Induction of a specific olfactory memory leads to a long-lasting activation of protein kinase C in the antennal lobe of the honeybee

Abstract

In this study we investigated the role of protein kinase C (PKC) in associative learning of Apis mellifera. Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes, a brain structure involved in associative learning. Multiple conditioning trials inducing a memory different from that induced by a single conditioning trial specifically cause an increase in PKC activity. This increase begins 1 hr after conditioning, lasts up to 3 d, and is attributable to an increased level of constitutive PKC. The increased level of constitutive PKC consists of an early proteolysis-dependent phase and a late phase that requires RNA and protein synthesis. Inhibition of the pathways resulting in constitutive PKC selectively impairs distinct phases of multiple-trial induced memory. The inhibition of the proteolytic mechanism has an instant effect on an early phase of multiple-trial induced memory but does not affect acquisition and the late phase of memory. Blocking of the transient PKC activation during conditioning does not affect the induction of memory formation. Thus, the constitutive PKC in the antennal lobe seems to contribute to the early phase of memory that is induced by multiple-trial conditioning.

Fig. 1.

Comparison of the distribution of PKC in the honeybee brain with the neuropils critical to olfactory learning. The distribution of PKC in the adult honeybee brain, as analyzed by immunohistochemistry (A), is compared with the neuropils that are involved in olfactory learning (schematic).B, The antennal lobes (al), which receive mainly chemosensory input, show strong staining in the central core (asterisk), in the central regions of the glomeruli (g), and in the somata of the interneurons (arrow). Strong labeling is found throughout the mushroom bodies (mb), which process multimodal input and are innervated by AL interneurons. The lateral protocerebral lobes (lp) are stained also. In contrast, the optical lobes (ol) are stained very weakly. Scale bar, 100 μm.

Fig. 2.

PKC activation induced by stimuli used in olfactory conditioning. Animals received either a singleCS or US, a single forward (CS-US) or backward pairing (US-CS), or three forward or backward pairings delivered with an intertrial interval (ITI) of 2 min. Animals were dissected for the determination of PKC activity in the antennal lobes (AL) either immediately (0), 1.5, or 10 min after stimulation. In each experiment, values were normalized to PKC activity in unstimulated animals (−). Each column represents the mean ± SEM of n measurements as indicated by thenumbers on the bars. For all stimulations PKC activity at 0 is significantly different from PKC activity in unstimulated animals (p < 0.05;t test).

Fig. 3.

Time course of PKC activity after one and three conditioning trials. PKC activity in the AL was determined at different times after one or three conditioning trials (ITI, 2 min). In each experiment, values were normalized to PKC activity in unstimulated naive animals (dashed line). Each pointrepresents the mean ± SEM of n measurements. Values that are significantly different from PKC activity in unstimulated naive animals are marked by an asterisk(p < 0.05; t test).

Fig. 4.

Characterization of the long-lasting PKC activation induced by three-trial conditioning. A, PKC activity in the AL was measured at 3 and 18 hr after three-trial conditioning (ITI, 2 min) in the presence or absence of calcium and diacylglycerol (DAG). In a control group of untrained animals the PKC activity was measured by following the same procedure as in trained animals. In each experiment, values were normalized with respect to PKC activity in untrained animals, measured in the presence of calcium and DAG. B, Animals were dissected at 3 and 18 hr after three-trial conditioning. Conditioned animals were each injected with 1 μl of either Gö 7874 (1 mm) or PBS 20 min before dissection. Untrained animals were handled in parallel in the same way as trained animals. In all experimental groups the PKC activity was measured in the presence of calcium and DAG. In each experiment, values were normalized with respect to PKC activity in PBS-injected untrained animals. Each column represents the mean ± SEM of n measurements as indicated by the numbers on the bars(*p < 0.05; t test).

Fig. 5.

Effect of E 64, actinomycin D, and anisomycin/cycloheximide on conditioning-induced PKC activation.A, Animals were injected with 1 μl of the thiol protease inhibitor E 64 (1 mm) or PBS 20 min before three-trial conditioning (ITI, 2 min). PKC activity was measured at 3 and 18 hr after conditioning. B, Animals were injected with 1 μl of actinomycin D (2 mg/ml), anisomycin/cycloheximide (CHX; 2.5 respectively, 5 mg/ml), or PBS 1 hr after three-trial conditioning. PKC activity was measured at 3 and 18 hr after conditioning. PKC activity was determined in parallel in groups of untrained animals, injected according to the protocol for trained animals. In each experiment, values were normalized to PKC activity in PBS-injected untrained animals. Each column represents the mean ± SEM of n measurements as indicated by the numbers on the bars(*p < 0.05; t test).

Fig. 6.

Dissection of memory phases by inhibition of thiol proteases or transcription. Animals were injected with 1 μl of PBS or 1 μl of the thiol protease inhibitor E 64 (1 mm) 20 min before conditioning, or they were injected with 1 μl of PBS or actinomycin D (2 mg/ml) 1 hr after conditioning. Animals received either one conditioning trial (A) or three conditioning trials (B). The probability of the CS-elicited proboscis extension response (PER) was tested during acquisition (acq) and at different times after conditioning (retr). The PER of animals that received PBS injection before or after conditioning did not differ between the groups. Thus the values were combined in the PBS control group. The data show the PER of n animals, as indicated. Sequence from top to bottom: PBS, actino, and E 64 (*p < 0.001; χ²test).