Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Abstract

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.

Introduction

Octopamine is a multifunctional naturally occurring biogenic amine that plays a major neuromodulatory role in invertebrates (David and Coulon, 1985). Octopamine modulates nonassociative learning, such as food-induced arousal (Braun and Bicker, 1992) and responses to conditioned olfactory stimuli (Mercer and Menzel, 1982, Hildebrandt and Müller, 1994). It enhances associative olfactory memory consolidation when injected to mushroom bodies (MB) or antennal lobes (AL) (Hammer and Menzel, 1998). Collective evidence suggests that octopamine plays an important role in olfactory learning and memory processes in the honeybee brain.

In the honeybee, an octopaminergic neuron (VUMmx1) mediates an interaction between an odor-sensitive (CS) and sucrose-sensitive (US) pathways in the AL (Hammer, 1993). The convergence of these two types of stimuli suggests that the AL is a site where at least a component of olfactory memory consolidates (Farooqui et al., 2003). VUMmx1 responds to odor only after pairing that odor with an unconditioned stimulus (sucrose). VUMmx1 releases octopamine into all or most glomeruli of the AL (Hammer, 1993). Its responsiveness to odor changes in a way correlated to associative changes in the behavioral response to odor in the conditioning paradigm (Hammer, 1993).

Multiple octopamine receptor subtypes have been pharmacologically characterized in locust nervous tissue (Evans and Robb, 1993, Roeder and Nathanson, 1993, Howell and Evans, 1998). Based on these studies at least two different types of octopamine receptors exist in the insect CNS: (1) OCTOPAMINE1 receptor inhibits myogenic rhythm through an increase in calcium levels and (2) OCTOPAMINE2 receptor modulates neuromuscular transmission by increasing the level of adenylate cyclase activity (Evans, 1993).

Recently, molecular cloning has identified two octopamine receptors in fruitfly. One of them was designated as a tyramine/octopamine receptor which has higher affinity for tyramine than octopamine in inhibiting adenylyl cyclase activity (Robb et al., 1994). Another octopamine receptor was designated as OAMB, which is highly expressed in the MB of the brain and has higher efficacy for octopamine than tyramine in activating cAMP accumulation (Han et al., 1998). Two alternatively spliced forms of octopamine receptor sequences have also been published from fruitfly (NCBI accession no: splice variant 1A, AJ007618; splice variant 1B, AJ007617).

An octopamine receptor from honeybee brain (AmOA1¹) has been recently isolated and characterized (Grohmann et al., 2003¹). AmOA1 encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. The AmOA1 gene is abundantly expressed in many somata of the honeybee brain, which suggests its involvement in the processing of sensory inputs, antennal motor outputs and high-order brain functions. Furthermore, its expression in the cells of the MB, AL, and optic lobes (OL) suggests its involvement in the processing of olfactory and visual information (Grohmann et al., 2003).

We have previously reported disruption of AmOA1 in the AL of the honeybee brain by blocking the receptor with mianserin and silencing specific gene expression by RNAi (Farooqui et al., 2003). Application of AmOA1–dsRNA in the AL produced 40% reduction in receptor (~78 kDa) protein expression in dsRNA-treated subjects (Farooqui et al., 2003). Both treatments inhibited olfactory acquisition and recall but did not affect odor discrimination. Thus, octopamine mediates consolidation of a component of olfactory memory at this early processing stage in the AL. Furthermore, after consolidation OA release becomes essential for recall, suggesting that the modulatory circuits become incorporated as essential components of neural representations that activate odor memory (Farooqui et al., 2003).

Our current results extend our earlier study by showing that our anti-AmOA1 antiserum recognizes four protein bands (48, 60, 72, and 78 kDa) in the brain. The 78 kDa band, which we reported earlier (Farooqui et al., 2003), as well as another smaller band (72 kDa) are the only ones expressed in the AL. The intensity of all four bands is reduced by AmOA1–dsRNA, and this reduction is a function of time and distance from a localized site of dsRNA treatment in the AL.