Cholinergic pesticides cause mushroom body neuronal inactivation in honeybees

Abstract

Pesticides that target cholinergic neurotransmission are highly effective, but their use has been implicated in insect pollinator population decline. Honeybees are exposed to two widely used classes of cholinergic pesticide: neonicotinoids (nicotinic receptor agonists) and organophosphate miticides (acetylcholinesterase inhibitors). Although sublethal levels of neonicotinoids are known to disrupt honeybee learning and behaviour, the neurophysiological basis of these effects has not been shown. Here, using recordings from mushroom body Kenyon cells in acutely isolated honeybee brain, we show that the neonicotinoids imidacloprid and clothianidin, and the organophosphate miticide coumaphos oxon, cause a depolarization-block of neuronal firing and inhibit nicotinic responses. These effects are observed at concentrations that are encountered by foraging honeybees and within the hive, and are additive with combined application. Our findings demonstrate a neuronal mechanism that may account for the cognitive impairments caused by neonicotinoids, and predict that exposure to multiple pesticides that target cholinergic signalling will cause enhanced toxicity to pollinators.

Figure 1. Kenyon cell recordings in acutely isolated honeybee brain.

(a, b) Images of an acutely isolated honeybee brain (mb, mushroom body) and a KC whole-cell recording with the fluorescent dye Lucifer Yellow in the patch electrode; to the left is a second micropipette used for pressure application of ACh. (c) Top, the current response to voltage steps (10 mV increments from a holding potential of −73 mV) showing activation of inward Na⁺ and outward K⁺ currents. Below, mean (±s.e.m.) I/V relationships (n=11) for the peak inward current (black circle), peak outward current (grey circle) and sustained outward current (open circle). The N-shape of the I/V relationship (open circle) indicates that Ca²⁺-activated K⁺ channels contribute to the sustained current. (d) Under current clamp, current injection (+20–+40 pA) evokes AP firing that exhibits adaptation in both frequency and amplitude, and a short delay between the current step and the first AP. Repetitive AP firing is therefore inhibited by sustained depolarization.

Figure 2. Cholinergic pesticides depolarize KCs at low concentrations.

The effect of neonicotinoids and coumaphos oxon on KC membrane potential (V_M) and AP firing were investigated under current clamp. (a) Bath application of clothianidin evokes sustained depolarization of KC V_M. Coapplication of the nAChR antagonist d-TC (500 μM, here and subsequently) reverses the depolarization, indicating that it is mediated by nAChR activation. (b) AP firing is transiently observed during the development of the depolarization (2 min after clothianidin application), but is inhibited during the subsequent sustained depolarization (20 min example trace). (c) Coumaphos oxon evokes a more slowly developing depolarization of KC V_M, which is reversed by d-TC. The slower time course is consistent with nAChR activation by accumulated ACh as a result of AChE inhibition. (d) AP firing is again transiently observed during the development of the depolarization (15 min after coumaphos oxon application) but not during the sustained phase (30 min example trace). (e) Dose dependence of the mean (±s.e.m.) depolarizing effects of the neonicotinoids clothianidin (black square, n=4) and imidacloprid (black circle, n=3-4), the metabolite imidacloprid–olefin (open circle, n=4) and the organophosphate coumaphos oxon (grey triangle, n=2–7; n numbers refer to each data point; for all data points ≥10 nM, P<0.05, paired t-test). (f) Mean (±s.e.m.) data showing the reversal by d-TC of the depolarizations evoked by clothianidin (10–100 nM, n=3), imidacloprid (50–500 nM, n=4), imidacloprid–olefin (500 nM, n=3) and coumaphos oxon (200 nM–1 μM, n=4). All four pesticides potently modulate KC excitability by causing sustained activation of nAChRs.

Figure 3. Coumaphos oxon potently inhibits AChE.

Mean (±s.e.m.) AChE activity measured in honeybee brain (n=3) and rat brain (n=3) by the Ellman assay, showing inhibition by coumaphos oxon but little effect of coumaphos. AChE activity was normalized to control measurements; IC₅₀ values were obtained from Hill equation fits of the data.

Figure 4. Properties of KC ACh responses.

(a) Local application of ACh (200 μM, 100 ms) to a voltage-clamped KC evokes a transient inward current that is blocked by bath application of d-TC, indicating that it is solely mediated by nAChRs. In the same KC under current clamp, ACh evokes a transient depolarization and a burst of APs (shown below). (b) Mean (±s.e.m.) data showing the inhibition of ACh-evoked currents by d-TC (n=5) and by α-bungarotoxin (α-BGT; n=5), which inhibits vertebrate α7 nAChRs (*P<0.05, paired t-test). (c) Mean (±s.e.m.) response size at different holding potentials, showing that the voltage-dependence of ACh responses exhibits slight inward rectification (n=5). (d) Example ACh responses showing the variability in amplitude and kinetics of the response between KCs, with distinct fast and slow components in some recordings.

Figure 5. Neonicotinoids evoke a tonic nAChR current and inhibit ACh responses.

