The distribution of dopamine in the brain and suboesophageal ganglion of the honeybee Apis mellifera was investigated by means of immunocytochemistry with a well-characterized antiserum against dopamine. The binding of the antiserum in paraffin serial sections was studied with the peroxidase-antiperoxidase method. Dopamine-like immunoreactive neurons are present in most parts of the brain and in the suboesophageal ganglion. Only the optic lobes are devoid of label. There are ca. 330 dopamine immunoreactive somata in each brain hemisphere plus respective suboesophageal hemiganglion, which is less than 0.1% of the entire neuronal population. Most of the labelled somata are situated within three clusters: one below the lateral calyx and two in the anterior-ventral protocerebrum. Other labelled somata lie dispersed or in small groups around the protocerebral bridge, below the optic tubercles, proximal to the ventral rim of the lobula, and in the lateral and ventral somatal rind of the suboesophageal ganglion. Similar to neurons that react with an antiserum against serotonin, the fine processes of dopamine immunoreactive fibers have a varicose appearance which is typical for aminergic neurons. In addition to the neuronal staining, dopamine-like immunoreactivity is also present in the sheath surrounding the brain and in the retina, where it is not restricted to any particular cell type. A detailed account is given for those neurons and groups of neurons that could be traced and reconstructed in some detail. A common feature of all dopamine immunoreactive fibers is that each fiber invades large volumes of neuropil, suggesting that dopamine is more important in mediating distant rather than local neural interactions.

The dynamic nature of neuronal growth cone filopodia led to the suggestion that the primary function of filopodia is to sample their immediate environment, responding to and transducing environmental signals that affect growth cone behaviour and shape. Filopodia seem well suited to serve as antenna-like sensors, their broad span allows sampling of information over a greatly enhanced radius, and forward-projecting filopodia encounter potential cues in the molecular terrain long before the advancing growth cone itself. Filopodia in culture can serve structural roles, exert mechanical tension and selectively adhere to their surrounding. Whether or not filopodia have a general sensory role has not been tested directly, largely because of their small size, which limits an electrophysiological approach, and their integral relationship with the parent growth cone, which prevents resolution of their different functions. Here we use surgical procedures to isolate individual filopodia from their parent growth cone and, by monitoring their morphology and calcium second messenger systems, we show that neuronal growth cone filopodia contain signal transduction mechanisms that allow autonomous responses and the transmission of distant environmental information to their parent growth cone.

Filopodia have been regarded as the sensory extensions of neuronal growth cones. As such, filopodia assay distant environments and are important for directing growth cones toward their targets. Since the territory encountered by a growth cone depends on the area spanned by the filopodia, changes in filopodial length or number result in the "exploration" of different-sized regions of the environment. The present study tests the potential regulatory role of intracellular calcium levels ([Ca2+]i) on filopodial morphology in identified neurons from the snail Helisoma. Experimentally evoked changes in [Ca2+]i were measured with the fluorescent calcium indicator fura-2 and directly correlated with growth cone filopodial morphology. A rise in [Ca2+]i caused two distinct, concentration-dependent effects separable by their different time courses: within the first 10 min, filopodia underwent significant elongation, while the second phase was characterized by a massive loss of filopodia. Both of these behaviors were increased in a calcium-dependent fashion. The magnitude of both filopodial elongation and filopodial loss correlated well with the transient peak values of [Ca2+]i reached during a given experimental treatment (r less than or equal to 0.98). In addition to the direct effect of the initial transient rise in [Ca2+]i, there is evidence for a form of adaptation of filopodial behavior to sustained calcium levels. A transient change in [Ca2+]i of as little as 30-50 nM reliably altered filopodial morphology. These results indicate that even small changes in intrinsic calcium homeostatic properties or extrinsic signals that alter intracellular calcium levels can act as regulators of the size of the environment sampled by an elongating growth cone.

The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT 2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT 2␤Pan , couples with the traditional G q pathway when expressed in HEK293 cells, but not to G s or G i/o . Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.

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