6 Effects of imidacloprid on the neural processes of memory in honey bees C. Armengaud, M. Lambin, and M. Gauthier Summary The cholinergic system in insects is the main target of insecticides. One class of molecules, the neonicotinoids, induces direct activation of the neu- ronal nicotinic acetylcholine receptors (nAChRs). In the honey bee these receptors are mainly distributed in the olfactory pathways that link sensory neurons to antennal lobes and mushroom bodies. These structures seem to play an important role in olfactory conditioning. We have previ- ously shown that cholinergic antagonists injected in different parts of the brain impaired the formation and retrieval of olfactory memory. We then advanced the hypothesis that, through the activation of the nAChR, the neonicotinoid imidacloprid (IMI) would lead to facilitation of the memory trace. To test this hypothesis, IMI was applied topically upon the thorax and the effects were tested on the habituation of the proboscis extension reflex induced by repeated sugar stimulation of the antennae. Animals treated with IMI to a dose that did not affect sensory or motor functions needed fewer trials than nontreated animals to show a reflex inhibition. This effect can be interpreted as a learning facilitation. We developed a functional histochemistry of cytochrome oxidase (CO) to reveal the brain targets of the drug in the honey bee brain. Following IMI injection, a CO staining increase, probably linked to an increase in metabolism, was observed in the antennal lobes. In integrative structures, in particular the calyces of mushroom bodies, IMI exerted a facilitatory or inhibitory effect on neuronal metabolism depending on the dose. The brain targets of nicotinic ligands, including pesticides, can be compared by using this technique. Introduction Two of the three main classes of insecticides exert their neurotoxic effects through action on the cholinergic system. This is the case for the new class of neonicotinoids, which are known to act on the nicotinic acetylcholine © 2002 Taylor & Francis receptor (nAChR) channel. Imidacloprid (IMI) {1–[(6-chloro-3- pyridinyl)methyl]-4,5-dihydro-N-nitro-1H-imidazol-2-amine} is one of these new molecules (Figure 6.1). According to the literature, IMI in insects acts at three pharmacologically distinct acetylcholine receptor sub- types inducing a dose-dependent depolarization [1]. Other electrophysio- logical effects of IMI have been described in different models. The single patch-clamp technique applied to the rat pheochromocytoma (PC 12) cells showed that the molecule may have both agonist and antagonist effects on the nAChR [2–4]. Binding experiments of [ 3 H]IMI to membranes from different species showed high-affinity binding sites in house fly head [5], and high- and low-affinity binding sites in the aphid Myzus persicae [6]. The nicotinic receptor subunit composition seems to exert a profound influence upon IMI binding affinity [7]. This brief review of the literature underlines the complex action of IMI on the nAChR. The neurotransmitter acetylcholine (ACh) is distributed largely in the honey bee brain [8]. Acetylcholinesterase and ACh receptors have been identified in the antennal lobes and in the mushroom bodies (MBs), particularly in the calycal part [9, 10]. In addition, Kenyon cells, which fill the calyces, express functional nAChRs in vitro [11]. The involvement of the cholinergic system in memory processes in the honey bee has been demonstrated by intracranial injections of cholinergic antagonists using a classical conditioning procedure [12–14]. Local brain injections have shown that the nAChR antagonist mecamylamine impaired the recall or the formation of the memory trace depending on the brain site injection and 86 C. Armengaud et al. Figure 6.1 Chemical structures of acetylcholine and nicotinic cholinoceptor ligands used in this study. © 2002 Taylor & Francis the moment of the injection relative to the conditioning trial. From these experiments, we postulated that ACh, as in vertebrates, exerts a facilitatory effect on memory processes. We made the hypothesis that activation of the cholinergic pathways with agonists like those molecules belonging to the neonicotinoids would facilitate the formation and/or the recall of memory. To test this hypothesis, we submitted honey bees to the habituation of the proboscis extension reflex (PER). This nonassociative learning para- digm can be easily used to detect the behavioral effect of different kinds of molecules. The PER is induced by antennal sucrose stimulation and involves activation of motor neurons situated in the subesophageal gan- glion and driving the mouthpart muscles. The repetition of this non- noxious stimulation leads to a decrease in the response occurrence. We postulated that IMI could reduce the number