Neuroanatomical Targets of the Organophosphate Chlorpyrifos by c-fos Immunolabeling
http://www.100md.com
《毒物学科学杂志》
Department of Neurociencia y Ciencias de la Salud, University of Almeria, 04120 Almeria, Spain
1 Correspondence should be addressed to Inmaculada Cubero, Ph.D., at Department of Neurociencia y Ciencias de la Salud, Universidad de Almería, 04120 Almería, Spain. Fax: 950–015473. Email: icubero@ual.es.
ABSTRACT
Chlorpyrifos (CPF) is an organophosphate widely used as an insecticide in agriculture which elicits short- and long-term neurobehavioral deficits after acute administration. Because little is known about the specific brain areas targeted by CPF, investigating for the location of its neuroanatomical targets could help to describe the brain systems involved in the neurobehavioral toxicity developed in CPF-exposed organisms. To meet this objective, in the present study we evaluated CPF-induced c-fos expression. In addition, locomotor behavior and cerebral cholinesterase level were evaluated. We found two main sets of results. First, no significant c-fos expression was found in cholinoceptive regions in CPF-treated rats 2 h or 24 h post-administration, despite the fact that 41% and 62% acetylcholinesterase inhibition, respectively, were present in brain homogenates. These results are consistent with previous reports showing CPF-induced activation of adaptive neural mechanisms re-establishing cholinergic tone. Second, 24 h post-intoxication CPF elicited c-fos expression in cytokine-related areas. Cytokines have been involved in anxiety-like responses and psychiatric stress syndromes. Taking into account that CPF triggers the synthesis of peripheral cytokines, the present data stress the need to further clarify functional relations between organophosphate-triggered peripheral cytokines and emotional disturbances reported in intoxicated organisms.
Key Words: chlorpyrifos; cytokines; c-fos; lithium chloride; cholinonoceptive areas.
INTRODUCTION
Chlorpyrifos (CPF) is an organophosphate compound widely used as insecticide in agriculture with both cholinergic and non-cholinergic activity (Pope, 1999; Richardson, 1995). Because it is slowly delivered in the organism when administered subcutaneously (Richardson, 1995), a single dose of CPF induces acetylcholinesterase (AchE) inhibition that peaks 5 days post-intoxication, followed by a progressive and slow enzymatic recovery rate that keeps AchE activity mildly inhibited for weeks (Pope, 1999).
Despite the fact that CPF exerts acute cholinergic activity, surprisingly, no overt toxicity signs are found after administration of high doses of the compound (Richardson, 1995). However, several reports have shown short- and long-term neurobehavioral and emotional deficits in animals (Abou-Donia et al., 2003; Richardson, 1995). In our lab, we have previously reported CPF-induced anxiogenic-like responses as measured in rats by the plus maze at 48 h post-intoxication (Sanchez-Amate et al., 2001). In addition, a long-lasting CPF generalization to pentylenetetrazol (PTZ), an anxiogenic compound, was found in a drug-discrimination task (Sanchez-Amate et al., 2002). Finally, 6 months after CPF intoxication, impaired spatial acquisition and disrupted amphetamine-induced place preference responses were observed (Sanchez-Santed et al., 2004).
Most of the basic research aimed toward describing cerebral mechanisms involved with organophosphate-induced neurotoxicity has centered its efforts at the molecular and neurochemical level (Abou-Donia et al., 2003; Bushnell et al., 1994; Chaudhuri et al., 1993; Gupta, 2004; Huff et al., 2001; Huff and Abou-Donia, 1995; Katz et al., 1997; Nostrandt et al., 1997; Ward et al., 1993), and little is known about the specific brain areas or neural circuits on which organophosphates exert their action. Investigating for specific brain targets has been recently proposed as the main tool for deeper understanding and/or prevention of emotional and cognitive impairments caused by organophosphate compounds (Gupta, 2004).
Several studies have successfully employed c-fos activity as a marker of neural activity (Thiele et al., 1996; Yamamoto et al., 1992) in such a way that low c-fos baseline levels are found in non-active neurons, whereas increased c-fos expression is indicative of neural activity. Moreover, c-fos expression has been involved with organophosphate administration (Kaufer et al., 1998). In the present study, regional c-fos expression in CPF exposed rats was quantified in order to search for specific neuroanatomical targets, with a double objective. First, recent reports have shown that organophosphates such as sarin (Henderson et al., 2002), soman (Svensson et al., 2001), or CPF (Gordon and Rowsey, 1999; Rowsey and Gordon, 1999) acutely stimulate cytokine synthesis, molecules that relay the inflammatory and immune message to the brain. Moreover, cytokines are involved with anxiety-like responses and psychiatric stress syndromes (Anisman and Merali, 2003; Dantzer, 2001; Kronfol and Remick, 2000), and it has been recently proposed that organophosphate-exposed soldiers in the Gulf War developed persistent psychological symptoms that closely correspond to the physiological and behavioral sequelae of a cytokine-mediated sickness response (Ferguson and Cassaday, 1999). Experimental evidence has shown a consistent pattern of regional c-fos expression in response to chemical compounds such as lithium chloride (LiCl), which is known to induce cytokine synthesis (Thiele et al., 1996; Yamamoto et al., 1992). Thus, the first goal in this study was to evaluate CPF-induced c-fos expression in brain regions targeted by cytokines (Konsman et al., 2002).
Second, unlike other organophosphates, acute high doses of CPF, inducing profound AChE inhibition, are not immediately followed by the neurobehavioral "cholinergic syndrome" classically associated with AchE inhibitors (Richardson, 1995). Several fast compensatory molecular mechanisms involving cholinergic receptors as well as pharmacodynamic properties intrinsic to the organophosphate CPF (Pope, 1999) seem to compensate for the increase in cholinergic tone. Moreover, a fast but long-lasting increase in AchE mRNA levels has been found in cellular neurites in response to 3 days of exposure to very low doses of the organophosphate diisopropylfluorophosphonate (Meshorer et al., 2002). Taken together, previous data strongly point to fast feedback cellular reactions re-establishing cholinergic activity in response to AchE compounds. Thus, in order to indirectly evaluate cholinergic tone in CPF-treated rats, the present study was designed to quantify c-fos expression in cholinoceptive areas as well as levels of cerebral AchE inhibition in response to CPF.
The CPF-induced c-fos regional pattern will be compared with that which emerged in response to the toxin LiCl, for several reasons. First, LiCl is a cytokine inductor (Maier et al., 1993, Nemeth et al., 2002) that activates transcriptional factors, and immunohistochemistry procedures have consistently shown a well-defined pattern of regional c-fos expression in cytokine-related areas (Thiele et al., 1996, Yamamoto et al., 1992), which provides a helpful comparative framework within which to discuss CPF-induced c-fos expression data. Second, LiCl-induced c-fos was used as a positive control in our immunohistochemistry procedure.
In addition to the labeling procedure, we assessed locomotor activity together with the biochemical AchE profile resulting from toxin administration.
MATERIALS AND METHODS
Animals.
Wistar male rats (Charles River Laboratories, Spain) weighing 300–350 g at the beginning of the experiments were housed 4/cage and maintained in an environmentally controlled room (22°C on a 12:12 h light–dark cycle). Food and water were provided ad libitum and all the manipulations were conducted during the light phase. Behavioral procedures and pharmacological techniques were in agreement with the animal care guidelines established by the Spanish Royal Decree 223/1988 for reducing animal pain and discomfort.
Behavioral procedure.
After 15 days of habituation to the laboratory conditions, the animals were weighed, homogeneously distributed into three groups ([n = 6], CPF, LiCl, and Veh), and then injected subcutaneously with chlorpyrifos (O,O'-diethyl-O-[3,5,6–trichloro-2-pyridyl] phosphorothioate, 99.5% [Riedel-de Han, Germany], dissolved in olive oil, 250 mg/kg in 1 ml/kg volume), or lithium chloride ip (LiCl, 0.15 M [Sigma, Spain], dissolved in isotonic saline [0.9%], 20 ml/kg) or olive oil sc, respectively, as vehicle. Immediately after the injections, the animals were put back in their home cages. Half of them remained there for 2 h and the rest remained there for 24 h. Once these pre-established temporal intervals were completed, a small group of animals (n = 4) belonging to each treatment group were decapitated to assess brain AchE activity (see procedural details below). The rest of animals
Thus, this experimental procedure would enable us to comparatively evaluate cerebral c-fos expression and locomotor activity as well as the AchE profile in rats pre-treated with CPF, LiCl, or vehicle, at two different intervals post-injection, 2 h and 24 h,.
Acetylcholinesterase assay.
