Nicotinic acetylcholine receptor ligands differently affect cytochrome oxidase in the Honeybee brain

Neuroscience Letters 304 (2001) 97±101

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Nicotinic acetylcholine receptor ligands differently affect cytochrome oxidase in the Honeybee brain Catherine Armengaud*, Jamila AõÈt-Oubah, Nicolas Causse, Monique Gauthier Laboratoire de Neurobiologie de l'Insecte, Universite de Toulouse III, 118 route de Narbonne, 31062 Toulouse Cedex, France Received 7 December 2000; received in revised form 13 March 2001; accepted 13 March 2001

Abstract The objective of this study was to determine if nicotinic receptor antagonists known for their ability to impair memory in the honeybee could induce changes in brain metabolism. We tested the effect of antagonists [hexamethonium, mecamylamine, a-bungarotoxin (a-BTX)] and agonist (nicotine) brain injections on cytochrome oxidase (CO) histochemistry. Within as little as 30 min following nicotine injection, an increase of the staining was observed in almost all the structures analyzed. The increase was limited to the a-lobe after a-BTX injection. In contrast, the antagonists hexamethonium and mecamylamine reduced CO staining in this structure that seems to be involved in information retrieval. These results suggest that the decrease of metabolism in the a-lobe obtained with hexamethonium and mecamylamine injections could be related to the impairment of retrieval processes previously observed with these drugs. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Honeybee; Cytochrome oxidase; Nicotinic acetylcholine receptor antagonists; Brain; Nicotine; Memory

It is well established that acetylcholine is present in high concentrations in the insect brain [5] and that nicotinic acetylcholine binding sites are largely present in the honeybee brain [12,13]. The involvement of the cholinergic system in memory processes in the honeybee has been studied by testing the effect of intracranial injections of cholinergic antagonists on the well-described one-trial olfactory conditioning of the proboscis extension re¯ex [8,9,10]. We have shown an impairment of acquisition and/or retrieval processes after intracranial injection of the nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine and hexamethonium, whereas no effect was obtained with abungarotoxin (a-BTX). To con®rm that the amnesic effect induced by mecamylamine and hexamethonium resulted from a preferential action of these antagonists on brain structures speci®cally involved in memory processes, we have evaluated the metabolic activity of the honeybee brain after nicotinic antagonist injections. The histochemistry of cytochrome oxidase (CO) is commonly used in vertebrates as an endogenous metabolic marker for neuronal activity: energy demand due to neuronal activity increases * Corresponding author. Tel.: 133-5-61556436; fax: 133-561558444. E-mail address: [email protected] (C. Armengaud).

oxidative energy production [19,20]. A signi®cant body of evidence suggests that a defect in the activity of CO is associated with learning de®cits. Chronic and selective inhibition of CO in rat by sodium azide impair spatial learning [2]. Moreover, a CO defect is found post-mortem in Alzheimer's disease brain tissue [15]. So the localisation of the metabolic variations in the neuronal circuitry is of general interest to determine the different targets of nAChR antagonists in the honeybee brain. Moreover, identifying discrete brain regions and neural pathways that are functionally altered following nAChR antagonist treatments is central to the elucidation of memory processes. In the present study we hypothesize that a decrease of neural activity in the a-lobe is associated with retrieval impairment. We test the effect of nicotinic agonist and antagonist injections using experimental conditions previously established for behavioural experiments [7]. Honeybee workers (Apis mellifera) were caught at the hive entrance and kept for a couple of hours with food (honey) ad lib. in a Plexiglas box. Then they were ®xed in small tubes as previously described [8]. After 1 h in the tube a small window was made in the head cuticle between the antenna and the median ocellus. The honeybees were allowed to recover from the operation for 1 h before drug injection. Nicotine, hexamethonium, mecamylamine and

