmunosuppression, it may antagonize the residual immunosuppression following initial UCS administration and also the conditioned immunosuppression occurring after CS reexposure. Therefore, conclusions drawn from studies of behaviourally conditioned immunomodulation where LiCI is used as part of either the CS or UCS may require reevaluation. Lithium chloride
Conditioned immunity
Antilymphocyte serum
IT is well established that when rats are presented with a benign but novel gustatory conditioned stimulus (CS) followed immediately by an unconditioned stimulus (UCS) which induces an immunological response, both aversion to the taste of the UCS and reenlistment of the immunological effects of the UCS occur upon reexposure to the CS alone. Such a response is an example of classical conditioning (2,3). In previous studies with rats it was shown that when a weak saccharin solution (the CS) was paired with rabbit anti-rat lymphocyte serum (ALS) as the UCS, both taste aversion and conditioned immunosuppression were elicited when rats were later exposed to the saccharin solution alone (9). Subsequent studies demonstrated that if LiCI, which has well-documented taste aversion inducing properties (13), was given with ALS as a compound UCS, the resultant conditioned behavioural response (taste aversion) was greater than that induced by either ALS or LiC1 alone (8). However, in those experiments the effect of this compound UCS on conditioned immune parameters was not determined. In conditioning studies, many reports have appeared in which LiC1 has been used on the assumption that it is an immunologically neutral stimulus but elicits strong taste aversion because of its noxious side effects (2, 6, 13). However, LiCI has been shown in some reports to induce immunoaugmentation (1,14) which can be reenlisted as a conditioned response when LiC1 is used as a UCS (4,5). Indeed, the effects of lithium on immune function appear to be most pronounced when immunity has been compromised (10,12), which is usually the case when an immu-
Immunomodulation
Taste aversion
nosuppressive UCS is used in conditioning experiments due to the residual effects of UCS administration. The purpose of the study reported here was to determine whether LiC1 could modify the conditioned immune response when given as a compound UCS with ALS. If LiC1 is indeed immunologically neutral, then it would be expected that there would be no change in the conditioned immune response. METHOD
Animals Ninety-six male inbred Wistar rats aged 100-110 days were individually housed in hanging wire cages in soundproof cubicles with ad lib access to food and water. A 12:12 h light:dark cycle was maintained with lights on at 0600 h.
Antilymphocyte Serum ALS was prepared in two New Zealand white rabbits. Rabbits received two intramuscular injections of mesenteric lymph node cells (obtained from Wistar rats, 1 × 107 cells per injection) given 14 days apart followed 7 days later by two intravenous injections given 7 days apart. The rabbits were bled weekly from the marginal ear vein on 4 consecutive weeks commencing 1 week after the last injection. They were exsanguinated by cardiac puncture on the fifth week. Sera were prepared, pooled and depleted of complement by heat inactivation at 56°C. Sera from
~Present address: Abteilung Medizinische Psychologie, Postfach 61 01 80, 3000 Ha[mover 61, Germany. 2Requests for reprints should be addressed to Dr. A. J. Husband, Faculty of Medicine, The University of Newcastle, NSW 2308, Australia.
207
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GAUCI, BULL, SCHEDLOWSKI. HUSBAND AND KING
4 uninjected rabbits were pooled and heat inactivated to produce control normal rabbit serum (NRS). ALS and NRS were tested for cytotoxic activity against rat mesenteric lymph node cells using a chromium release assay (11). At a dilution of 1:64, ALS produced 44% lysis of target cells while NRS produced only 3% lysis.