For AchE assays, a group of animals (n = 4/pre-treated group) randomly selected from pre-treated rats were anesthetized 2 h post-injection with sodium pentothal (80 mg/kg in 1 mg/kg volume) and then decapitated. The same protocol was followed 24 h later for a second group of pre-treated rats. The whole brain was removed and immediately homogenized with 1% Triton X-100 in 0.1 M Na phosphate buffer at pH 8 at a ratio of 1/10 (wt/vol). The homogenate was centrifuged at 1000xg for 10 min; then the pellet was discarded and the supernatant was kept for AChE assay. Acetylcholinesterase activity was determined by spectrophotometer (DU 530 Beckman spectrophotometer) by the Ellman method (Ellman et al., 1961) with tetraisopropyl pyrophosphoramide (iso-OMPA, specific inhibitors for BuChE) (50 μl; final concentration 50 μM), acetylthiocholine iodide (30 μl; final concentration 0.5 mM) as substrate, and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (200 μl; final concentration 0.33 mM). Assay tubes were completed to 1 ml with Na phosphate buffer, pH 8. Enzyme activity was calculated relative to protein concentration by the Bradford method (Bradford, 1976). For biochemical assays, acetylthiocoline iodide, iso-OMPA, and 5,5'-dithio-bis-nitrobenzoic acid (DTNB) were purchased from Sigma-Quimica, Madrid, Spain.
Immunostaining for c-fos procedure.
Immediately upon completion of the open field test the animals were euthanized with an overdose of sodium pentothal (80 mg/kg in 1 mg/kg volume) and transcardially perfused with phosphate buffered saline (PBS) followed by 0.1 M phosphate buffered paraformaldehyde 4% (pH 7.4). The brains were removed and immersed in PBS for 48 h at 4°C. Cerebral sections were cut in coronal sections 50 mm thick with a motorized vibratome. Following our experimental objectives, we focused on two different sets of brain regions: cytokine-related areas and cholinoceptive regions. Thus, a total of 11 different brain regions were collected based on Paxinos and Watson stereotaxic atlas coordinates (Paxinos and Watson, 1998): the nucleus of the solitary tract (NTS), bregma –13.3 mm; the area postrema (AP) bregma –14.08 to –13.68 mm; the central nucleus of the amygdala (CeA), bregma –3.14 to –2.30 mm; the lateral parabrachial area (lPB), bregma –9.16 mm; the hippocampus (HC), bregma –3.6 mm to –3.14 mm; the globus pallidus (GP), bregma –2.3 mm to –3.14 mm; the dorsomedial nucleus of the thalamus (DMT), bregma –2.3 mm to –3.14 mm; the interpeduncular nucleus (IP), bregma –5.8 mm; the posterior hypothalamus (PH), bregma –3 mm to –3.8 mm; the paraventricular nucleus of the hypothalamus (Pa), bregma –3.3 mm to –3.8 mm; and the locus coeruleus (LC), bregma –10.04 mm to –9.68 mm. Slices were rinsed (3x, PBS), incubated for 20 min in 0.3% H2O2 in absolute methanol to quench endogenous peroxidase, rinsed (3x, PBS), and incubated for 1 h in 3% goat serum in PBS. Slices were then transferred, without rinsing, to the primary antibody solution, which consisted of 1:10,000 c-fos polyclonal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes residues 3–16 of the c-fos protein. After 36 h incubation at 4°C, slices were rinsed (10x, PBS, 2 h) and processed with the ABC method (Vector Laboratories, Burlingame, CA). Briefly, slices were transferred to a solution containing biotinylated anti-rabbit IgG for 1 h, rinsed (10x PBS 1 h), transferred to avidin–biotin peroxidase for 1 h, rinsed (5x PBS 30 min, then 5x PB 30 min), and developed with nickel-intensified diaminobenzidine substrate (6 min). Following proper development, slices were rinsed (PBS, 30 min), mounted on slides, and coverslipped with Permount.
Stained sections were examined through a microscope (Olympus, B250) with 40x magnification; c-fos positive cells were scored through an attached camera lucida in selected brain regions (area 100 x 100 microns) by an observer blind to the experimental conditions.
Because all cardiac perfusions were performed 2 h or 24 h from drug administration but only 5 min separated them from locomotor testing, this procedure will allow us to specifically correlate c-fos expression with drug injection.
RESULTS
Locomotor Activity in Open-field
Total distance traveled, mean velocity, and total rearing recorded in the open field chamber over a 5-min measurement period are represented in Figure 1. Data obtained 2 h and 24 h post-injection were analyzed through independent one-way analysis of variance (ANOVAs) with a single between-subject factor, "Drug," which compared locomotor activity in CPF-, LiCl-, and vehicle-administered rats. The statistical analysis performed for each dependent variable showed no significant effects for the factor Drug, at 2 h or 24 h post-intoxication, (F 1, p > 0.05). Thus, the present data suggest that under the experimental conditions and doses we employed, neither LiCl nor CPF significantly altered locomotor activity.
Brain Acetylcholinesterase Profile
Data obtained 2 h and 24 h post-injection were analyzed through independent one-way ANOVAs with the single between-subject factor Drug, which compared AchE activity in CPF, LiCl, and vehicle administered rats. At 2 h post-administration, the analysis revealed a statistically significant effect for the main factor, Drug (F [2, 9] = 26.70, p < 0.05) in cholinesterase activity (CPF 0.028 ± 0.0033 μM/mg/min, LiCl 0.048 ± 0.0018 μM/mg/min, Veh 0.048 ± 0.0006 μM/mg/min). Posterior post-hoc Newman-Keuls test showed decreased AchE activity in CPF-treated rats (p < 0.0005), being 41% inhibited when compared with the AchE level in the control group (Fig. 2).
In addition, the ANOVA conducted on data obtained 24 h post-intoxication, revealed a significant effect for the factor Drug, (F [2,9] = 60.70, p < 0.01) in cholinesterase activity (CPF 0.014 ± 0.0020 μM/mg/min, LiCl 0.043 ± 0.0013 μM/mg/min, Veh 0.038 ± 0.0025 μM/mg/min), and a posterior Newman-Keuls tests showed that CPF-treated rats had reduced AchE activity (p < 0.0002), being 62% inhibited when compared to vehicle-administered rats (Fig. 2). No differences were found at any evaluated interval, when cholinesterase activity in LiCl- and vehicle-treated rats was compared.
In summary, AchE activity was similar in vehicle- and LiCl-injected rats. By contrast and as expected, CPF-treated rats showed a time-dependent decrease in AchE activity, being 41% and 62% inhibited when it was compared with the level of enzymatic activity in the control group at 2 h and 24 h post-administration, respectively.
Immunostaining Data: Regional c-fos Brain Expression
Data from c-fos expression obtained in brain regions after drug treatment 2 h or 24 h post-injection, were analyzed by independent one-way ANOVAs with the single between-subject factor, Drug, which compared, in each scored region, total c-fos expression in CPF-, LiCl-, and vehicle-administered rats.
C-fos expression in cytokine-related regions.
The cytokine-related region included the area postrema, nucleus of the solitary tract, lateral parabrachial area, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala. ANOVAs conducted on c-fos data obtained 2 h post-administration revealed a significant main effect for the factor Drug in the area postrema, AP (F [2, 9] =16.84, p < 0.05), nucleus of the solitary tract, NTS (F [2, 13] = 9.25; p < 0.05), lateral parabrachial area, lPB (F [2, 12] = 4.99, p < 0.05), and the central nucleus of the amygdala, CeA (F [2, 13] = 9.83, p < 0.05). No differences were found in the number of c-fos positive cells in the Pa, (p > 0.05). Consistent with previous reports (Konsman et al., 2002; Thiele et al., 1996; Yamamoto et al., 1992), subsequent post hoc analyses with the Newman-Keuls test showed that 2 h post-administration LiCl administration led to significant increases in c-fos expression in the AP (p < 0.001), NTS (p < 0.004), lPB (p < 0.05) and the CeA (p < 0.005). By contrast, no significant increases in regional c-fos expression were found in response to CPF or vehicle administrations (Table 1).
Independent one-way ANOVAs conducted on c-fos data collected 24 h after experimental treatment revealed statistical significance for the factor Drug in the nucleus of the solitary tract, NTS (F [2, 13] = 11.50, p < 0.05), lateral parabrachial area, lPB (F [2, 14] = 6.84, p < 0.05), and the central nucleus of the amygdala, CeA (F [2, 13] = 15.06, p < 0.05). No differences were found in the number of c-fos positive cells in the AP, (p > 0.05). Post-hoc Newman-Keuls tests showed that c-fos expression in these areas in response to LiCl were similar to that induced by vehicle administration. However, CPF-treated rats showed a significant increase, different from that exhibited by LiCl and vehicle treatments, in the number of c-fos positive cells in the NTS (p < 0.003), the lPB (p < 0.01), and the CeA (p < 0.001. In addition, because a reduced number of subjects were finally scored in the Pa (n = 4), a non-parametric U-Mann Whitney test was conducted, revealing that the CPF group also expressed a higher level of c-fos positive cells when compared to vehicle-treated rats (p < 0.05) (Table 1).