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C. Armengaud et al. / Neuroscience Letters 304 (2001) 97±101

a-BTX from Sigma (St Quentin Fallavier, France) were dissolved in bee saline solution containing KCl: 2.01 g/l, NaCl: 2.24 g/l, MgCl2: 0.101 g/l, CaCl2: 1.99 g/l, sucrose: 34 g/l. The volume (0.5 ml) injected and the concentration of nAChR antagonists were determined in earlier behavioural experiments [7]. We have chosen a time interval that had induced the most pronounced memory impairment. Thirty minutes after a nicotine (10 28, 10 26, 10 24 M), hexamethonium (10 22 M), mecamylamine (10 22 M), a-BTX (10 23 M) or saline injection the animals were sacri®ced by rapid decapitation. Brains were rapidly dissected in 4% paraformaldehyde ®xative in 0.1 M phosphate buffer (PB). After 1.5 h the ®xative solution was replaced by 25% sucrose (in PB) overnight. Cryostat frontal sections (16 mm) from the whole brain were prepared for CO histochemistry according to our previous report [1]. Densitometric analysis was performed under £ 20 magni®cation (Zeiss microscope). Quanti®cation was performed by computer-aided densitometry of CO histochemistry intensity using Visilog (5.0, Noesis) image analysis software. The grey level of three to six sections of each analyzed structure was calculated for each brain from saline and treated groups of an experiment comprising no more than 12 animals. Statistical analysis was performed by two-way ANOVA (treatment factor and day of experiment factor) on pooled sections of a minimum of three separated experiments. When the F value obtained by ANOVA was signi®cant (P , 0:05), the Fisher PLSD (protected least signi®cant difference) was used to compare cholinergic treatments and saline mean values. P , 0:05 was considered as signi®cant. To reduce variation in the intensity of CO staining from one experiment to another, changes in response amplitude were expressed as the percent change from the saline value. CO staining was observed throughout most of the brain neuropiles and in some somata (Fig. 1a). Analysis of the staining was performed in two neuropiles essentially involved in olfactory learning: the antennal lobes and the mushroom bodies and in a third neuropile: the central body. At the periphery of the antennal lobe, the glomeruli contain a dense area of synaptic contacts. Because the cortical area of glomeruli is darker than the medullar part (Fig. 1a), these two regions were analyzed separately. In mushroom bodies, the staining concentrated in lip and basal ring neuropiles was analyzed as indicated in Fig. 1b. As a result of the ordered distribution of intrinsic and extrinsic neurones, the a-lobe appears strati®ed. Only the largest stained layers were analyzed. We called them B1, B2, and B3 (Fig. 1a). Within the central body, the two divisions were analyzed separately because AChR-like immunocytochemistry in the lower division (LD) of the central body appears denser than in the upper division (UD) [13]. Before testing the antagonists we investigated if stimulation of nAChRs by nicotine induced an increase of neural metabolism (Table 1 and Fig. 2). Analyses of data concerning antennal lobe cortical and medullar areas by two-way ANOVA, indicate that overall effects of treatment

(FT ˆ 7:31 and 4.43, P , 0:01 for both) and experiment (FE ˆ 93:39 and 39.19, P ˆ 0:0001 for both) are statistically signi®cant without signi®cant interaction (FTE ˆ 1:54 and 1.25, P , 0:20 for both). CO was stimulated by nicotine in a dose-dependent manner in these brain regions. The medullar part of the antennal lobe in particular,

Fig. 1. CO histochemistry in frontal sections of saline-injected brain. (a) In a-lobe the labelling appeared in three main bands (B1, B2, B3); in the glomeruli of the antennal lobe (AL) the cortical neuropil (arrow head) shows a higher metabolism than the medullar one (*); stained somata group dorsally to the antennal lobe (arrow). (b) Calyces of the mushroom bodies. The labelling is con®ned mainly to lip and basal ring (BR), whereas the soma of Kenyon cells (*) shows no labelling. The arrow indicates a ®ne stained pro®le within the pedunculus. The central body presents strong metabolism in its two divisions: upper (UD) and lower (LD) division. scale bars: 100 mm.