of stimulations needed to observe the response decrease. However, given the neurotoxic action of IMI, the absence of the PER could indicate a problem of gustatory per- ception or a central motor disruption. In preliminary experiments, we defined the IMI dose that did not induce modifications of the gustatory threshold or a perturbation of motor activity. The involvement of mushroom bodies in memory processes is well established in insects. Consequently imidacloprid brain targets were inves- tigated using cytochrome oxidase (CO) histochemistry. CO activity is com- monly used in vertebrates as an endogenous metabolic marker of neuronal activity. Energy demand due to neuronal activity increases the production of oxidative energy [15]. Classically, CO histochemistry is used in verte- brates to identify a pathological modification [16, 17] or the effect of chronic surgical and pharmacological treatments [18–20]. We attempted to develop a functional histochemistry of CO in honey bee brain that allowed the analysis of the short-term effect of cholinergic ligands including IMI on the metabolism of the different brain structures [21]. Materials and methods Worker honey bees (Apis mellifera) were caught at the hive entrance and maintained with food and water ad libitum in small Plexiglas boxes until the beginning of the experiments. To evaluate the gustatory threshold and for learning and metabolism experiments, honey bees were immobilized individually in small plastic tubes with a drop of wax–collophane mixture laid between the dorsal part of the thorax and the tube. The head and the prothoracic legs were free to move, allowing the honey bee to clean its antennae from the repeated sucrose stimulations. Honey bees underwent a 2-hour starvation period before the beginning of the experiments. Imidacloprid (Cluzeaux, France; molecular weight: 255.7; degree of purity 98 percent) was dissolved in dimethyl sulfoxide (DMSO; Sigma) to obtain a 10 Ϫ1 M solution. Lower concentrations were obtained with successive dilutions in saline. Control groups were treated with DMSO Neuronal effect of imidacloprid in the honey bee 87 © 2002 Taylor & Francis dissolved in saline (vehicle) in the same proportions. For behavioral experiments, the drug or vehicle was used in topical applications (1l) to the thorax. Doses ranging from 1.25 to 5ng/bee were used which were below the DL50 value (10 to 20ng/bee, defined for thoracic application to Apis mellifera at 24h; unpublished observations from L.P. Belzunces). For CO experiments, we tested the effect of intracranial injection of saline or IMI on worker honey bees receiving an injection of 0.5l saline or IMI (10 Ϫ4 , 10 Ϫ6 , or 10 Ϫ8 M) at the brain surface. Nicotine (10 Ϫ8 , 10 Ϫ6 , and 10 Ϫ4 M) and mecamylamine (10 Ϫ2 M) were also tested as nAChR agonist and antagonist, respectively. Behavioral tests Gustatory threshold The aim of this experiment was to study the effect of IMI on the gustatory perception. The gustatory threshold was defined as the lowest concentra- tion of a sucrose solution applied to the antennae able to elicit a proboscis extension. The threshold was defined twice for each honey bee: first before any treatment and then after IMI or vehicle application. Several doses of IMI were used with several time-intervals between the application of the drug and the test. The gustatory threshold was determined as follows. Fasted honey bees were submitted to antennal stimulations (1-minute intertrial interval) with increasing concentrations of sucrose solutions ranging from M/1024 to 4M and following a geometric progression (M/1024, M/512, M/256, etc.). The range of increasing sucrose concentrations was applied twice separated by a 5-minute interval. The lowest concentration of sucrose that elicits the PER was defined as the gustatory threshold. Honey bees that failed to respond to one of the sucrose solutions were discarded. The remaining honey bees were fed with two drops of 50 percent (w/v) sucrose solution and fasted for 2 hours. This was done to ensure that the gustatory thresh- old determination under IMI application was made under the same moti- vational state. The thoracic application of vehicle or IMI at a dose of 1.25, 2.50, or 5ng/bee was performed during the starvation period, 15 min, 30 min or 60 min before the second gustatory threshold determination. This second determination was done like the first one. For data quantification, any modification of the gustatory threshold between the two determina- tions from one sucrose concentration to the one immediately lower or higher was respectively quantified as Ϫ1 or ϩ1 arbitrary unit. Locomotion Locomotion of honey bees was tested in an open-field-like apparatus con- sisting of a white Plexiglas box (30ϫ 30ϫ4cm) with a glass front for obser- 88 C. Armengaud et al. © 2002 Taylor & Francis vation. The back surface was divided into 5-cm 2 squares and the box was illuminated from above. The box did not allow the honey bees to fly. A hole made in the bottom right-hand side of the box allowed the introduc- tion of a single honey bee for a 5-min observation period. The position of the honey bee was recorded every 5s. The duration of successive 5-s periods in the same square was reported as immobility as the locomotor activity of the honey bee in the same square was very low, if nonexistent. Otherwise, the honey bee was moving around. The effect of the drug on locomotor activity was studied 15, 30, and 60 min after application of a dose of 1.25, 2.50, and 5ng/bee and was compared to the effect of vehicle. Habituation Fasted honey bees were stimulated repeatedly with a 50 percent (w/v) sucrose solution applied to one antenna at 1-min intervals. The habitua- tion criterion was defined as three consecutive sucrose stimulations without proboscis extension. When this criterion was reached, the sucrose solution was applied to the controlateral antenna to rule out the eventual- ity of motor tiredness. Honey bees that did not respond to the 50 percent sucrose solution and to the restoration test of the reflex were discarded. IMI was applied at 1.25ng/bee and the drug effect was tested after 15 min, 30 min or 1 hour in three independent groups. A group receiving no treat- ment and a solvent-treated group were also added. CO histochemistry Thirty minutes after injection of the drug, the animals were killed by rapid decapitation. Frontal sections (16m) from the whole brain were pre- pared for CO histochemistry, according to Wong-Riley [20]. Quantifica- tion of staining was performed by computer-aided densitometry of CO histochemistry intensity. We focused our investigations on antennal lobes, calyces, and ␣-lobes of MBs because it was previously shown that 30 min after an injection of AChR antagonists in these structures, memory processes were impaired [12, 13]. IMI was tested at concentrations of 10 Ϫ8 , 10 Ϫ6 , and 10 Ϫ4 M, corresponding to doses ranging from 1.28pg to 12.8ng per honey bee. At higher doses IMI induced toxic and lethal effects. Data analysis Data sets were analyzed using a two-population independent two-tailed t-test or an analysis of variance ( ANOVA). Figures show meansϮ s.e.m. In all cases, P-values less than 0.05 were considered as significant. Neuronal effect of imidacloprid in the honey bee 89 © 2002 Taylor & Francis Results Behavioral tests Gustatory threshold An increase in the gustatory threshold was observed between the first and the second determinations whatever the treatment (Figure 6.2). A very slight increase of less than one-half unit was found for the vehicle and for the lowest doses of IMI (1.25 and 2.50ng/bee). Animals treated with the vehicle (controls) were not different from those receiving no treatment (data not shown). Groups that received 1.25 and 2.50ng IMI were not dif- ferent from controls, so in subsequent habituation experiments, both doses could have been used. A loss of sensitivity was noticed for the dose of 5ng after 1 hour. This delayed effect seems not to be related to the time needed by imidacloprid to reach the brain from the thoracic application 90 C. Armengaud et al. 15 min (n ϭ 20) 3 2 1 0 30 min (n ϭ 10) 60 min (n ϭ 10) DMSO 1.25 ng 2.50 ng 5 ng Treatment Gustatory threshold (mean arbitrary units Ϯ s.e.m.) * Figure 6.2 Variations of the gustatory threshold (arbitrary units) 15 min, 30 min, or 1h after thoracic application of DMSO (nϭ20 for each time) or imidacloprid at different doses (1.25, 2.5, 5ng/bee). The number of imidacloprid-treated animals is indicated on the graph. *PϽ0.05. © 2002 Taylor & Francis site since the high dose of 20ng induces the same sensitivity loss as soon as 15 min after application (data not shown). Locomotion Opposite effects of IMI on motor displacements were observed depending on the dose (Figure 6.3). Compared to the vehicle, the lowest dose of IMI (1.25ng/bee) induced an increase in displacements independently of time (shown as a decrease in immobility in Figure 6.3). A significant increase in locomotion was also observed for 2.5ng/bee at 15 min. With 5ng, IMI induced a decrease in the honey bee displacements in the box as soon as 30 min after application. The decrease in displacements was explained by a loss of motor coordination. The honey bees fell down on their backs, showing leg movements and body and wing shaking. Additional observa- tions up to 2 hours after drug application showed that there was no behav- ioral recovery. Unlike the previous experiment on gustatory perception, we did not observe a dose–effect relationship in this experiment, as there were more Neuronal effect of imidacloprid in the honey bee 91 0 * * * * * 15 min 30 min 60 min 100 200 300 * * * * * * * * * * * DMSO 1.25 ng 2.50 ng 5 ng Treatment Mean duration of immobility Ϯ s.e.m. (sec) Figure 6.3 Time spent in immobility (seconds) in honeybees treated with DMSO or imidacloprid at different doses (1.25, 2.5, 5ng/bee), 15 min, 30 min, or 1h before the test. nϭ 10 in each group. *PϽ0.05; **PϽ0.01; ***PϽ 0.001. © 2002 Taylor & Francis numerous displacements at the lowest dose (1.25ng/bee). This dose was retained to test the effect of IMI on habituation. Habituation Under IMI treatment (1.25ng/bee), honey bees needed fewer trials to display PER habituation than honey bees receiving the vehicle or receiv- ing no treatment (statistics highly significant in both cases, see Figure 6.4). There was no effect of time on the facilitating effect. This observation is closer to the enhancing effect of 1.25ng of IMI on displacements, which is also independent of time. Dilute DMSO induced a slight but significant reduction in the number of trials compared to the groups receiving no treatment (statistics shown in Figure 6.4). CO histochemistry Histological modifications induced by IMI were of weak amplitude but very reproducible: for example in the antennal lobe, IMI 10 Ϫ4 M induced a 92 C. Armengaud et al. 15 min 0 10 20 30 40 50 60 Number of trials to reach habituation (mean Ϯ s.e.m) 30 min 60 min Time interval between treatment and test ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ * * * ϩ ϩ ϩ * * * ϩ ϩ ϩ ϩ ϩ ϩ * * * No treatment DMSO Imidacloprid Figure 6.4 Number of trials required to reach habituation in animals receiving no treatment or animals treated topically with DMSO or imidacloprid (1.25ng/bee) 15 min, 30 min, or 1h before learning. n ϭ 20 in each group. ϩϩϩcomparison to ‘No treatment’, P Ͻ 0.001. ***comparison to DMSO, PϽ0.001. © 2002 Taylor & Francis staining increase in all the experiments performed. The intensity of stain- ing was analyzed in the cortical layer and in the internal area of the glomeruli. At the concentrations of 10 Ϫ4 , 10 Ϫ6 , and 10 Ϫ8 M, a significant increase in staining was obtained for the two regions of the glomeruli. The increment ranged from ϩ8 percent to ϩ17 percent. A dose–response effect was observed for this structure (Figure 6.5A). The greatest modifications of CO labeling induced by IMI were observed in the ␣-lobe stratification, corresponding for the dorsal layer B1, to ϩ23 percent of the saline group labeling (Figure 6.5B). For 10 Ϫ4 M the increment was significant in the dorsal, intermediate and ventral layers (B1, B2, and B3). In the calyces the 10 Ϫ8 M IMI injection induced a significant reduction in the labeling (Figure 6.5C). In the basal ring the mean gray level of the 10 Ϫ6 M group was significantly lower compared to the saline group. In the 10 Ϫ4 M IMI-treated group, the CO staining in the upper (UD) and lower (LD) divisions of the central body was significantly greater than that of the saline group; the opposite effect was observed for 10 Ϫ8 M (Figure 6.5D). In subsequent experiments other nAChR ligands were tested. CO was stimulated by nicotine in a dose-dependent manner in many brain regions (data not shown). In particular, the internal part of the glomeruli exhibited significant increases of 19 percent at 10 Ϫ4 M nicotine (Figure 6.6A). The effects of nicotine were statistically significant in the B1, B2, and B3 layers of the ␣-lobe (Figure 6.6B). The greatest stimulation by 10 Ϫ4 M nicotine administration was obtained for the B3 layer (ϩ23 percent). Moreover, for the ventral layer a significant increase was still present after 10 Ϫ8 M (data not shown). In calyces, whatever the concentration of nicotine tested, no significant differences were found between the saline and nicotine groups whereas 10 Ϫ4 M IMI induced an increase in labeling. Moreover, 30 min after 10 Ϫ8 M IMI injection, a decrease in brain metabolism was observed in the central body, calyces, and ␣-lobe which was not observed with nicotine injection to the same concentration and at the same interval. Changes in the metabolic activity of the honey bee brain were exam- ined following nAChR antagonist (mecamylamine) administration to high concentration (10 Ϫ2 M) inducing an impairment of retrieval processes [13]. Comparison between saline- and antagonist-treated brains indicates that mecamylamine induced a significant decrease in neural metabolism in the ␣-lobe (Figure 6.6B) and no effect in the other structures (Figure 6.6A, C, D). Like IMI and nicotine, mecamylamine has a significant effect on the ␣-lobe. Neuronal effect of imidacloprid in the honey bee 93 © 2002 Taylor & Francis Figure 6.5 Relative variation of CO histochemistry induced by imidacloprid. (A) Antennal lobe: glomeruli cortical area, glomeruli internal area. (B) ␣-Lobe: B1, dorsal layer; B2, intermediate layer; B3, ventral layer. (C) Calyces: lip area, basal ring area. (D) Central body: UD, upper division of central body; LD, lower division © 2002 Taylor & Francis [...]