ToxSci Advance Access originally published online on February 13, 2006
Toxicological Sciences 2006 91(1):210-217; doi:10.1093/toxsci/kfj126
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Lateral Parabrachial Lesions Disrupt Paraoxon-Induced Conditioned Flavor Avoidance
* Departamento de Neurociencia y Ciencias de la Salud, University of Almeria, 04120 Almeria, Spain; and Bowles Center for Alcohol Studies, Department of Psychiatry, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
1 To whom correspondence should be addressed. Fax: +34 950 015 473. E-mail: icubero{at}ual.es.
Received November 18, 2005; accepted February 2, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preliminary clinical evidence obtained in Gulf War veterans and patients suffering multiple chemical sensitivity points to the existence of a potential link between environmental exposure to organosphosphates (OPs) and the emergence of unspecific sickness syndromes in which associative Pavlovian conditioning might be partly involved. A laboratory animal model might be a useful tool for analyzing the involvement of conditioning in sickness syndromes potentially linked to OP poisoning. The first objective in the present study was to determine if paraoxon (PX), the neuroactive metabolite of the OP parathion, elicits a conditioned avoidance response to a novel stimulus (a taste-odor compound) in a conditioned flavor aversion procedure. Data obtained in Experiment 1 show conditioned flavor avoidance, demonstrative of the associative nature of the sickness properties of PX. The second objective was to characterize the nature of the specific physiological cue serving as the unconditioned stimulus in PX-induced conditioned avoidance. Despite PX administration did induce cholinergic hyperactivity, as measured by body hypothermia and increased jaw movements, lesions of the lateral parabrachial area (lPB) disrupted PX-elicited flavor avoidance responses, indicating that cholinergic signs were not sufficient as unconditioned stimului supporting avoidance responses. Given that lPB neural integrity is necessary to process aversive interoceptive information, disruption of conditioned flavor avoidance as a result of lPB lesions is consistent with a central interruption of interoceptive processing in PX-poisoned animals. Data are discussed under the light of the hypothesis claiming the importance of associative processes and noncholinesterase targets in sickness syndromes potentially induced by OP exposure.
Key Words: paraoxon; organophosphates; associative sickness properties; conditioned flavor aversion; lateral parabrachial area.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organophosphates (OPs) are neurotoxic compounds widely used as pesticides in agriculture, wartime, and domestic insect control (Pope, 1999
). Cholinesterase inhibition is the main mechanism of OP toxicity; however, alternative noncholinesterase targets have been recently described (Casida and Quistad, 2004; Gupta, 2004).
Preliminary clinical evidence obtained in Gulf War veterans (Ferguson and Cassaday, 1999, 2001; Ferguson et al., 2004) and patients suffering multiple chemical sensitivity (Devriese et al., 2000; Miller, 2000; Siegel, 1999) has lead some authors to propose the existence of a potential link between OP exposure and the emergence of unspecific sickness syndromes in vulnerable organisms (Ferguson and Cassaday, 1999, 2001; Miller, 2000; Overstreet and Djuric, 1999). Moreover, it has been suggested that Pavlovian conditioning might be partly involved in the development of such responses (Devriese et al., 2000; Ferguson and Cassaday, 1999, 2001; Ferguson et al., 2004). The authors propose that physiological reactions elicited by OP poisoning might become accidentally linked to environmental "odors" by associative learning, inducing unspecific sickness responses when the olfactory cue is later presented in the habitual environment. To date, clinical evidence supporting the participation of associative processes is scarce; however, experimental studies conducted by Van den Bergh and others in humans have shown conditioned somatic symptoms in response to chemical substances (Devriese et al., 2000; Van den Bergh et al., 2001; Winters et al., 2001, 2003).