C. Armengaud et al. / Neuroscience Letters 304 (2001) 97±101

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Table 1 Densitometric analysis of cytochrome oxidase histochemistry in saline- and nicotine-injected brains a Relative optical density (mean ^ SEM) Brain region Antennal Lobe Cortical area Medullar area Alpha Lobe B1 layer B2 layer B3 layer Calyces Lip Basal ring Protocerebral lobe Central body Upper division Lower division

Saline (n ˆ 6)

10 28 M (n ˆ 6)

Nicotine 10 26 M (n ˆ 7)

10 24 M (n ˆ 7)

42 ^ 1.15 27.86 ^ 1.08

45.43 ^ 1.56² 31.37 ^ 1.32*

46.56 ^ 1.22* 32 ^ 1.02*

49.37 ^ 1.12*** 33.11 ^ 1.02***

19.67 ^ 0.67 18.09 ^ 0.58 32.79 ^ 1.03

21.20 ^ 1.13 19.48 ^ 1.06² 36.48 ^ 1.86*²

24.81 ^ 0.87*** 22.42 ^ 0.67*** 39.73 ^ 1.15***

23.65 ^ 0.93*** 22.24 ^ 0.77** 40.19 ^ 0.85***

33.29 ^ 1.2 41.04 ^ 1.28 33.29 ^ 0.84

32.56 ^ 1.49² 38.52 ^ 1.82²² 35.97 ^ 1.60

34.14 ^ 1.62 42.63 ^ 2.04 37.79 ^ 1.06**

36.93 ^ 1.27 45.11 ^ 1.42 38.32 ^ 0.83***

36.38 ^ 1.6 43.56 ^ 2.12

39.57 ^ 2.29 45.17 ^ 2.3

40.86 ^ 2.06 47.05 ^ 2.25

42.25 ^ 2.11 49.54 ^ 1.98

a Staining intensity is indicated by grey level. Each value represents the mean ^ SEM of 29±38 sections from six to seven injected animals. *P , 0:05 versus the corresponding saline-treated group, (PLSD from Fisher).**P , 0:01 versus the corresponding salinetreated group, (PLSD from Fisher).***P , 0:001 versus the corresponding saline-treated group, (PLSD from Fisher) ²,P , 0:05 versus the 10 24 M nicotine-treated group, one-way ANOVA. ²², P , 0:01 versus the 10 24 M nicotine-treated group, one-way ANOVA.

exhibited signi®cant increases of 11% after 10 28 M nicotine (PLSD ˆ 3:20 P , 0:05), 17% after 10 26 M (PLSD ˆ 3:29 P , 0:05) and 19% after 10 24 M (PLSD ˆ 5:17 P , 0:001) (Table 1). For the highest concentration of nicotine a substantial stimulation (115%) was also obtained in the cortical part of the antennal lobe. The effects of treatment (nicotine) and experiment factors were statistically signi®cant in the B1, B2 and B3 layers of the a-lobe (P , 0:0001 for all) with no signi®cant interaction between treatment and experiment for layers B1 and B2 (P . 0:05) and an interaction (P ˆ 0:03) for layer B3. A signi®cant increase of metabolism in layers B1 and B2 was observed for 10 26 M (PLSD ˆ 4:28 and 3.66, respectively; P , 0:001 for both). The greatest stimulations by nicotine administration were obtained for layers B1 and B3 (123%) with 10 26 M and 10 24 M, respectively. Moreover for the B3 layer a signi®cant increase was still present after 10 28 M (PLSD ˆ 3:51, P , 0:05). In the calyces, two-way ANOVA revealed a signi®cant effect of treatment in lip (FT ˆ 2:75 P ˆ 0:047) and basal ring (FT ˆ 4:14, P ˆ 0:008) and a signi®cant treatment £ experiment interaction (P , 0:05 for both). However, for these neuropiles, whatever the concentration of nicotine tested, no signi®cant differences were found between saline and nicotine groups. In addition, a signi®cant difference was found between 10 28 M and 10 24 M nicotine groups (PLSD ˆ 4:44 for basal ring and PLSD ˆ 3:77 for lip P , 0:05 for both). The protocerebral lobe exhibited a signi®cant effect for treatment (FT ˆ 5:75, P ˆ 0:001) and experiment (FE ˆ 95:24, P ˆ 0:0001) and a signi®cant interaction (FTE ˆ 5:41, P ˆ 0:0016). The increase of metabolic activity reached signi®cant levels after 10 26 M (PLSD ˆ 3:97,