... reduced the amplitude of the ACh responses recorded on SAD2 hybrid receptors expressed in Xenopus oocytes [22] Using [3H]IMI, high- and low-affinity nAChR-like binding sites have been characterized in the aphid Myzus periscae [6] The dual agonist/antagonist effects of IMI on CO histochem- © 2002 Taylor & Francis Neuronal effect of imidacloprid in the honey bee 99 istry could be linked to the presence of. .. 4] Finally, the dual effect of IMI can be explained by the presence in the central nervous system of the honey bee of two types of nicotinic receptors as shown in the cockroach nervous system [1] Stimulating effects, such as depolarization of the cockroach cercal afferent giant interneuron and inward currents with activation of the hybrid nAChR, were obtained with concentration up to 10 6 M [1, 22]... depends on the physiological function tested It seems that gustatory perception is less sensitive to the insecticide than motor function or learning processes The first manifestation of the drug on the gustatory threshold appeared at 5 ng/bee after 1 hour whereas a strong effect of the drug at the dose of 1.25 ng/bee was observed on the other functions after the shortest interval We do not retain the possibility... could indicate the specific effect of the drug linked to nicotinic activation whereas the higher doses would induce a nonspecific toxic effect The complex effect of IMI on neuronal metabolism has also been observed after intracranial injection of the insecticide in the honey bee All of the 10 regions analyzed showed a significant staining increase for the highest concentration (10Ϫ4 M) For the lower concentrations... Modulation of the neuronal nicotinic acetylcholine receptor-channel by the nitromethylene heterocycle imidacloprid J Pharmacol Exp Ther 285, 731–738 5 Liu, M.-Y and Casida, J.E (1993) High affinity binding of [3H]imidacloprid in the insect acetylcholine receptor Pestic Biochem Physiol 46, 40– 46 6 Lind, R.J., Clough, M.S., Reynolds, S.E and Early, F.G.P (1998) [3H]Imidacloprid labels high- and low-affinity... conditioning retrieval in the honey bee Behav Brain Res 63 , 145–149 Cano Lozano, V., Bonnard, E., Gauthier, M and Richard, D (19 96) Mecamylamine-induced impairment of acquisition and retrieval of olfactory conditioning in the honey bee Behav Brain Res 81, 215–222 Cano Lozano, V and Gauthier, M (1998) Effects of the muscarinic antagonists atropine and pirenzepine on olfactory conditioning in the honey bee Pharmacol... (nicotine-like) of IMI, observed with the highest concentration (10Ϫ4 M), may contribute to the toxicity of this insecticide In conclusion, these results support our hypothesis that the cholinergic system is involved in learning processes in insects We have previously shown that cholinergic antagonists impair the formation and recall of olfactory memory [12–14] We show now that activation of the cholinergic... the drug needs more time to diffuse from the thorax to the brain than to the ventral nerve cord The high doses of 10 and 20 ng induced a large increase in the gustatory threshold as soon as 30 min after application (results not shown) An interesting finding in this work is the dual effects of IMI on motor displacements and brain metabolism depending on the dose The locomotor activation observed at the. .. localization of 125I ␣-bungarotoxin binding sites in the honeybee brain Brain Res 534, 332–335 Goldberg, F., Grünewald, B., Rosenboom, H and Menzel, R (1999) Nicotinic acetylcholine currents of cultured Kenyon cells from the mushroom bodies of the honeybee Apis mellifera J Physiol 514, 759– 768 Gauthier, M., Cano Lozano, V., Zajoual, A and Richard, D (1994) Effects of intracranial injections of scopolamine... concentrations (10 6 and 10Ϫ8 M) a regional sensitivity and specificity were observed The effect of IMI in the antennal lobes, the first relay of the olfactory information, was always an increase in metabolism In integrative structures, in particular the calyces, the action of IMI is more complex Low concentrations induced inhibition of CO histochemistry whereas high concentrations resulted in activation of CO In . channel. Imidacloprid (IMI) {1–[ ( 6- chloro- 3- pyridinyl)methyl ]-4 ,5-dihydro-N-nitro-1H-imidazol-2-amine} is one of these new molecules (Figure 6. 1). According to the literature, IMI in insects acts. box allowed the introduc- tion of a single honey bee for a 5-min observation period. The position of the honey bee was recorded every 5s. The duration of successive 5-s periods in the same square. reported as immobility as the locomotor activity of the honey bee in the same square was very low, if nonexistent. Otherwise, the honey bee was moving around. The effect of the drug on locomotor