Thus, a similar but delayed pattern of increased regional c-fos expression emerged in LiCl- and CPF-treated rats, respectively, at 2 h and 24 h post-administration, suggesting cellular activity in brain regions known to be targeted by cytokines.
c-fos Expression in cholinoceptive areas.
The cholinoceptive areas include the posterior hypothalamus, central nucleus of the amygdala, interpeduncular nucleus, globus pallidus, dorsomedial nucleus of the thalamus, hippocampus, locus coeruleus.
Regional c-fos data obtained in cholinoceptive regions 2 h and 24 h post-intoxication were analyzed through independents one-way ANOVAs, with the single between-subject factor Drug. No statistically significant differences were found in any cholinoceptive area scored (p > 0.05), except the CeA (see above under c-fos Expression in cytokine-related regions, for CeA analyses; see also Table 2).
Thus, the present preliminary data obtained in cholinoceptive areas suggests that, despite significant levels of AchE inhibition in CPF-treated rats, surprisingly, no correlative significant increases in CPF-induced cholinergic neural activity were detected as measured by c-fos immunostaining.
DISCUSSION
c-fos Expression in cytokine-related regions.
Results for the area postrema, nucleus of the solitary tract, lateral parabrachial area, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala were consistent with previously reported evidence (Thiele et al., 1996; Yamamoto et al., 1992). They show that 2 h after LiCl is delivered in the organism, an acute response is elicited in the AP, the NTS, or the lPB (Konsman et al., 2002), as well as in brain areas organizing sickness behaviors, such as the CeA (Buller and Day, 2002). As expected, no c-fos expression was found 24 h post-treatment in the LiCl group in any scored region, probably because of LiCl metabolism. In contrast, CPF administration did not seem to induce significant c-fos activity 2 h post-administration in any scored cerebral region.
However, 24 h after CPF injection an interesting pattern of c-fos expression emerged, partially matching that elicited by LiCl 2 h post-injection. The analysis of the pattern of regional c-fos expression induced by LiCl 2 h post-administration, and that evoked by CPF 24 h after exposure, revealed some interesting similarities, suggesting, in both cases, cellular activity in cytokine-related areas involved with the "sickness behavior" cerebral system (Konsman et al., 2002). It is known that this system triggers an "alert response" when potentially dangerous chemicals gain access to the organism (Dantzer, 2001), enabling neural adaptive responses to be properly organized. The toxicity message is mediated by peripheral cytokine synthesis, represented by interleukin 1 (IL1) and tumor necrosis factor-alpha (TNF-), which activate their target structures via humoral and neural pathways projecting to specific brain areas (Konsman et al., 2002). In this context, several potentially dangerous stimuli such as aversive and sickness-inducing chemical stimuli (Maier et al., 1993; Nemeth et al., 2002), proteins from virus membranes (lipopolysaccharides, LPP), and some organophosphate compounds (Gordon and Rowsey, 1999; Henderson et al., 2002; Rowsey and Gordon, 1999; Svensson et al., 2001) induce a cytokine relay acute signal that conveys the immune and inflammatory message toward the brain-sickness system.
Interestingly, behavioral studies have suggested a strong relationship between cytokines and some emotion-based psychiatric syndromes (Anisman and Merali, 2003; Kronfol and Remick, 2000; Pollmacher et al., 2002). I.c.v. administration of the cytokine TNF- elicits anxiety-like responses as measured by the Plus maze test, even without otherwise noticeable behavioral or physiological overt symptoms of sickness (Connor et al., 1998). On the other hand, immunostaining data have revealed that TNF- influences stressor-reactive brain regions and also induces expression of c-fos in the CeA (Buller and Day, 2002). In light of these results, it has been proposed that increased activity in the sickness-behavior system due to stimuli triggering peripheral cytokines could also sustain disease-associated hyper-reactivity, emotional disturbances, and anxiety-like responses (Anisman and Merali, 2003; Kronfol and Remick, 2000; Pollmacher et al., 2002).
This is the first study, to our knowledge, showing delayed cellular activity after CPF administration in cytokine-related brain areas, as measured by regional c-fos expression. Given the fact that CPF triggers TNF- synthesis (Gordon and Rowsey, 1999; Rowsey and Gordon, 1999), and that CPF induces anxiety-like responses in the plus maze at 48 h, the present data extend previous results and provide c-fos evidence of CPF-induced activity in cytokine-related brain areas. Future studies aimed to co-localize TNF receptors and c-fos expression in the brain of CPF-treated rats will further characterize the phenotype of activated cells. Our study stresses the need for future research aimed at gaining an understanding of potential interactions between CPF-induced cytokines, chronic activation in the brain sickness–behavior system, and delayed development of emotional disturbances.
Nonetheless, the present results cannot rule out direct toxic actions of the organophosphate on the reported c-fos–expressing areas. Whether our data are demonstrative of indirect actions of CPF in the "sickness–behavior" cerebral system modulated by peripheral cytokine synthesis or whether they represent a direct toxic action of that compound needs further investigation.
c-fos Expression in cholinoceptive areas.
The cholinoceptive areas include the posterior hypothalamus, central nucleus of the amygdala, interpeduncular nucleus, globus pallidus, dorsomedial nucleus of thalamus, hippocampus, and locus coeruleus.
To indirectly assess CPF-induced cholinergic activity, the second objective in the study was to evaluate c-fos expression in these cholinoceptive regions (Zhu et al., 2001). Although measures obtained in brain homogenates revealed a significant level of AchE inhibition both 2 h and 24 h post-intoxication, no significant CPF-induced neural activity appeared in any (but the CeA) cholinoceptive region scored. Moreover, although decreased locomotor activity resulting from cholinesterase inhibition is a consistent effect reported in the literature (Moser, 2000; Nostrandt et al., 1997; Timofeeva and Gordon, 2002), preliminary data from the present study, together with previous studies conducted in our lab (Sanchez-Amate et al., 2001; Sanchez-Amate et al., 2002; Sanchez-Santed et al., 2004), suggest that no significant changes would be observed in this behavior after CPF exposure unless a 70% level of AchE inhibition is reached in brain homogenates.
Several compensatory neural mechanisms could help to explain these apparently contradictory results. It has been proposed that adaptive presynaptic and postsynaptic mechanisms triggered by organophosphate intoxication successfully prevent acute increases in cholinergic tone (Chaudhuri et al., 1993; Huff et al., 2001; Huff and Abou-Donia, 1995; Meshorer et al., 2002; Pope, 1999; Ward et al., 1993). Chlorpyrifos acts as muscarinic agonist at the m2 and m4 receptors, where AchE release is decreased through adenilcyclase inhibition (Huff et al., 2001; Huff and Abou-Donia, 1995). In addition, more recently it has been reported that chronic treatment with low doses of CPF or nicotine can induce increased AchE levels at the synaptic cleft. This process is immediately followed by desensitization of the nicotinic receptor (Abou-Donia et al., 2003; Fenster et al., 1999; Katz et al., 1997), a cellular mechanism not associated with transcriptional factors and c-fos expression. Finally, new synthesis of the rare AchE-R has been described in cellular neurites as a mechanism re-establishing cholinergic communication in response to organophosphate insults (Meshorer et al., 2002). Thus, present behavioral and molecular data are consistent with active CPF-triggered presynaptic and postsynaptic adaptive mechanisms preventing acute increases in cholinergic tone.
The central nucleus of the amygdala is a cholinoceptive region, although, in this study a different pattern of c-fos expression to that observed in other cholinoceptive scored regions emerged, with significant increases at 24 h post-CPF intoxication. Some explanations could account for the obtained data. First, because receptor densities differ in brain cholinergic areas (Chaudhuri et al., 1993; Gupta, 2004; Mesulam, 1995; Nostrandt et al., 1997), regulatory molecular mechanisms successfully modulating the cholinergic tone in some brain regions could be missed at the CeA. Second, CPF could be directly interacting with specific amygdala cells. Finally, the hypothesis holding amygdala activity as the final neural relay in the cytokine-induced activation of the sickness–behavior circuit is also consistent with present data.
In summary, the obtained results reveal delayed c-fos activity in the brain 24 h post-CPF intoxication and strongly suggest that a single dose of this organophosphate, whether administered directly or indirectly, can target specific brain regions. Moreover, our data clearly show that c-fos immunostaining may be a useful experimental approach in evaluating whether CPF exerts delayed actions targeting specific cerebral circuits. However, because loss of c-fos expression is not definitively demonstrative of reduced neural activity, with the present data we cannot rule out other early-immediate genes as involved in cellular activation induced by CPF. Further research is needed to clarify functional relations between CPF exposition, subsequent cholinergic activity, cytokine synthesis, and development of behavioral/emotional disturbances.