P , 0:01) and 10 24 M nicotine injection (PLSD ˆ 4:97, P , 0:001); this stimulating effect of nicotine was 13 and 16%, respectively compared to saline. A moderate increase of staining (18%) was observed in the central body. However, in UD and LD of the central body two-way ANOVA neither revealed a signi®cant treatment effect (FT ˆ 2:14 and 2.47; P ˆ 0:10 and 0.06, respectively) nor a signi®cant treatment £ experiment interaction (FTE ˆ 1:31 and 0.88; P ˆ 0:27 and 0.46, respectively). Changes in the metabolic activity of honeybee brain were examined following nAChR antagonist administration at high concentrations (10 22,10 23 M) inducing impairment of the retrieval processes [8,10]. In antennal lobes no significant effect of a-BTX was obtained as indicated by two-way ANOVA (FT ˆ 1:63, P ˆ 0:20 for cortical area, FT ˆ 1:3, P ˆ 0:25 for medullar area). An absence of effect of hexamethonium (FT ˆ 0:69, P ˆ 0:41 for cortical area, FT ˆ 2:12, P ˆ 0:15 for medullar area) was also observed in these glomerular structures (Fig. 2a). However, a signi®cant difference was found between saline and mecamylamine groups as indicated by two-way ANOVA (FT ˆ 12:60, P ˆ 0:006 for cortical area FT ˆ 24:78, P ˆ 0:001 for medullar area) and PLSD's Fischer analysis (PLSD ˆ 4:80 for cortical area and PLSD ˆ 3:23 for medullar area P , 0:05 for both). Comparison between saline- and antagonist-treated brains indicates that mecamylamine and hexamethonium both induce a decrease of neural metabolism in the a-lobe (Fig. 2b). After mecamylamine injection, a signi®cant decrease of the staining was observed in layers B1 and B3 (FTB1 ˆ 5:97, PB1 ˆ 0:02; FTB3 ˆ 9:87, PB3 ˆ 0:002, two-way ANOVA). However in the B2 layer, two-way ANOVA analysis did not indicate a signi®cant effect of mecamylamine when PLSDs Fisher

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Fig. 2. Effect of nicotinic AChR antagonists and nicotine on cytochrome oxidase histochemistry in the antennal lobes (a), alpha lobes (b), calyces (c) and central body (d). UD, upper division; LD, lower division; a-BTX, a-bungarotoxin. Results represent mean ^ SEM of calculated brain values (nicotine: n ˆ 7; mecamylamine: n ˆ 9; hexametonium: n ˆ 5; a-BTX: n ˆ 11). The reference 100% is the mean saline value of each experiment.

analysis (2.80) indicates a signi®cant effect. The metabolism in layers B1, B2 and B3 was decreased by 11, 12 and 12% respectively under saline. After hexamethonium injection, the decrease was signi®cant only in B1 (FT ˆ 4:71, P ˆ 0:04) and B3 layers (FT ˆ 8:13, P ˆ 0:009). Surprisingly the irreversible nAChR antagonist a-BTX induced a signi®cant increase of staining in B1, B2 and B3 by 20, 25 and 18%, respectively as shown by two-way ANOVA and PLSD (P , 0:05 for all). In the calyces (Fig. 2c) two-way ANOVA did not reach a signi®cant level for a-BTX treatment in lip (FT ˆ 0:033, P ˆ 0:86) or in basal ring (FT ˆ 0:065; P ˆ 0:80). Mecamylamine and hexamethonium were without effect on lip and basal ring metabolism too (P . 0:05 for both). No signi®cant modi®cations were obtained for UD and LD (P . 0:05 for both) after nAChR antagonist injections (Fig. 2d). In the present study, we show that in the a-lobe, the effects of nicotine on CO opposed the effects of the nAChR antagonists mecamylamine and hexamethonium and were identical to that of the nAChR antagonist aBTX. These results are partly consistent with our hypothesis that the nAChR agonist nicotine stimulates honeybee brain metabolism and antagonists decrease it. Stimulation of honeybee brain metabolism after a 10 24 M nicotine injection was observed in almost all the structures analyzed. We previously described a similar effect of a neonicotinoid, imidacloprid [1]. However, 10 24 M imidacloprid was more potent in the calyces than nicotine. Nicotine stimulated CO in the antennal lobe and the a-lobe but not in the calyces and no signi®cant effect was found in this neuropile after nAChR antagonist injections. However, a high density