SACC/NRS SACC/NRS+LiCI -13.- WatlNRS -O- Wat/NRS+LiC1 12
Experimental Protocol
.-II.. SACC/AL5 ~ SACC/ALS+LiC1 -0-O-
WaYALS Wat/ALS+LtC1
°
Following a 2-week acclimatisation period, rats were placed on an 8-day water restriction regimen during which water intake was restricted to one 15 min period commencing at 0600 h each day. Rats were then allocated to one of 8 treatment groups (12 rats/group). On the eighth day (Day 0 of the conditioning protocol), rats were presented with either normal bottled water (Wat) or 0.2% saccharin in water (Sacc). Immediately following the 15 min drinking period, rats were injected with either 0.2 ml ALS or 0.2 ml NRS followed immediately by injection of either 0.3 ml LiCl (50 mg/kg) or 0.3 ml sterile phosphate buffered (pH 7.4) saline (PBS). Animals were then returned to their cages. Six days after the conditioning day rats were again exposed to their respective CS (either Wat or Sacc), and fluid intake was monitored by comparing bottle weights before and after drinking. Rats were sacrificed by decapitation 24 h after CS reexposure (Day 7). Trunk blood was collected into heparinised tubes and peripheral blood leukocytes counted using a haemocytometer. Spleens were removed and cell suspensions prepared as previously described (9). The resulting suspensions were washed and leukocyte counts performed using a haemocytometer.
Data Analysis Fluid consumption data for Days 0 and 6 were analysed using a two-way ANOVA with repeated measures for Day effects. Peripheral blood and spleen leukocyte counts on Day 7 were analysed using one-way ANOVAs. Post hoc comparisons were performed where appropriate using the Fisher Least Significant Difference (LSD) (7). RESULTS
Behavioural Responses Fluid consumption on Day 0 and Day 6 for all treatment groups is shown in Fig. 1. There were significant Group effects, F(7,88) = 22.6, p
0031-9384/92 $5.00 + .00
BRIEF COMMUNICATION
Lithium Chloride and Immunomodulation in Taste Aversion Conditioning M. G A U C I , * D. F. B U L L , t M. S C H E D L O W S K I , t I A. J. H U S B A N D .2 A N D M. G. K I N G t
*Faculty of Medicine and ~Department of Psychology, The University of Newcastle, NSW, 2308, Australia R e c e i v e d 27 M a r c h 1991 GAUCI, M., D. F. BULL, M. SCHEDLOWSKI, A. J. HUSBAND AND M. G. KING. Lithium chloride and immunomodulation in taste aversion conditioning. PHYSIOL BEHAV 51(1) 207-210, 1992.--Lithium chloride has been used in many studies of conditioning to induce taste aversion behaviour, and in some experiments investigating conditioning effects on immunity it has been used on the assumption that it is immunologically neutral. The studies reported here, however, indicate that LiC1 is not immunologically neutral and when used to endow a UCS with noxious properties to enhance the behavioural response in taste aversion conditioned irnmunosuppression, it may antagonize the residual immunosuppression following initial UCS administration and also the conditioned immunosuppression occurring after CS reexposure. Therefore, conclusions drawn from studies of behaviourally conditioned immunomodulation where LiCI is used as part of either the CS or UCS may require reevaluation. Lithium chloride
Conditioned immunity
Antilymphocyte serum
IT is well established that when rats are presented with a benign but novel gustatory conditioned stimulus (CS) followed immediately by an unconditioned stimulus (UCS) which induces an immunological response, both aversion to the taste of the UCS and reenlistment of the immunological effects of the UCS occur upon reexposure to the CS alone. Such a response is an example of classical conditioning (2,3). In previous studies with rats it was shown that when a weak saccharin solution (the CS) was paired with rabbit anti-rat lymphocyte serum (ALS) as the UCS, both taste aversion and conditioned immunosuppression were elicited when rats were later exposed to the saccharin solution alone (9). Subsequent studies demonstrated that if LiCI, which has well-documented taste aversion inducing properties (13), was given with ALS as a compound UCS, the resultant conditioned behavioural response (taste aversion) was greater than that induced by either ALS or LiC1 alone (8). However, in those experiments the effect of this compound UCS on conditioned immune parameters was not determined. In conditioning studies, many reports have appeared in which LiC1 has been used on the assumption that it is an immunologically neutral stimulus but elicits strong taste aversion because of its noxious side effects (2, 6, 13). However, LiCI has been shown in some reports to induce immunoaugmentation (1,14) which can be reenlisted as a conditioned response when LiC1 is used as a UCS (4,5). Indeed, the effects of lithium on immune function appear to be most pronounced when immunity has been compromised (10,12), which is usually the case when an immu-
Immunomodulation
Taste aversion
nosuppressive UCS is used in conditioning experiments due to the residual effects of UCS administration. The purpose of the study reported here was to determine whether LiC1 could modify the conditioned immune response when given as a compound UCS with ALS. If LiC1 is indeed immunologically neutral, then it would be expected that there would be no change in the conditioned immune response. METHOD
Animals Ninety-six male inbred Wistar rats aged 100-110 days were individually housed in hanging wire cages in soundproof cubicles with ad lib access to food and water. A 12:12 h light:dark cycle was maintained with lights on at 0600 h.
