The organization of putative octopaminergic pathways in the brain and subesophageal ganglion of the honeybee was investigated with a well-defined polyclonal antiserum against octopamine. Five prominent groups of just over 100 immunoreactive (IR) somata were found in the cerebral ganglion: Neurosecretory cells in the pars intercerebralis innervating the corpora cardiaca via NCC I, one cluster mediodorsal to the antennal lobe, one scattered on both sides of the midline of the protocerebrum, one between the lateral protocerebral lobes and the dorsal lobes, and a single soma on either side of the central body. With the exception of the pedunculi and beta-lobes of the mushroom bodies, varicose immunoreactive fibers penetrate all parts of the cerebral ganglion. Strong labelling was found in the central complex and the protocerebral bridge. Fine networks of labelled processes invade the antennal lobes, the calyces and a small part of the alpha-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia. In the subesophageal ganglion, one labelled cell body was found in the lateral soma layer of the mandibular segment. Each of the three neuromeres contains a group of six to ten somata in the ventral median parts. Most of the ventral median cells send their neurites dorsally through the midline tracts, whereas the neurites of a few cells follow the ventral cell body neurite tracts. Octopamine-IR was demonstrated in all neuropils that contain pathways for proboscis extension learning in honeybees. Because octopaminergic mechanisms seem to be involved in the behavioral plasticity of the proboscis extension reflex, our study provides anatomical data on the neurochemical organization of an appetitive learning paradigm.

The distribution of GABA-like immunoreactivity in the brain of the honeybee was investigated with antisera generated against GABA protein conjugates. The binding of the antisera in paraffin serial sections was studied with the peroxidase-antiperoxidase method. GABA-like immunoreactive fibers appeared in all main neuropile areas. The staining of the optic lobes showed pronounced stratification. The receptor cells of compound eyes, ocelli, and antennae were not labelled. Several prominent fiber tracts showed GABA-like immunoreactivity, whereas other tracts were devoid of staining. There are no major immunoreactive commissures linking the two brain hemispheres with the exception of small commissures that bridge short distances between the beta-lobes and the antennal lobes. Several fibers in the cervical connective were also labelled; some of those may descend from the suboesophageal ganglion to the thoracic ganglia. The dense reactivity seen in the optic and antennal neuropiles implies that GABA is more important in mediating local rather than more distant neural interactions.

1. The proboscis extension reflex is an appetitive component of the bee's feeding behavior that is elicited by touching one antenna with a droplet of sugar water. Repetitive stimulation leads to a decrement and finally to the disappearance of the response, which can be restored by stimulating the contralateral antenna. This behavioral plasticity conforms to essential parametric characteristics for habituation. 2. The response was quantified by recording extracellularly from a muscle involved in proboscis movement, by measuring the duration of the proboscis extension, or by determining the number of trials necessary to abolish any visible response. 3. Because habituation was restricted to the repetitively stimulated antenna and did not generalize to the contralateral hemisphere, the neural circuits mediating habituation may be confined to one hemisphere. 4. State dependence of habituation could be demonstrated by showing that hungry animals exhibited a smaller response decrement and required more trials until disappearance of the response compared with satiated animals. The initial response and the subsequent response decrement are separate components determined by satiation level and stimulus strength. 5. Depleting the nervous system of monoamines by the use of reserpine abolished the reflex in 30% of the animals and reduced responsiveness in the remainder. Injection of octopamine or its metabolic precursor tyramine restored the reflex in reserpinized unresponsive animals, and tyramine also enhanced the muscle-spike discharge of reserpinized, responsive animals. In undepleted animals, tyramine application also accelerated the rate of habituation of the reflex. We therefore propose that octopaminergic neurons participate in mediating food arousal and the state dependence of habituation and, in a separate process, influence the response decrement during habituation. 6. Application of an acetylcholine esterase (AChE) inhibitor and a cholinergic receptor blocker confirmed histochemical data that implicate cholinergic transmission in the reflex pathways. 7. The combined pharmacological dissection of the reflex and immunocytochemical investigations of its chemical architecture provide evidence that the proboscis extension reflex is mediated by nonaminergic and monoaminergic pathways operating in parallel.

Neuromodulators and hormones elicit and modify well-defined behaviours. Their mode of action can be studied to advantage in arthropods, where the natural releasing cells and neuronal target circuits are concisely identified. The coordinated actions of biogenic amines and peptides on both central and peripheral neural activity and metabolic processes bias the whole organism to perform a coherent behavioural routine.