The effect of neonicotinoids on baseline I_M and on ACh responses were investigated under voltage clamp. (a) Bath application of imidacloprid (1 μM) evokes an inward shift in the baseline current (tonic I_M) and inhibits responses evoked by local application of ACh (200 μM, 100 ms). (b) The time course of the effect of imidacloprid on I_M (○) and ACh responses (). The tonic current (increase in I_M) develops rapidly and then declines slowly due to nAChR desensitization; ACh responses are rapidly inhibited and remain inhibited for the duration of imidacloprid application. (c) In a different KC, imidacloprid evokes a tonic current that exhibits little desensitization. Coapplication of d-TC reverses the tonic current, showing that it is due to sustained nAChR activation. (d) Mean (±s.e.m.) data showing that imidacloprid (pooled 1–10 μM) also evokes an increase in I_M variance (current noise; n=16, *P<0.01, paired t-test) that is reversed by d-TC (2 μM imidacloprid, n=3, *P<0.05, unpaired t-test), consistent with increased nAChR channel activity. (e) The dose dependence of the peak tonic current evoked by imidacloprid (mean±s.e.m., n=3–7 for each concentration, *P<0.05, paired t-test) and the effect of clothianidin (200 nM, n=3) for comparison. (f) The dose dependence of ACh response inhibition by imidacloprid (mean±s.e.m., n=3–7) with a Hill equation fit, and the effect of clothianidin (200 nM, n=3) for comparison. ACh response inhibition was measured after at least 10 min of neonicotinoid application. The neonicotinoids reduce KC responsiveness to ACh by tonically activating and desensitizing nAChRs.

Figure 6. Coumaphos oxon has a biphasic effect on ACh responses.

The effects of coumaphos oxon on baseline I_M and ACh responses were investigated under voltage clamp. (a) Coumaphos oxon (200 nM) initially potentiates ACh responses (10 min after application), consistent with AChE inhibition. Subsequently, coumaphos oxon evokes a tonic current and inhibits ACh responses (30 min after application). (b) The time course of the effect of coumaphos oxon on I_M in a different KC. The tonic current develops slowly and is reversed by d-TC, consistent with nAChR activation by accumulated endogenous ACh. (c) Mean (±s.e.m.) data showing the biphasic effect (initial potentiation followed by sustained inhibition) of coumaphos oxon (n=11, *P<0.01, paired t-test) on ACh responses. Included for comparison is the effect of the widely used AChE inhibitor donepezil (n=7, *P<0.01, paired t-test). (d) Mean (±s.e.m.) data showing the effects of coumaphos oxon (n=11) and donepezil (n=7) on I_M (*P<0.01, paired t-test). Inhibition of ACh responses is associated with an increase in I_M (tonic current). (e) Mean (±s.e.m.) data showing that the time course of the biphasic effect of coumaphos oxon on ACh responses is dependent on concentration (0.05 μM, n=4; 0.2 μM, n=3; 1 μM, n=4; *P<0.01, unpaired t-test). (f) Mean (±s.e.m.) data showing that the parent compound coumaphos inhibits ACh responses at concentrations ≥10 μM (1 μM, n=3; 10 μM, n=5; 50 μM, n=3; *P<0.05, paired t-test). The effect of 0.5% ethanol, the vehicle for the highest coumaphos concentration, is included as a control. (g) Example ACh responses before and after coumaphos (10 μM) application. (h) The time course of the effect of coumaphos on ACh responses in a different KC. Coumaphos (10–50 μM) inhibits ACh responses without significantly changing I_M (−1.3±1.5 pA, n=8) or ACh response kinetics, consistent with a lack of AChE inhibition.

Figure 7. Cholinergic pesticides have additive effects on KC V_M and inhibit ACh-evoked depolarizations.

As honeybees are likely to be exposed to both neonicotinoids and miticides, the effect of coapplication on KC function were determined. (a) Under current clamp, application of coumaphos oxon evokes depolarization of KC V_M. Subsequent coapplication of imidacloprid evokes a further concentration-dependent depolarization. (b) Comparison of the sum of the depolarizations evoked by coumaphos oxon and imidacloprid alone (imidacloprid data from Fig. 1e), with the depolarization evoked by the coapplied pesticides (mean±s.e.m.; 10 nM, n=5; 50 nM, n=3). The depolarizations are very similar in magnitude, indicating that the effects of imidacloprid and coumaphos oxon on KC function are additive. (c) The effect of combined pesticide exposure on the response of KCs to ACh was examined under current clamp. Local application of ACh (200 μM, 100 ms) evokes a transient depolarization and burst of APs. Coapplication of imidacloprid (10 nM) plus coumaphos oxon (50 nM) initially slows the decay of ACh responses (25 min after application) due to AChE inhibition. Subsequently, the pesticides evoke sustained depolarization of KC V_M and inhibit ACh-evoked responses (50 min after application). (d) Mean (±s.e.m.) data showing the sustained effect of imidacloprid plus coumaphos oxon on the size of ACh-evoked depolarizations (n=5, *P<0.05, paired t-test). Cholinergic pesticides reduce the responsiveness of KCs to ACh by causing tonic depolarization.