of nAChR-immunoreactivity has been shown and a-BTX binding sites are present in the antennal lobe, a-lobe and also in the calyces [13,18]. Mecamylamine or hexamethonium injected in the brain hemolymph induced a severe impairment of memory retrieval [7]; the same kind of effect was observed with an injection of mecamylamine or scopolamine in the a-lobes of the mushroom bodies (unpublished data). To con®rm that the amnesic effect induced by mecamylamine and hexamethonium resulted in a preferential action in the a-lobe, we evaluated honeybee brain metabolism after injection of nicotinic antagonists. The regional effects of mecamylamine and hexamethonium observed with CO histochemistry were consistent with mecamylamine and hexamethonium-induced memory dysfunctions. Unlike mecamylamine, the intracranial injections of aBTX (10 28 ±10 26 M) had no effect on olfactory conditioning or on recall processes. This was previously attributed to the reduced access of the high-molecular-weight toxin to brain structures and/or to the low concentrations used compared to those used with mecamylamine. For this reason we chose a higher concentration of a-BTX (10 23 M) for histochemistry experiments. Unlike mecamylamine and hexamethonium, a-BTX injection did not decrease neuronal metabolism in the a-lobe. The increase of staining obtained with a-BTX is dif®cult to explain because many studies con®rm that a-BTX is a potent nAChR antagonist in the honeybee brain. The speci®c block by a-BTX of ACh- and nicotine-induced intracellular Ca 21 increase provided physiological evidence that Kenyon cells express nAChR [4]. Patch clamp experiments in Kenyon cells of the mushroom bodies indicated that a-BTX blocked 80% of the

C. Armengaud et al. / Neuroscience Letters 304 (2001) 97±101

ACh-induced inward current [11]. With an in vitro whole bee brain preparation, a-BTX caused a 62% attenuation of the calycal population spikes induced by the stimulation of the antennal lobe [16]. The incomplete blockade of electrical activity by a-BTX and the opposite effect of the toxin compared to other nAChR antagonists strongly suggest the existence of different subpopulations of nAChRs in honeybee brain as in other insects [11,14]. Their pharmacological pro®le distinguished on the basis of their sensitivities to aBTX [3,6]. These nAChRs probably act in different pathways. We show a pronounced effect of a-BTX and other antagonists in the a-lobe, which represents the output region of calyces and neuropile for extrinsic neurones [17]. None of the nicotinic treatment used had any effect on calyx metabolism; this result is surprising because Kenyon cells arborized into the calyces and electrophysiological and biochemical studies indicated that the activity of cultured Kenyon cells can be modulated by nicotinic ligands [4,11]. In conclusion, the biochemical effects (modulation of CO activity) elicited by the modi®cation of neuronal activity with mecamylamine and hexametonium, are consistent with behavioural effects of these antagonists. These ®ndings are consistent with our hypothesis: an impairment of recall is associated with a decrease of CO activity in the a-lobe. This neurochemical study on insect brain metabolism has implications concerning targets or the mode of action of cholinergic ligands including insecticides. We thank Dr V. Cano Lozano for her great help during the hexamethonium experiment and Dr P. Winterton for English language improvement. This research was supported by the ReÂgion Midi PyreÂneÂes (Grant 99902824). [1] Armengaud, C., Causse, N., AIÈt-Oubah, J., Ginolhac, A. and Gauthier, M., Functional cytochrome oxidase histochemistry in the honeybee brain, Brain Res., 24 (2000) 3690±3693. [2] Bennet, M.C., Mlady, G.W., Fleshner, M. and Rose, G.M., Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning de®cit and inhibiting cytochrome oxidase activity, Proc. Natl. Acad. Sci. USA, 93 (1996) 1330±1334. [3] Bertrand, D., Ballivet, M., Gomez, M., Bertrand, S., Phannavong, B. and Gundel®nger, E.D., Physiological properties of neuronal nicotinic receptors reconstituted from the vertebrate b2 subunit and drosophila a subunits, Eur. J. Neurosci, 6 (1994) 869±875. [4] Bicker, G., Transmitter-induced calcium signalling in cultured neurons of insect brain, J. Neurosc. Meth., 69 (1996) 33±41. [5] Breer, H., Neurochemical aspects of cholinergic synapses

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