ACKNOWLEDGMENTS
This work was supported by the Spanish grant MCYT PM//99–1046, Spain. We thank Simon Peter K. Smith for reviewing the English version of the manuscript.
REFERENCES
Abou-Donia, M. B., Abdel-Rahman, A., Goldstein, L., Dechkovskaia, A., Shah, D. U., Bullman, S. L., Khan, W. A. (2003). Sensoriomotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats. Arch. Toxicol. 77, 452–458.
Abou-Donia, M. B. (2003). Organophosphorus ester-induced chronic neurotoxicity. Arch. Environ. Health 58, 484–497.
Anisman, H., Merali, Z. (2003). Cytokines, stress and depressive illness: Brain-immune interactions. Ann. Med. 35, 2–11.
Bradford, M. M. (1976). A rapid a sensitive method for the quantification of microgram of protein utilizing the principle of protein-dye binding. Ann. Biochem. 72, 248–254.
Buller, K. M., Day, T. A. (2002). Systemic administration of interleukin-1 beta activates selectively populations of central amygdala afferents. J. Comp. Neurol. 452, 288–296.
Bushnell, P. J., Kelly, K. L., Ward, T. R. (1994). Repeated inhibition of acetylcholinesterase by chlorpyrifos in rats: Behavioral, neurochemical and pharmacological indices of tolerance. J. Pharmacol. Exp. Ther. 270, 15–25.
Chaudhuri, J., Chakraborti, T. K., Chanda, S., Pope, C. N. (1993). Differential modulation of organophosphate-sensitive muscarinic receptors in rat brain by parathion and chlorpyrifos. J. Biochem. Toxicol. 8, 207–216.
Connor, T. J., Song, C., Leonard, B. E., Merali, Z., Anisman, H. (1998). An assessment of the effects of central interleukin-1, –2, –6, and tumor necrosis factor- administration on some behavioural, neurochemical, endocrine and immune parameters in the rat. Neuroscience 84, 923–933.
Dantzer, R. (2001). Cytokine-induced sickness behaviour: Mechanisms and implications. Ann. N. Y. Acad. Sci. 933, 222–234.
Ellman, G. L., Coutney, K. D., Andres, V. J., Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Fenster, C. P., Whitworth, T. L., Shefield, E. B., Quick, M. W., Lester, R. A. J. (1999). Upregulation of surface 42 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J. Neurosci. 19, 4804–4814.
Ferguson, E., Cassaday, H. J. (1999). The Gulf War and illness by association. Br. J. Psychol. 90, 459–475.
Gordon, C. J., Rowsey, P. J. (1999). Are circulating cytokines interleukin-6 and tumor necrosis factor involved in chlorpyrifos-induced fever Toxicology 134, 9–17.
Gupta, R. C. (2004). Brain regional heterogeneity and toxicological mechanism of organophosphates and carbamates. Toxicol. Mech. Methods 14, 103–143.
Henderson, R. F., Barr, E. B., Blackwell, W. B., Clark, C. R., Conn, C. A., Kalra, R., March, T. H., Sopori, M. L., Tesfaigzi, Y., Menache, M. G., Mash, D. C. (2002). Response of rats to low levels of sarin. Toxicol. Appl. Pharmacol. 184, 67–76.
Huff, R. A., Abu-Qare, A. W., Abou-Donia, M. B. (2001). Effects of sub-chronic in vivo chlorpyrifos exposure on muscarinic receptors and adenylate cyclase of rat striatum. Arch. Toxicol. 75, 480–486.
Huff, R. A., Abou-Donia, M. B. (1995). In vitro effect of chlorpyrifos oxon on muscarinic receptors and adenylate cyclase. Neurotoxicology 16, 281–290.
Katz, E. J., Cortes, V. I., Eldefrawi, M. E., Eldefrawi, A. T. (1997). Chlorpyrifos, parathion and their oxons bind to and desensitize a nicotinic acetylcholine receptor: Relevance to their toxicities. Toxicol. Appl. Pharmacol. 146, 227–236.
Kaufer, D., Friedman, A., Seidman, S. Soreq, H. (1998) Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393, 373–377.
Konsman, J. P., Parnet, P., Dantzer, R. (2002). Cytokine-induced sickness behaviour: Mechanisms and implications. Trends Neurosci. 25, 154–159.
Kronfol, Z., Remick, D. G. (2000). Cytokines and the brain: Implications for clinical psychiatry. Am. J. Psychiatry 157, 683–694.
Maier, S. F., Wiertelak, E. P., Martin, D., Watkins, L. R. (1993). Interleukin-1 mediates the behavioral hyperalgesia produced by lithium chloride and endotoxin. Brain Res. 623, 321–324.
Meshorer, E., Erb, C., Gazit, R., Pavlovsky, L., Kaufer, D., Friedman, A., Glick, D., Ben-Arie, N., Soreq, H. (2002) Alternative splicing and neuritic translocation under long-term neuronal hypersensitivity. Science 295, 508–512.
Mesulam, M. M. (1995). Structure and function of cholinergic pathways in the cerebral cortex, limbic system, basal ganglia, and thalamus of the human brain. In: Psychopharmachology: The Fourth Generation of Progress (F. E. Bloom, D. J. Kupfer, Eds.), pp. 125–134, Raven Press. New York .
Moser, V. C. (2000). Dose-response and time-course of neurobehavioral changes following oral chlorpyrifos in rats of different ages. Neurotoxicol. Teratol. 22, 713–723.
Nostrandt, A. C., Padilla, S., Moser, V. C. (1997). The relationship of oral chlorpyrifos effects on behavior, acetylcholinesterase inhibition, and muscarinic receptor density in rat. Pharmacol. Biochem. Behav. 58, 15–23.
Nemeth, Z. H., Deitch, E. A., Szabo, C., Fekete, Z., Hauser, C. J., Hasko, G. (2002). Lithium induces NF-kappa B activation and interleukin-8 production in human intestinal epithelial cells. J. Biol. Chem. 277, 7713–7719.
Paxinos, G., Watson, C. (1998). The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press, San Diego.
Pollmacher, T., Haack, M., Schuld, A., Reichenberg, A., Yirmiya, R. (2002). Low levels of circulating inflammatory cytokines—Do they affects human brain functions Brain. Behav. Immun. 16, 525–532.
Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity J. Toxicol. Environ. Health, Part B Crti. Rev. 2, 161–181.
Richardson, R. J. (1995). Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature. J. Toxicol. Environ. Health 44, 135–165.
Rowsey, P. J., and Gordon, C. J. (1999). Tumor necrosis factor is involved in chlorpyrifos. Induced changes in core temperature in the female rat. Toxicol. Lett. 109, 51–59.
Sánchez-Amate, M. C., Flores, P., Sanchez-Santed, F. (2001). Effects of chlorpyrifos in the plus-maze model of anxiety. Behav. Pharmacol. 12, 285–292.
Sánchez-Amate, M. C., Dávila, E., Caadas, F., Flores, P., Sanchez-Santed, F. (2002). Chlorpyrifos shares stimulus properties with pentylenetetrazol as evaluated by an operant drug discrimination task. Neurotoxicology 23, 795–803.
Sánchez-Santed, F., Caadas, F., Flores, P., López-Grancha, M., Cardona, D. (2004). Long-term functional neurotoxicity of paraoxon and chlorpyrifos: Behavioural and pharmacological evidence. Neurotoxicol. Teratol. 26, 305–317.
Svensson, I., Waara, L., Johansson, L., Butch, A., Cassel, G. (2001). Soman-induced interleukin-1 beta mRNA and protein in rat brain. Neurotoxicology 22, 355–362.
Thiele, T. E., Roitman, M. F., Bernstein, I. L. (1996). C-fos induction in rat brainstem in response to ethanol- and lithium chloride- induced conditioned taste aversions. Alcohol Clin. Exp. Res. 20, 1023–1028.
Timofeeva, O. A., and Gordon, C. J. (2002). EEG spectra, behavioral states and motor activity in rats exposed to acetylcholinesterase inhibitor chlorpyrifos. Pharmacol. Biochem. Behav. 72, 669–679.
Ward, T. R., Ferris, D. J., Tilson, H. A., Mundy, W. R. (1993). Correlation of the antiacetylcholinesterase activity of a series of organophosphaes with their ability to compete with agonist binding to muscarinic receptors. Toxicol. Appl. Pharmacol. 122, 300–307.
Yamamoto, T., Shimura, T., Sako, N., Azuma, S., Bai, W., Wakisaka, S. (1992). C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. NeuroReport 3, 1049–1052.