A laboratory animal model might be useful for analyzing the involvement of odor conditioning in sickness syndromes potentially linked to OP poisoning. The conditioned taste aversion (CTA) learning paradigm is a valuable tool for characterizing the associative nature of the sickness properties of a noxious compound (Bures et al., 1998). In CTA learning, organisms become ill after sampling a new substance (often a taste) and develop a conditioned aversion that is expressed as avoidance of that substance in subsequent presentations (Bures et al., 1998). Interestingly, previous data on conditioned taste avoidance responses to OP exposure (Bignami et al., 1985; Roney et al., 1986) suggest that sickness signs elicited by OPs might work as unconditioned stimuli (US) inducing conditioned taste responses. Given that environmental odors associated with OP might work as conditioned stimuli (CS) triggering conditioned sickness and/or avoidance responses, experimental studies that explore avoidance responses to tastes/odors after OP poisoning would provide a useful tool to further characterize sickness syndromes potentially linked to OP exposure.
The first objective in the present study was to determine if paraoxon (PX), the neuroactive metabolite of the OP parathion (Pope, 1999), elicits a conditioned avoidance response when previously paired with a novel taste-odor compound (a flavor) in a discriminative procedure (Cubero and Puerto, 2000; Cubero et al., 2001). Acquisition and expression of conditioned flavor avoidance responses would be demonstrative of the associative nature of the sickness properties of PX.
The second objective was to identify the specific physiological cues serving as US in PX-induced conditioned flavor avoidance. PX elicits a well-characterized pattern of cholinergic signs indicative of acute toxicity, such as marked body hypothermia and jaw movements (McDaniel and Moser, 1993; Moser, 1995). On the other hand, CTA studies have shown that many noxious substances supporting CTA responses elicit aversive interoceptive stimulation (Bernstein et al., 1992; Navarro and Cubero, 2003), which is processed by specific brain circuits (Konsman et al., 2002; Mediavilla et al., 2005). Thus, there is the possibility that OPs might generate cholinergic stimulation and also interoceptive sensation of visceral distress and malaise.
The lateral parabrachial area (lPB) is a pivotal region in the cerebral circuit that processes noxious visceral information (Mediavilla et al., 2005; Saper, 2002). Experimental evidence has shown that lesions of the lPB disrupt visceral function (Navarro and Cubero, 2003) and subsequent expression of flavor avoidance responses induced by cholinergic (Cubero and Puerto, 2000; Cubero et al., 2001) and noncholinergic compounds (Mediavilla et al., 2005; Yamamoto et al., 1995). Interestingly, a single dose of the OP chlorpyrifos (CPF) induces c-fos expression in the lPB 24 h postadministration, in the absence of both cholinergic manifestation and c-fos expression in cholinoceptive regions (Carvajal et al., 2005).
In Experiment 2 we explore, through a lesion approach, which are the sickness cues (cholinergic vs interoceptive malaise) working as US in the PX-induced flavor avoidance learning task. lPB neural integrity is necessary to process aversive interoceptive information and CTA acquisition (Cubero and Puerto, 2000; Navarro and Cubero, 2003; Saleh and Cechetto, 1995; Saper, 2002); consequently, disruption of conditioned flavor avoidance resulting from lPB lesions would be consistent with a central interruption of interoceptive processing in PX-poisoned animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Male Wistar rats weighing 350–400 g obtained from the University of Granada (Spain) were individually housed in methacrylate cages that also served as experimental training cages. The rats were maintained in an environmentally controlled room (22°C temperature and a 12/12-h light-dark cycle). Food and water were provided ad libitum except when noted otherwise. All the manipulations were conducted during the light phase. Behavioral procedures as well as pharmacological techniques were conducted in agreement with the animal care guidelines established by the Spanish Royal decree 223/1988 for reducing animal pain and discomfort and the guiding principles in the use of animals in toxicology, adopted by the Society of Toxicology.
Experiment 1. PX-Induced Conditioned Flavor Avoidance: A Dose-Response Study
Behavioral Procedure
Conditioned flavor avoidance.