Antilymphocyte Serum ALS was prepared in two New Zealand white rabbits. Rabbits received two intramuscular injections of mesenteric lymph node cells (obtained from Wistar rats, 1 × 107 cells per injection) given 14 days apart followed 7 days later by two intravenous injections given 7 days apart. The rabbits were bled weekly from the marginal ear vein on 4 consecutive weeks commencing 1 week after the last injection. They were exsanguinated by cardiac puncture on the fifth week. Sera were prepared, pooled and depleted of complement by heat inactivation at 56°C. Sera from
~Present address: Abteilung Medizinische Psychologie, Postfach 61 01 80, 3000 Ha[mover 61, Germany. 2Requests for reprints should be addressed to Dr. A. J. Husband, Faculty of Medicine, The University of Newcastle, NSW 2308, Australia.
207
208
GAUCI, BULL, SCHEDLOWSKI. HUSBAND AND KING
4 uninjected rabbits were pooled and heat inactivated to produce control normal rabbit serum (NRS). ALS and NRS were tested for cytotoxic activity against rat mesenteric lymph node cells using a chromium release assay (11). At a dilution of 1:64, ALS produced 44% lysis of target cells while NRS produced only 3% lysis.
SACC/NRS SACC/NRS+LiCI -13.- WatlNRS -O- Wat/NRS+LiC1 12
Experimental Protocol
.-II.. SACC/AL5 ~ SACC/ALS+LiC1 -0-O-
WaYALS Wat/ALS+LtC1
°
Following a 2-week acclimatisation period, rats were placed on an 8-day water restriction regimen during which water intake was restricted to one 15 min period commencing at 0600 h each day. Rats were then allocated to one of 8 treatment groups (12 rats/group). On the eighth day (Day 0 of the conditioning protocol), rats were presented with either normal bottled water (Wat) or 0.2% saccharin in water (Sacc). Immediately following the 15 min drinking period, rats were injected with either 0.2 ml ALS or 0.2 ml NRS followed immediately by injection of either 0.3 ml LiCl (50 mg/kg) or 0.3 ml sterile phosphate buffered (pH 7.4) saline (PBS). Animals were then returned to their cages. Six days after the conditioning day rats were again exposed to their respective CS (either Wat or Sacc), and fluid intake was monitored by comparing bottle weights before and after drinking. Rats were sacrificed by decapitation 24 h after CS reexposure (Day 7). Trunk blood was collected into heparinised tubes and peripheral blood leukocytes counted using a haemocytometer. Spleens were removed and cell suspensions prepared as previously described (9). The resulting suspensions were washed and leukocyte counts performed using a haemocytometer.
Data Analysis Fluid consumption data for Days 0 and 6 were analysed using a two-way ANOVA with repeated measures for Day effects. Peripheral blood and spleen leukocyte counts on Day 7 were analysed using one-way ANOVAs. Post hoc comparisons were performed where appropriate using the Fisher Least Significant Difference (LSD) (7). RESULTS
Behavioural Responses Fluid consumption on Day 0 and Day 6 for all treatment groups is shown in Fig. 1. There were significant Group effects, F(7,88) = 22.6, p