Zhu, W., Umegaki, H., Yoshimura, J., Tamaya, N., Suzuki, Y., Miura, H., Iguchi, A. (2001). The elevation of plasma adrenocorticotrophic hormone and expression of c-fos in hypothalamic paraventricular nucleus by microinjection of neostigmine into the hippocampus in rats: Comparation with acute stress responses. Brain Res. 892, 391–395.(F. Carvajal, M. C. Sánche)
1 Correspondence should be addressed to Inmaculada Cubero, Ph.D., at Department of Neurociencia y Ciencias de la Salud, Universidad de Almería, 04120 Almería, Spain. Fax: 950–015473. Email: icubero@ual.es.
ABSTRACT
Chlorpyrifos (CPF) is an organophosphate widely used as an insecticide in agriculture which elicits short- and long-term neurobehavioral deficits after acute administration. Because little is known about the specific brain areas targeted by CPF, investigating for the location of its neuroanatomical targets could help to describe the brain systems involved in the neurobehavioral toxicity developed in CPF-exposed organisms. To meet this objective, in the present study we evaluated CPF-induced c-fos expression. In addition, locomotor behavior and cerebral cholinesterase level were evaluated. We found two main sets of results. First, no significant c-fos expression was found in cholinoceptive regions in CPF-treated rats 2 h or 24 h post-administration, despite the fact that 41% and 62% acetylcholinesterase inhibition, respectively, were present in brain homogenates. These results are consistent with previous reports showing CPF-induced activation of adaptive neural mechanisms re-establishing cholinergic tone. Second, 24 h post-intoxication CPF elicited c-fos expression in cytokine-related areas. Cytokines have been involved in anxiety-like responses and psychiatric stress syndromes. Taking into account that CPF triggers the synthesis of peripheral cytokines, the present data stress the need to further clarify functional relations between organophosphate-triggered peripheral cytokines and emotional disturbances reported in intoxicated organisms.
Key Words: chlorpyrifos; cytokines; c-fos; lithium chloride; cholinonoceptive areas.
INTRODUCTION
Chlorpyrifos (CPF) is an organophosphate compound widely used as insecticide in agriculture with both cholinergic and non-cholinergic activity (Pope, 1999; Richardson, 1995). Because it is slowly delivered in the organism when administered subcutaneously (Richardson, 1995), a single dose of CPF induces acetylcholinesterase (AchE) inhibition that peaks 5 days post-intoxication, followed by a progressive and slow enzymatic recovery rate that keeps AchE activity mildly inhibited for weeks (Pope, 1999).
Despite the fact that CPF exerts acute cholinergic activity, surprisingly, no overt toxicity signs are found after administration of high doses of the compound (Richardson, 1995). However, several reports have shown short- and long-term neurobehavioral and emotional deficits in animals (Abou-Donia et al., 2003; Richardson, 1995). In our lab, we have previously reported CPF-induced anxiogenic-like responses as measured in rats by the plus maze at 48 h post-intoxication (Sanchez-Amate et al., 2001). In addition, a long-lasting CPF generalization to pentylenetetrazol (PTZ), an anxiogenic compound, was found in a drug-discrimination task (Sanchez-Amate et al., 2002). Finally, 6 months after CPF intoxication, impaired spatial acquisition and disrupted amphetamine-induced place preference responses were observed (Sanchez-Santed et al., 2004).
Most of the basic research aimed toward describing cerebral mechanisms involved with organophosphate-induced neurotoxicity has centered its efforts at the molecular and neurochemical level (Abou-Donia et al., 2003; Bushnell et al., 1994; Chaudhuri et al., 1993; Gupta, 2004; Huff et al., 2001; Huff and Abou-Donia, 1995; Katz et al., 1997; Nostrandt et al., 1997; Ward et al., 1993), and little is known about the specific brain areas or neural circuits on which organophosphates exert their action. Investigating for specific brain targets has been recently proposed as the main tool for deeper understanding and/or prevention of emotional and cognitive impairments caused by organophosphate compounds (Gupta, 2004).
Several studies have successfully employed c-fos activity as a marker of neural activity (Thiele et al., 1996; Yamamoto et al., 1992) in such a way that low c-fos baseline levels are found in non-active neurons, whereas increased c-fos expression is indicative of neural activity. Moreover, c-fos expression has been involved with organophosphate administration (Kaufer et al., 1998). In the present study, regional c-fos expression in CPF exposed rats was quantified in order to search for specific neuroanatomical targets, with a double objective. First, recent reports have shown that organophosphates such as sarin (Henderson et al., 2002), soman (Svensson et al., 2001), or CPF (Gordon and Rowsey, 1999; Rowsey and Gordon, 1999) acutely stimulate cytokine synthesis, molecules that relay the inflammatory and immune message to the brain. Moreover, cytokines are involved with anxiety-like responses and psychiatric stress syndromes (Anisman and Merali, 2003; Dantzer, 2001; Kronfol and Remick, 2000), and it has been recently proposed that organophosphate-exposed soldiers in the Gulf War developed persistent psychological symptoms that closely correspond to the physiological and behavioral sequelae of a cytokine-mediated sickness response (Ferguson and Cassaday, 1999). Experimental evidence has shown a consistent pattern of regional c-fos expression in response to chemical compounds such as lithium chloride (LiCl), which is known to induce cytokine synthesis (Thiele et al., 1996; Yamamoto et al., 1992). Thus, the first goal in this study was to evaluate CPF-induced c-fos expression in brain regions targeted by cytokines (Konsman et al., 2002).
Second, unlike other organophosphates, acute high doses of CPF, inducing profound AChE inhibition, are not immediately followed by the neurobehavioral "cholinergic syndrome" classically associated with AchE inhibitors (Richardson, 1995). Several fast compensatory molecular mechanisms involving cholinergic receptors as well as pharmacodynamic properties intrinsic to the organophosphate CPF (Pope, 1999) seem to compensate for the increase in cholinergic tone. Moreover, a fast but long-lasting increase in AchE mRNA levels has been found in cellular neurites in response to 3 days of exposure to very low doses of the organophosphate diisopropylfluorophosphonate (Meshorer et al., 2002). Taken together, previous data strongly point to fast feedback cellular reactions re-establishing cholinergic activity in response to AchE compounds. Thus, in order to indirectly evaluate cholinergic tone in CPF-treated rats, the present study was designed to quantify c-fos expression in cholinoceptive areas as well as levels of cerebral AchE inhibition in response to CPF.
The CPF-induced c-fos regional pattern will be compared with that which emerged in response to the toxin LiCl, for several reasons. First, LiCl is a cytokine inductor (Maier et al., 1993, Nemeth et al., 2002) that activates transcriptional factors, and immunohistochemistry procedures have consistently shown a well-defined pattern of regional c-fos expression in cytokine-related areas (Thiele et al., 1996, Yamamoto et al., 1992), which provides a helpful comparative framework within which to discuss CPF-induced c-fos expression data. Second, LiCl-induced c-fos was used as a positive control in our immunohistochemistry procedure.
In addition to the labeling procedure, we assessed locomotor activity together with the biochemical AchE profile resulting from toxin administration.
MATERIALS AND METHODS
Animals.
Wistar male rats (Charles River Laboratories, Spain) weighing 300–350 g at the beginning of the experiments were housed 4/cage and maintained in an environmentally controlled room (22°C on a 12:12 h light–dark cycle). Food and water were provided ad libitum and all the manipulations were conducted during the light phase. Behavioral procedures and pharmacological techniques were in agreement with the animal care guidelines established by the Spanish Royal Decree 223/1988 for reducing animal pain and discomfort.
Behavioral procedure.
After 15 days of habituation to the laboratory conditions, the animals were weighed, homogeneously distributed into three groups ([n = 6], CPF, LiCl, and Veh), and then injected subcutaneously with chlorpyrifos (O,O'-diethyl-O-[3,5,6–trichloro-2-pyridyl] phosphorothioate, 99.5% [Riedel-de Han, Germany], dissolved in olive oil, 250 mg/kg in 1 ml/kg volume), or lithium chloride ip (LiCl, 0.15 M [Sigma, Spain], dissolved in isotonic saline [0.9%], 20 ml/kg) or olive oil sc, respectively, as vehicle. Immediately after the injections, the animals were put back in their home cages. Half of them remained there for 2 h and the rest remained there for 24 h. Once these pre-established temporal intervals were completed, a small group of animals (n = 4) belonging to each treatment group were decapitated to assess brain AchE activity (see procedural details below). The rest of animals
Thus, this experimental procedure would enable us to comparatively evaluate cerebral c-fos expression and locomotor activity as well as the AchE profile in rats pre-treated with CPF, LiCl, or vehicle, at two different intervals post-injection, 2 h and 24 h,.
Acetylcholinesterase assay.