In order to induce conditioned avoidance responses, we employed a behavioral discriminative procedure (see Cubero and Puerto, 2000; Cubero et al., 2001) involving two different flavored [taste + odor] solutions as CS and PX (paraoxon-ethil(O,O-diethyl-O-4-nitrophenyl Phosphate) [Riedel-de Häen, Seelze, Germany]) as the unconditioned stimulus. The experimental procedure was conducted in methacrylate chambers (measuring 30 x 15 x 30 cm) with two orifices located at the same height and distance from the midline. The animals had access to spouts attached to graduated burettes through which flavored solutions and water were delivered. After 2 weeks of habituation to the laboratory conditions, animals were randomly assigned to one of five groups receiving different doses of PX (0.05, 0.1, 0.2, 0.3 mg/kg [n = 8], and 0.4 mg/kg [n = 9]) as US. For 4 days, a pretraining session that habituated animals to consume water in a restricted schedule was conducted. The animals were water deprived for 23 h 20 min and allowed to daily drink tap water for 10 min from graduated burettes presented simultaneously (to avoid positional preferences) through the frontal holes in the cages. Six hours after the training, rats had access to water for 30 min for an appropriate hydration. Then, the experimental procedure began (see Table 1 for a resume of procedural details). On day 1, animals were presented for 10 min with one of two possible novel flavored solutions, 0.5% strawberry or coconut extract (McCormick and Co. Inc., Baltimore, MD) diluted in water. After 10 min, the burettes were removed and half of the animals were injected with a given dose of PX sc (0.05, 0.1, 0.2, 0.3, or 0.4 mg/kg) dissolved in olive oil, 1 mg/ml (paired condition). The other half of the animals received olive oil sc (vehicle [VEH] matching drug volume for paired condition) as nonaversive stimulus associated with a flavored solution (unpaired condition). Following 24 h, on day 2, the second flavor solution was presented and the same procedure as that described for day 1 was repeated. The sequence of the experimental conditions was properly balanced in such a way that all the animals experienced both flavored solutions but only one of them had been paired, in a single trial, with the toxic PX (paired condition).
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Day 3 was a test day. The animals were tested for conditioned flavor avoidance by using a two-bottle choice test. For that, the animals were allowed to drink freely for 10 minutes from two burettes, each one containing one flavoured solution (strawberry or coconut). The total amount ingested from each solution was recorded. Those animals developing conditioned flavor avoidance drank a lower amount from the flavored solution previously paired with PX during the training.
Home-cage screening test.
PX induces cholinesterase inhibition (Moser, 1995). Thus, the cholinergic status in poisoned animals was evaluated following PX or VEH administration (during the conditioning days), through sensitive parameters of cholinesterase inhibition, namely, body temperature and jaw movements (Moser, 1995). These variables were quantified for 5 min, at three different time points (1, 4, and 8 h postintoxication), by an observer blind to the experimental conditions (see Table 1).
Biochemical Assays: Brain ChE Determination
The main mechanism of PX toxicity is cholinesterase (ChE) inhibition. Thus, we evaluated the biochemical profile of cerebral ChE in PX-poisoned versus vehicle-treated rats. During the training phase, a group of animals (n = 4 per group) was randomly selected from three groups (VEH, 0.2 mg/kg, and 0.4 mg/kg of PX), anesthetized with pentothal sodium (80 mg/kg in 1 ml/kg volume), and then decapitated for cholinesterase assays, 4 h posttreatment. The whole brain was removed and immediately homogenized with 1% Triton X-100 in 0.1M Na phosphate buffer at pH 8 at a ratio of 1/10 (wt/vol). The homogenate was centrifuged at 1000 x g for 10 min; then the pellet was discarded and the supernatant was kept for ChE assay. ChE activity was determined spectrophotometrically (DU 530 Beckman spectrophotometer) by the Ellman method (Ellman et al., 1961), using acetylthiocholine iodide (Sigma-Quimica, Madrid, Spain) (30 µl; final concentration = 0.5mM) as substrate and 5,5'-dithiobis-2-nitrobenzoic acid (Sigma-Quimica) (200 µl; final concentration = 0.33mM). One milliliter Na phosphate buffer, pH 8, was added to the assay tubes. Enzyme activity was calculated relative to protein concentration by the Bradford method (Bradford, 1976).
Experiment 2. Effects of lPB Lesions in PX-Induced Conditioned Flavor Avoidance
Stereotaxic Surgery
In this study, the animals were randomly assigned to one of two groups: an lPB-lesioned group (n = 8) that received lPB bilateral electrolytic lesions, and a sham-lesioned control group (n = 8) that received an identical surgical procedure, except that no current was administered. Surgery was performed under general anesthesia with Equitesin (2.4 mg/kg).