For AchE assays, a group of animals (n = 4/pre-treated group) randomly selected from pre-treated rats were anesthetized 2 h post-injection with sodium pentothal (80 mg/kg in 1 mg/kg volume) and then decapitated. The same protocol was followed 24 h later for a second group of pre-treated rats. The whole brain was removed and immediately homogenized with 1% Triton X-100 in 0.1 M Na phosphate buffer at pH 8 at a ratio of 1/10 (wt/vol). The homogenate was centrifuged at 1000xg for 10 min; then the pellet was discarded and the supernatant was kept for AChE assay. Acetylcholinesterase activity was determined by spectrophotometer (DU 530 Beckman spectrophotometer) by the Ellman method (Ellman et al., 1961) with tetraisopropyl pyrophosphoramide (iso-OMPA, specific inhibitors for BuChE) (50 μl; final concentration 50 μM), acetylthiocholine iodide (30 μl; final concentration 0.5 mM) as substrate, and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) (200 μl; final concentration 0.33 mM). Assay tubes were completed to 1 ml with Na phosphate buffer, pH 8. Enzyme activity was calculated relative to protein concentration by the Bradford method (Bradford, 1976). For biochemical assays, acetylthiocoline iodide, iso-OMPA, and 5,5'-dithio-bis-nitrobenzoic acid (DTNB) were purchased from Sigma-Quimica, Madrid, Spain.
Immunostaining for c-fos procedure.
Immediately upon completion of the open field test the animals were euthanized with an overdose of sodium pentothal (80 mg/kg in 1 mg/kg volume) and transcardially perfused with phosphate buffered saline (PBS) followed by 0.1 M phosphate buffered paraformaldehyde 4% (pH 7.4). The brains were removed and immersed in PBS for 48 h at 4°C. Cerebral sections were cut in coronal sections 50 mm thick with a motorized vibratome. Following our experimental objectives, we focused on two different sets of brain regions: cytokine-related areas and cholinoceptive regions. Thus, a total of 11 different brain regions were collected based on Paxinos and Watson stereotaxic atlas coordinates (Paxinos and Watson, 1998): the nucleus of the solitary tract (NTS), bregma –13.3 mm; the area postrema (AP) bregma –14.08 to –13.68 mm; the central nucleus of the amygdala (CeA), bregma –3.14 to –2.30 mm; the lateral parabrachial area (lPB), bregma –9.16 mm; the hippocampus (HC), bregma –3.6 mm to –3.14 mm; the globus pallidus (GP), bregma –2.3 mm to –3.14 mm; the dorsomedial nucleus of the thalamus (DMT), bregma –2.3 mm to –3.14 mm; the interpeduncular nucleus (IP), bregma –5.8 mm; the posterior hypothalamus (PH), bregma –3 mm to –3.8 mm; the paraventricular nucleus of the hypothalamus (Pa), bregma –3.3 mm to –3.8 mm; and the locus coeruleus (LC), bregma –10.04 mm to –9.68 mm. Slices were rinsed (3x, PBS), incubated for 20 min in 0.3% H2O2 in absolute methanol to quench endogenous peroxidase, rinsed (3x, PBS), and incubated for 1 h in 3% goat serum in PBS. Slices were then transferred, without rinsing, to the primary antibody solution, which consisted of 1:10,000 c-fos polyclonal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes residues 3–16 of the c-fos protein. After 36 h incubation at 4°C, slices were rinsed (10x, PBS, 2 h) and processed with the ABC method (Vector Laboratories, Burlingame, CA). Briefly, slices were transferred to a solution containing biotinylated anti-rabbit IgG for 1 h, rinsed (10x PBS 1 h), transferred to avidin–biotin peroxidase for 1 h, rinsed (5x PBS 30 min, then 5x PB 30 min), and developed with nickel-intensified diaminobenzidine substrate (6 min). Following proper development, slices were rinsed (PBS, 30 min), mounted on slides, and coverslipped with Permount.
Stained sections were examined through a microscope (Olympus, B250) with 40x magnification; c-fos positive cells were scored through an attached camera lucida in selected brain regions (area 100 x 100 microns) by an observer blind to the experimental conditions.
Because all cardiac perfusions were performed 2 h or 24 h from drug administration but only 5 min separated them from locomotor testing, this procedure will allow us to specifically correlate c-fos expression with drug injection.
RESULTS
Locomotor Activity in Open-field
Total distance traveled, mean velocity, and total rearing recorded in the open field chamber over a 5-min measurement period are represented in Figure 1. Data obtained 2 h and 24 h post-injection were analyzed through independent one-way analysis of variance (ANOVAs) with a single between-subject factor, "Drug," which compared locomotor activity in CPF-, LiCl-, and vehicle-administered rats. The statistical analysis performed for each dependent variable showed no significant effects for the factor Drug, at 2 h or 24 h post-intoxication, (F 1, p > 0.05). Thus, the present data suggest that under the experimental conditions and doses we employed, neither LiCl nor CPF significantly altered locomotor activity.
Brain Acetylcholinesterase Profile
Data obtained 2 h and 24 h post-injection were analyzed through independent one-way ANOVAs with the single between-subject factor Drug, which compared AchE activity in CPF, LiCl, and vehicle administered rats. At 2 h post-administration, the analysis revealed a statistically significant effect for the main factor, Drug (F [2, 9] = 26.70, p < 0.05) in cholinesterase activity (CPF 0.028 ± 0.0033 μM/mg/min, LiCl 0.048 ± 0.0018 μM/mg/min, Veh 0.048 ± 0.0006 μM/mg/min). Posterior post-hoc Newman-Keuls test showed decreased AchE activity in CPF-treated rats (p < 0.0005), being 41% inhibited when compared with the AchE level in the control group (Fig. 2).
In addition, the ANOVA conducted on data obtained 24 h post-intoxication, revealed a significant effect for the factor Drug, (F [2,9] = 60.70, p < 0.01) in cholinesterase activity (CPF 0.014 ± 0.0020 μM/mg/min, LiCl 0.043 ± 0.0013 μM/mg/min, Veh 0.038 ± 0.0025 μM/mg/min), and a posterior Newman-Keuls tests showed that CPF-treated rats had reduced AchE activity (p < 0.0002), being 62% inhibited when compared to vehicle-administered rats (Fig. 2). No differences were found at any evaluated interval, when cholinesterase activity in LiCl- and vehicle-treated rats was compared.
In summary, AchE activity was similar in vehicle- and LiCl-injected rats. By contrast and as expected, CPF-treated rats showed a time-dependent decrease in AchE activity, being 41% and 62% inhibited when it was compared with the level of enzymatic activity in the control group at 2 h and 24 h post-administration, respectively.
Immunostaining Data: Regional c-fos Brain Expression
Data from c-fos expression obtained in brain regions after drug treatment 2 h or 24 h post-injection, were analyzed by independent one-way ANOVAs with the single between-subject factor, Drug, which compared, in each scored region, total c-fos expression in CPF-, LiCl-, and vehicle-administered rats.
C-fos expression in cytokine-related regions.
The cytokine-related region included the area postrema, nucleus of the solitary tract, lateral parabrachial area, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala. ANOVAs conducted on c-fos data obtained 2 h post-administration revealed a significant main effect for the factor Drug in the area postrema, AP (F [2, 9] =16.84, p < 0.05), nucleus of the solitary tract, NTS (F [2, 13] = 9.25; p < 0.05), lateral parabrachial area, lPB (F [2, 12] = 4.99, p < 0.05), and the central nucleus of the amygdala, CeA (F [2, 13] = 9.83, p < 0.05). No differences were found in the number of c-fos positive cells in the Pa, (p > 0.05). Consistent with previous reports (Konsman et al., 2002; Thiele et al., 1996; Yamamoto et al., 1992), subsequent post hoc analyses with the Newman-Keuls test showed that 2 h post-administration LiCl administration led to significant increases in c-fos expression in the AP (p < 0.001), NTS (p < 0.004), lPB (p < 0.05) and the CeA (p < 0.005). By contrast, no significant increases in regional c-fos expression were found in response to CPF or vehicle administrations (Table 1).
Independent one-way ANOVAs conducted on c-fos data collected 24 h after experimental treatment revealed statistical significance for the factor Drug in the nucleus of the solitary tract, NTS (F [2, 13] = 11.50, p < 0.05), lateral parabrachial area, lPB (F [2, 14] = 6.84, p < 0.05), and the central nucleus of the amygdala, CeA (F [2, 13] = 15.06, p < 0.05). No differences were found in the number of c-fos positive cells in the AP, (p > 0.05). Post-hoc Newman-Keuls tests showed that c-fos expression in these areas in response to LiCl were similar to that induced by vehicle administration. However, CPF-treated rats showed a significant increase, different from that exhibited by LiCl and vehicle treatments, in the number of c-fos positive cells in the NTS (p < 0.003), the lPB (p < 0.01), and the CeA (p < 0.001. In addition, because a reduced number of subjects were finally scored in the Pa (n = 4), a non-parametric U-Mann Whitney test was conducted, revealing that the CPF group also expressed a higher level of c-fos positive cells when compared to vehicle-treated rats (p < 0.05) (Table 1).