The animals were placed in the head holder of the stereotaxic apparatus (Stoelting Stereotaxic Instruments [Wood Dale, IL] Mod. 51.600), and two small trephine holes were bilaterally drilled at the parabrachial anatomical coordinates obtained from the Paxinos and Watson (1986) stereotaxic atlas: anterior-posterior = 9.16 mm posterior to bregma; lateral = ± 2.4 mm; and ventral to the skull surface = –6.5 mm. The electrode was placed on the lPB and then a 1.5-mA current was passed for 20 s. A Cibertec (Spain) lesion maker was employed, which supplied direct negative current through a monopolar electrode, approximately 200 µm in diameter, insulated throughout its length except for the last 0.5 mm. At the end of the surgical procedure, the electrode was removed and the wound was sutured.
Behavioral Procedure
PX-induced conditioned flavor avoidance.
Once the animals recovered from stereotaxic surgery (about 2 weeks), conditioned flavor avoidance training began (see Table 1 for procedural details). Given that lPB lesion might induce a slight weight loss secondary to decreased food intake (Sakai and Yamamoto, 1999), we employed an index of fluid consumption (ml/kg) that corrected for individual differences in the weight of rats. In the present study, we chose a dose of PX that triggered the strongest conditioned avoidance response during Experiment 1, namely 0.4 mg/kg. Acquisition of conditioned avoidance responses will be indicative of accurate sensorial processing of noxious interoceptive information elicited by PX during the training stage.
Home-cage screening tests.
Following the same experimental protocol as that described in Experiment 1, the animals were home-cage screened during the conditioning trials for body temperature and jaw movements. Given that the strongest cholinergic response in Experiment 1 was observed 4 h postintoxication, in this test we limited the screening tests to that time point.
Taste-preference study.
In Experiments 1 and 2, flavors employed as CS have been odor-taste compounds. The parabrachial region seems to be involved in gustatory but not in olfactory processing (Reilly and Trifunovic, 2001). Given that disrupted performance during the free-choice test in Experiment 2 could be due to impairments in interoceptive processing but also might be the result of impaired gustatory sensitivity, we decided to specifically evaluate sensorial gustatory function in lPB-lesioned animals. For this purpose, 10 days after the conclusion of flavor conditioning, we employed an experimental discriminative procedure assessing taste preferences (Thiele et al., 1998). Briefly, the rats were individually housed and then tested for free daily consumption of sucrose or quinine, given at two different concentrations (sucrose, Sigma Chemical Co., St. Louis, MO; quinine hydrobromure, Sigma Chemical Co.). The order of the taste compounds was as follows: sucrose solutions (1.70 and 4.25%) followed by quinine solutions (0.03 and 0.10 mM). The rats had free access to each solution for 48 h, and the position of the solution was counterbalanced between animals. The preference ratio for each solution was assessed by dividing the volume of the taste solution consumed by the total volume of fluid consumed (water + solution).
Histology: Placement of lPB Lesions
Once the taste-preference study concluded, the animals were decapitated and the brainstem was removed and stored in PBS/4% formaldehyde for at least 48 h. Then, coronal 50-µm sections were obtained by a motorized vibratome (WPI, Sarasota, FL). Placement of electrolytic lesions was verified with cresyl violet staining under a light microscope. In all the animals, the electrolytic lesions were centered in the lPB (Fig. 1) with minimum damage, if any, to the brachium or the medial-ventral parabrachial area. Lesions expanded, on average, approximately 8.9–9.5 mm posterior to the bregma, 1.6–2.6 mm lateral to the medial line, and 6–7 mm ventral to the skull surface, according to Paxinos and Watson's (1986) atlas.
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Analysis of Data
Independent one-way analysis of variance (ANOVA) with a within-subject factor, "Treatment" (paired vs. unpaired flavored solution), was conducted for data obtained during the choice test after conditioned flavor avoidance training. For each home-cage-screened variable (body temperature and jaw movements), two-way ANOVA was employed with a between-subject factor, Treatment, that compared PX versus VEH, and a within-subject factor, "Time" (1, 4, and 8 h), that evaluated the temporal profile of cholinergic manifestations. ChE activity was analyzed through a one-way ANOVA with a single between-subject factor, "Dose" (0, 0.2, and 0.4 mg/kg of PX). Flavor avoidance acquisition in Experiment 2 was analyzed through a two-way ANOVA with a between-subject factor, "Group" (lPB-lesioned vs. sham-lesioned animals), and a within-subject factor, Treatment (paired vs. unpaired flavored solution), that compared the consumption of paired versus unpaired flavor in lPB-lesioned and control animals. Data obtained in the taste-preference test were analyzed using a two-way ANOVA with a between-subject factor, Group (lesioned vs. sham-lesioned animals), and two intrasubject factors, "Taste" (sucrose and quinine) and "Concentration."
When appropriate, post hoc comparisons were conducted by Tukey tests. All analyses were computed by the Statistical 6.0 software package. Statistically significant determinations were made at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment 1. PX-Induced Conditioned Flavor Avoidance: A Dose-Response Curve
The ANOVAs conducted on data obtained during the choice test revealed a significant main effect of Treatment only in the group trained with the higher dose of PX, 0.4 mg/kg [F(1,8) = 12.24, p < 0.0081], and a clear, but not statistically significant, trend for the group administered 0.3 mg/kg (Fig. 2). A conditioned avoidance response was considered to be acquired when consumption of the paired solution was significantly lower than consumption of the unpaired solution.
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Present data show that the OP PX is a noxious compound triggering conditioned avoidance responses to odor-taste solutions previously paired with the OP even after one single pairing trial.
Home-Cage Screening
Body temperature.
The ANOVAs conducted for each group revealed significant effects for the factors Treatment and Time as follows: Treatment [F(1,6) = 8.03, p < 0.05] and Time [F(2,12) = 5.29, p < 0.05] when PX was administered at a dose equal to 0.2 mg/kg, and Treatment [F(1,6) = 11.2, p < 0.05] and Time [F(2,12) = 5.84, p < 0.05] when the dose of PX was 0.3 mg/kg. Treatment [F(1,7) = 7.41, p < 0.05], Time [F(4,14) = 8.51, p < 0.01], and "Treatment x Time" interaction [F(2,14) = 7.57, p < 0.01] attained statistical significance when the dose of PX employed was 0.4 mg/kg.
Post hoc analysis revealed a temporal profile for PX-induced hypothermia. We found significant body hypothermia 1 h postadministration of 0.2 mg/kg of PX. This effect was still observed 4 h postintoxication when animals received 0.3 and 0.4 mg/kg of PX and disappeared 8 h posttreatment (see Table 2).
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Jaw repetitive movements.
ANOVAs conducted for the variable repetitive jaw movements indicated a significant main effect for the factor Treatment [F(1,7) = 54.27, p < 0.01] and the factor Time [F(2,14) = 11.83, p < 0.01], only in the group that was poisoned with the highest dose of PX (0.4 mg/kg). In addition, a significant effect of Treatment x Time interaction [F(2,14) = 11.83, p < 0.01] emerged. Post hoc Tukey tests indicated a temporal profile. Rats injected with 0.4 mg/kg of PX exhibited a high frequency of this measure 1 and 4 h postadministration; however, no signs of altered jaw movements were detected 8 h posttreatment, indicating a fast recovery in ChE activity and/or adaptive neural mechanisms (see Table 2).
Brain ChE Inhibition Induced by PX Intoxication
Four hours after PX or VEH administration, ANOVA conducted to assess brain ChE levels revealed a statistically significant effect for the main factor Dose [F (2,38) = 73.68, p < 0.001]. As expected, Tukey post hoc test revealed a dose-dependent cholinesterase inhibition in response to PX (Fig. 3).
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Experiment 2. Effects of lPB Lesions in PX-Induced Conditioned Flavor Avoidance
During Experiment 2, one animal died in the lesioned group immediately after PX administration and a second one was eliminated from statistical analysis due to lesion misplacement revealed by histological verification.