Thus, a similar but delayed pattern of increased regional c-fos expression emerged in LiCl- and CPF-treated rats, respectively, at 2 h and 24 h post-administration, suggesting cellular activity in brain regions known to be targeted by cytokines.
c-fos Expression in cholinoceptive areas.
The cholinoceptive areas include the posterior hypothalamus, central nucleus of the amygdala, interpeduncular nucleus, globus pallidus, dorsomedial nucleus of the thalamus, hippocampus, locus coeruleus.
Regional c-fos data obtained in cholinoceptive regions 2 h and 24 h post-intoxication were analyzed through independents one-way ANOVAs, with the single between-subject factor Drug. No statistically significant differences were found in any cholinoceptive area scored (p > 0.05), except the CeA (see above under c-fos Expression in cytokine-related regions, for CeA analyses; see also Table 2).
Thus, the present preliminary data obtained in cholinoceptive areas suggests that, despite significant levels of AchE inhibition in CPF-treated rats, surprisingly, no correlative significant increases in CPF-induced cholinergic neural activity were detected as measured by c-fos immunostaining.
DISCUSSION
c-fos Expression in cytokine-related regions.
Results for the area postrema, nucleus of the solitary tract, lateral parabrachial area, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala were consistent with previously reported evidence (Thiele et al., 1996; Yamamoto et al., 1992). They show that 2 h after LiCl is delivered in the organism, an acute response is elicited in the AP, the NTS, or the lPB (Konsman et al., 2002), as well as in brain areas organizing sickness behaviors, such as the CeA (Buller and Day, 2002). As expected, no c-fos expression was found 24 h post-treatment in the LiCl group in any scored region, probably because of LiCl metabolism. In contrast, CPF administration did not seem to induce significant c-fos activity 2 h post-administration in any scored cerebral region.
However, 24 h after CPF injection an interesting pattern of c-fos expression emerged, partially matching that elicited by LiCl 2 h post-injection. The analysis of the pattern of regional c-fos expression induced by LiCl 2 h post-administration, and that evoked by CPF 24 h after exposure, revealed some interesting similarities, suggesting, in both cases, cellular activity in cytokine-related areas involved with the "sickness behavior" cerebral system (Konsman et al., 2002). It is known that this system triggers an "alert response" when potentially dangerous chemicals gain access to the organism (Dantzer, 2001), enabling neural adaptive responses to be properly organized. The toxicity message is mediated by peripheral cytokine synthesis, represented by interleukin 1 (IL1) and tumor necrosis factor-alpha (TNF-), which activate their target structures via humoral and neural pathways projecting to specific brain areas (Konsman et al., 2002). In this context, several potentially dangerous stimuli such as aversive and sickness-inducing chemical stimuli (Maier et al., 1993; Nemeth et al., 2002), proteins from virus membranes (lipopolysaccharides, LPP), and some organophosphate compounds (Gordon and Rowsey, 1999; Henderson et al., 2002; Rowsey and Gordon, 1999; Svensson et al., 2001) induce a cytokine relay acute signal that conveys the immune and inflammatory message toward the brain-sickness system.
Interestingly, behavioral studies have suggested a strong relationship between cytokines and some emotion-based psychiatric syndromes (Anisman and Merali, 2003; Kronfol and Remick, 2000; Pollmacher et al., 2002). I.c.v. administration of the cytokine TNF- elicits anxiety-like responses as measured by the Plus maze test, even without otherwise noticeable behavioral or physiological overt symptoms of sickness (Connor et al., 1998). On the other hand, immunostaining data have revealed that TNF- influences stressor-reactive brain regions and also induces expression of c-fos in the CeA (Buller and Day, 2002). In light of these results, it has been proposed that increased activity in the sickness-behavior system due to stimuli triggering peripheral cytokines could also sustain disease-associated hyper-reactivity, emotional disturbances, and anxiety-like responses (Anisman and Merali, 2003; Kronfol and Remick, 2000; Pollmacher et al., 2002).
This is the first study, to our knowledge, showing delayed cellular activity after CPF administration in cytokine-related brain areas, as measured by regional c-fos expression. Given the fact that CPF triggers TNF- synthesis (Gordon and Rowsey, 1999; Rowsey and Gordon, 1999), and that CPF induces anxiety-like responses in the plus maze at 48 h, the present data extend previous results and provide c-fos evidence of CPF-induced activity in cytokine-related brain areas. Future studies aimed to co-localize TNF receptors and c-fos expression in the brain of CPF-treated rats will further characterize the phenotype of activated cells. Our study stresses the need for future research aimed at gaining an understanding of potential interactions between CPF-induced cytokines, chronic activation in the brain sickness–behavior system, and delayed development of emotional disturbances.
Nonetheless, the present results cannot rule out direct toxic actions of the organophosphate on the reported c-fos–expressing areas. Whether our data are demonstrative of indirect actions of CPF in the "sickness–behavior" cerebral system modulated by peripheral cytokine synthesis or whether they represent a direct toxic action of that compound needs further investigation.
c-fos Expression in cholinoceptive areas.
The cholinoceptive areas include the posterior hypothalamus, central nucleus of the amygdala, interpeduncular nucleus, globus pallidus, dorsomedial nucleus of thalamus, hippocampus, and locus coeruleus.
To indirectly assess CPF-induced cholinergic activity, the second objective in the study was to evaluate c-fos expression in these cholinoceptive regions (Zhu et al., 2001). Although measures obtained in brain homogenates revealed a significant level of AchE inhibition both 2 h and 24 h post-intoxication, no significant CPF-induced neural activity appeared in any (but the CeA) cholinoceptive region scored. Moreover, although decreased locomotor activity resulting from cholinesterase inhibition is a consistent effect reported in the literature (Moser, 2000; Nostrandt et al., 1997; Timofeeva and Gordon, 2002), preliminary data from the present study, together with previous studies conducted in our lab (Sanchez-Amate et al., 2001; Sanchez-Amate et al., 2002; Sanchez-Santed et al., 2004), suggest that no significant changes would be observed in this behavior after CPF exposure unless a 70% level of AchE inhibition is reached in brain homogenates.
Several compensatory neural mechanisms could help to explain these apparently contradictory results. It has been proposed that adaptive presynaptic and postsynaptic mechanisms triggered by organophosphate intoxication successfully prevent acute increases in cholinergic tone (Chaudhuri et al., 1993; Huff et al., 2001; Huff and Abou-Donia, 1995; Meshorer et al., 2002; Pope, 1999; Ward et al., 1993). Chlorpyrifos acts as muscarinic agonist at the m2 and m4 receptors, where AchE release is decreased through adenilcyclase inhibition (Huff et al., 2001; Huff and Abou-Donia, 1995). In addition, more recently it has been reported that chronic treatment with low doses of CPF or nicotine can induce increased AchE levels at the synaptic cleft. This process is immediately followed by desensitization of the nicotinic receptor (Abou-Donia et al., 2003; Fenster et al., 1999; Katz et al., 1997), a cellular mechanism not associated with transcriptional factors and c-fos expression. Finally, new synthesis of the rare AchE-R has been described in cellular neurites as a mechanism re-establishing cholinergic communication in response to organophosphate insults (Meshorer et al., 2002). Thus, present behavioral and molecular data are consistent with active CPF-triggered presynaptic and postsynaptic adaptive mechanisms preventing acute increases in cholinergic tone.
The central nucleus of the amygdala is a cholinoceptive region, although, in this study a different pattern of c-fos expression to that observed in other cholinoceptive scored regions emerged, with significant increases at 24 h post-CPF intoxication. Some explanations could account for the obtained data. First, because receptor densities differ in brain cholinergic areas (Chaudhuri et al., 1993; Gupta, 2004; Mesulam, 1995; Nostrandt et al., 1997), regulatory molecular mechanisms successfully modulating the cholinergic tone in some brain regions could be missed at the CeA. Second, CPF could be directly interacting with specific amygdala cells. Finally, the hypothesis holding amygdala activity as the final neural relay in the cytokine-induced activation of the sickness–behavior circuit is also consistent with present data.
In summary, the obtained results reveal delayed c-fos activity in the brain 24 h post-CPF intoxication and strongly suggest that a single dose of this organophosphate, whether administered directly or indirectly, can target specific brain regions. Moreover, our data clearly show that c-fos immunostaining may be a useful experimental approach in evaluating whether CPF exerts delayed actions targeting specific cerebral circuits. However, because loss of c-fos expression is not definitively demonstrative of reduced neural activity, with the present data we cannot rule out other early-immediate genes as involved in cellular activation induced by CPF. Further research is needed to clarify functional relations between CPF exposition, subsequent cholinergic activity, cytokine synthesis, and development of behavioral/emotional disturbances.
ACKNOWLEDGMENTS
This work was supported by the Spanish grant MCYT PM//99–1046, Spain. We thank Simon Peter K. Smith for reviewing the English version of the manuscript.