The ANOVA conducted for data obtained during the discriminative choice test revealed no significant effects for the factor Group, showing that the total amount of fluid ingested by lesioned and sham-lesioned animals was equivalent, which eliminates motor or motivational disturbances in lesioned animals. A significant effect for the factor Treatment [F(1,12) = 12.35, p < 0.01] and a "Group x Treatment" interaction effect [F(1,12) = 8.35, p < 0.05] were detected. Independent ANOVAs aimed to further analyze the interaction effect yielded statistical significance for Treatment in the sham-lesioned group [F(1,6) = 14.6, p < 0.01], showing acquisition and expression of conditioned avoidance to the flavor previously paired with PX in sham-lesioned but not in lPB-lesioned animals (Fig. 4).
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Thus, consistent with previous reports linking the lPB to aversive visceral processing (Cubero and Puerto, 2000
; Navarro and Cubero, 2003; Reilly, 1999), present data suggest that lPB-lesioned animals were impaired in processing noxious information, which prevented the normal acquisition of conditioned avoidance responses.
Home-Cage Screening
Body temperature.
Data representing core body temperature 4 h after conditioning trials are represented in Figure 5A. The ANOVA showed significant effects only for the factor Treatment [F(1,10) = 7.09, p < 0.05]. Tukey test indicated that lesioned and sham-lesioned rats showed a significant decline in body temperature in response to PX. Thus, PX elicited the expected decline in body temperature in response to doses equal to 0.4 mg/kg, an effect that persisted after lPB lesions. Present data are consistent with previous reports showing that toxin-induced hypothermia is dependent on the area postrema but not on the lPB (Bernstein et al., 1992; Navarro and Cubero, 2003).
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Jaw movements.
Figure 5B depicts the frequency of repetitive jaw movements. The ANOVA performed on the data yielded significant effects for the factor Treatment [F(1,10) = 20.34, p < 0.001]. Thus, lPB-lesioned and sham-lesioned animals injected with 0.4 mg/kg of PX exhibited a significant high frequency of repetitive jaw movements 4 h posttreatment. One more time, lPB lesions did not block signs of cholinergic toxicity.
Taste-Preference Study
In order to rule out any potential gustatory impairment explanative of conditioned flavor aversion disruption in lPB-lesioned animals, we tested gustatory processing in IPB-lesioned animals through a two-bottle taste preference task. Fluid preference associated with each gustatory stimulus and solution concentration was calculated by an index (consumption [ml/kg] of gustatory stimulus/total volume consumed [ml/kg]) indicative of the relative consumption of each gustatory solution during the taste-preference test.
Data obtained are depicted in Figure 6. The ANOVA yielded a significant effect for the factor Taste [F(1,12) = 79.18, p < 0.001] revealing that lPB-lesioned and sham-lesioned animals showed a clear preference for sucrose versus quinine; the factor Concentration was significant [F(1,12) = 132.41, p < 0.001], demonstrating a great preference for the solution with the higher concentration of sucrose, and a lower preference for quinine solutions; the "Group x Taste" [F(1,12) = 5.50, p < 0.05] and "Taste x Concentration" [F(1,12) = 59.81, p < 0.001] interactions were also statistically significant. As shown in Figure 6, lPB lesions did not impair gustatory processing. Both lesioned and sham-lesioned animals exhibited the same pattern of fluid consumption in response to the different tastes and concentrations given during the preference task.
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Thus, regardless of lPB damage, all the animals drank more sucrose than water and showed a reduced ingestion of quinine. These results suggest that animals with lPB lesions maintained undisturbed their gustatory sensorial system and were able to discriminate between different flavored stimuli.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first objective in the present study was to determine if PX, the neuroactive metabolite of the OP parathion (Pope, 1999
), elicits a conditioned avoidance response when previously paired with a novel flavor (a taste-odor compound) (Cubero and Puerto, 2000; Cubero et al., 2001). Consistent with previous results (Bignami et al., 1985; Roney et al., 1986), PX elicited avoidance responses correlative with the expected brain ChE inhibition and subsequent signs of acute cholinergic hyperactivity, as measured by body hypothermia and increased jaw movements.
Acquisition of conditioned flavor avoidance responses indicated that sickness cues triggered by PX poisoning were associated in a single trial with the sensorial stimulus (an odor-taste compound). Given that the same dose of PX inducing the strongest cholinergic signs of malaise was the one generating the strongest conditioned avoidance response (4 mg/kg), there is a possibility that a common cholinesterase-dependent mechanism might underlie both physiological disturbances and behavioral manifestations. However, CTA studies have revealed that several noxious substances elicit aversive interoceptive stimulation, which is processed in the brain through a sensorial visceral pathway (Bernstein et al., 1992; Bures et al., 1998; Navarro and Cubero, 2003). Consistent with this, PX might have generated unobservable interoceptive sensation of visceral distress and malaise.
Experiment 2 tested, through a lesion approach, which was the effective US (cholinergic vs interoceptive malaise) working as US in the PX-induced flavor avoidance learning task. Given that the lPB plays a critical role in the cerebral sickness behavior system by processing ascending aversive interoceptive information (Cubero and Puerto, 2000; Konsman et al., 2002; Navarro and Cubero, 2003; Saleh and Cechetto, 1995; Saper, 2002), we hypothesized that disruption of conditioned avoidance responses after lPB damage might indirectly be indicative of impaired visceral processing. Experiment 2 demonstrated that lPB-lesioned animals do not develop avoidance responses to flavors previously paired with PX, despite observable evidence of cholinergic hyperstimulation. Taking into account that in Experiment 1 we showed that doses of PX unable to induce conditioned flavor avoidance triggered cholinergic indexes of poisoning, it is tempting to suggest that lPB lesions disrupted noxious interoceptive processing, preventing the formation of PX-flavor associations and the subsequent expression of conditioned avoidance responses. In other words, PX-induced cholinergic signs such as body hypothermia and increased jaw movements, did not serve as US during the flavor avoidance acquisition process; rather, aversive interoceptive information might have been the critical cue serving as the unconditioned stimulus supporting conditioned avoidance of PX-paired flavors.
This assertion has some theoretical implications for the hypothesis claiming a role for associative processes in sickness syndromes linked to OP exposure (Ferguson et al., 2004). Recent reports have shown that OPs such as sarin (Henderson et al., 2002), soman (Svensson et al., 2001), and CPF (Gordon and Rowsey, 1999; Rowsey and Gordon, 1999) acutely stimulate synthesis of cytokines, molecules that relay the inflammatory and immune message to the brain system of sickness behavior by targeting the area postrema and the lPB (Konsman et al., 2002). In addition, CPF, an OP sharing intoxicant chemical properties with PX (the active metabolite Oxon), elicits c-fos expression indicative of neural activity in the lPB (Carvajal et al., 2005). Taking together these data, it is tempting to propose that OPs might elicit interoceptive information of visceral distress and malaise. This sensorial information would target, either directly or indirectly through peripheral cytokine synthesis, the sickness behavior brain system, serving as the US supporting the acquisition of conditioned avoidance responses to environmental odors-tastes. Moreover, given the generalization process that follows a learned aversive response (Devriese et al., 2000), present results showing conditioned avoidance to flavors paired with PX may be indicative of a role for associative processes in the development of delayed sickness syndromes in OP-exposed organisms (Devriese et al., 2000; Ferguson and Cassaday, 2001; Miller, 2000).
In conclusion, the data presented here provide a useful animal model to study the implication of associative processes in the development of OP-related sickness syndromes. This study represents the first step in obtaining further clarification about whether measurable sickness responses indicative of conditioned interoceptive malaise are triggered in the presence of environmental CS. Adapted CTA testing procedures allowing a controlled exposure to the CS (odor-taste compounds) might be employed to accurately test conditioned sickness.
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ACKNOWLEDGMENTS |
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This work was supported by the Spanish grant MCYT PM//99-1046. We thank Simon Peter K. Smith for reviewing the English version of the manuscript.
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