REFERENCES
Abou-Donia, M. B., Abdel-Rahman, A., Goldstein, L., Dechkovskaia, A., Shah, D. U., Bullman, S. L., Khan, W. A. (2003). Sensoriomotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats. Arch. Toxicol. 77, 452–458.
Abou-Donia, M. B. (2003). Organophosphorus ester-induced chronic neurotoxicity. Arch. Environ. Health 58, 484–497.
Anisman, H., Merali, Z. (2003). Cytokines, stress and depressive illness: Brain-immune interactions. Ann. Med. 35, 2–11.
Bradford, M. M. (1976). A rapid a sensitive method for the quantification of microgram of protein utilizing the principle of protein-dye binding. Ann. Biochem. 72, 248–254.
Buller, K. M., Day, T. A. (2002). Systemic administration of interleukin-1 beta activates selectively populations of central amygdala afferents. J. Comp. Neurol. 452, 288–296.
Bushnell, P. J., Kelly, K. L., Ward, T. R. (1994). Repeated inhibition of acetylcholinesterase by chlorpyrifos in rats: Behavioral, neurochemical and pharmacological indices of tolerance. J. Pharmacol. Exp. Ther. 270, 15–25.
Chaudhuri, J., Chakraborti, T. K., Chanda, S., Pope, C. N. (1993). Differential modulation of organophosphate-sensitive muscarinic receptors in rat brain by parathion and chlorpyrifos. J. Biochem. Toxicol. 8, 207–216.
Connor, T. J., Song, C., Leonard, B. E., Merali, Z., Anisman, H. (1998). An assessment of the effects of central interleukin-1, –2, –6, and tumor necrosis factor- administration on some behavioural, neurochemical, endocrine and immune parameters in the rat. Neuroscience 84, 923–933.
Dantzer, R. (2001). Cytokine-induced sickness behaviour: Mechanisms and implications. Ann. N. Y. Acad. Sci. 933, 222–234.
Ellman, G. L., Coutney, K. D., Andres, V. J., Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Fenster, C. P., Whitworth, T. L., Shefield, E. B., Quick, M. W., Lester, R. A. J. (1999). Upregulation of surface 42 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J. Neurosci. 19, 4804–4814.
Ferguson, E., Cassaday, H. J. (1999). The Gulf War and illness by association. Br. J. Psychol. 90, 459–475.
Gordon, C. J., Rowsey, P. J. (1999). Are circulating cytokines interleukin-6 and tumor necrosis factor involved in chlorpyrifos-induced fever Toxicology 134, 9–17.
Gupta, R. C. (2004). Brain regional heterogeneity and toxicological mechanism of organophosphates and carbamates. Toxicol. Mech. Methods 14, 103–143.
Henderson, R. F., Barr, E. B., Blackwell, W. B., Clark, C. R., Conn, C. A., Kalra, R., March, T. H., Sopori, M. L., Tesfaigzi, Y., Menache, M. G., Mash, D. C. (2002). Response of rats to low levels of sarin. Toxicol. Appl. Pharmacol. 184, 67–76.
Huff, R. A., Abu-Qare, A. W., Abou-Donia, M. B. (2001). Effects of sub-chronic in vivo chlorpyrifos exposure on muscarinic receptors and adenylate cyclase of rat striatum. Arch. Toxicol. 75, 480–486.
Huff, R. A., Abou-Donia, M. B. (1995). In vitro effect of chlorpyrifos oxon on muscarinic receptors and adenylate cyclase. Neurotoxicology 16, 281–290.
Katz, E. J., Cortes, V. I., Eldefrawi, M. E., Eldefrawi, A. T. (1997). Chlorpyrifos, parathion and their oxons bind to and desensitize a nicotinic acetylcholine receptor: Relevance to their toxicities. Toxicol. Appl. Pharmacol. 146, 227–236.
Kaufer, D., Friedman, A., Seidman, S. Soreq, H. (1998) Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393, 373–377.
Konsman, J. P., Parnet, P., Dantzer, R. (2002). Cytokine-induced sickness behaviour: Mechanisms and implications. Trends Neurosci. 25, 154–159.
Kronfol, Z., Remick, D. G. (2000). Cytokines and the brain: Implications for clinical psychiatry. Am. J. Psychiatry 157, 683–694.
Maier, S. F., Wiertelak, E. P., Martin, D., Watkins, L. R. (1993). Interleukin-1 mediates the behavioral hyperalgesia produced by lithium chloride and endotoxin. Brain Res. 623, 321–324.
Meshorer, E., Erb, C., Gazit, R., Pavlovsky, L., Kaufer, D., Friedman, A., Glick, D., Ben-Arie, N., Soreq, H. (2002) Alternative splicing and neuritic translocation under long-term neuronal hypersensitivity. Science 295, 508–512.
Mesulam, M. M. (1995). Structure and function of cholinergic pathways in the cerebral cortex, limbic system, basal ganglia, and thalamus of the human brain. In: Psychopharmachology: The Fourth Generation of Progress (F. E. Bloom, D. J. Kupfer, Eds.), pp. 125–134, Raven Press. New York .
Moser, V. C. (2000). Dose-response and time-course of neurobehavioral changes following oral chlorpyrifos in rats of different ages. Neurotoxicol. Teratol. 22, 713–723.
Nostrandt, A. C., Padilla, S., Moser, V. C. (1997). The relationship of oral chlorpyrifos effects on behavior, acetylcholinesterase inhibition, and muscarinic receptor density in rat. Pharmacol. Biochem. Behav. 58, 15–23.
Nemeth, Z. H., Deitch, E. A., Szabo, C., Fekete, Z., Hauser, C. J., Hasko, G. (2002). Lithium induces NF-kappa B activation and interleukin-8 production in human intestinal epithelial cells. J. Biol. Chem. 277, 7713–7719.
Paxinos, G., Watson, C. (1998). The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press, San Diego.
Pollmacher, T., Haack, M., Schuld, A., Reichenberg, A., Yirmiya, R. (2002). Low levels of circulating inflammatory cytokines—Do they affects human brain functions Brain. Behav. Immun. 16, 525–532.
Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity J. Toxicol. Environ. Health, Part B Crti. Rev. 2, 161–181.
Richardson, R. J. (1995). Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature. J. Toxicol. Environ. Health 44, 135–165.
Rowsey, P. J., and Gordon, C. J. (1999). Tumor necrosis factor is involved in chlorpyrifos. Induced changes in core temperature in the female rat. Toxicol. Lett. 109, 51–59.
Sánchez-Amate, M. C., Flores, P., Sanchez-Santed, F. (2001). Effects of chlorpyrifos in the plus-maze model of anxiety. Behav. Pharmacol. 12, 285–292.
Sánchez-Amate, M. C., Dávila, E., Caadas, F., Flores, P., Sanchez-Santed, F. (2002). Chlorpyrifos shares stimulus properties with pentylenetetrazol as evaluated by an operant drug discrimination task. Neurotoxicology 23, 795–803.
Sánchez-Santed, F., Caadas, F., Flores, P., López-Grancha, M., Cardona, D. (2004). Long-term functional neurotoxicity of paraoxon and chlorpyrifos: Behavioural and pharmacological evidence. Neurotoxicol. Teratol. 26, 305–317.
Svensson, I., Waara, L., Johansson, L., Butch, A., Cassel, G. (2001). Soman-induced interleukin-1 beta mRNA and protein in rat brain. Neurotoxicology 22, 355–362.
Thiele, T. E., Roitman, M. F., Bernstein, I. L. (1996). C-fos induction in rat brainstem in response to ethanol- and lithium chloride- induced conditioned taste aversions. Alcohol Clin. Exp. Res. 20, 1023–1028.
Timofeeva, O. A., and Gordon, C. J. (2002). EEG spectra, behavioral states and motor activity in rats exposed to acetylcholinesterase inhibitor chlorpyrifos. Pharmacol. Biochem. Behav. 72, 669–679.
Ward, T. R., Ferris, D. J., Tilson, H. A., Mundy, W. R. (1993). Correlation of the antiacetylcholinesterase activity of a series of organophosphaes with their ability to compete with agonist binding to muscarinic receptors. Toxicol. Appl. Pharmacol. 122, 300–307.
Yamamoto, T., Shimura, T., Sako, N., Azuma, S., Bai, W., Wakisaka, S. (1992). C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. NeuroReport 3, 1049–1052.
Zhu, W., Umegaki, H., Yoshimura, J., Tamaya, N., Suzuki, Y., Miura, H., Iguchi, A. (2001). The elevation of plasma adrenocorticotrophic hormone and expression of c-fos in hypothalamic paraventricular nucleus by microinjection of neostigmine into the hippocampus in rats: Comparation with acute stress responses. Brain Res. 892, 391–395.(F. Carvajal, M. C. Sánche)