Cell culture experiments have been used to examine the effects of serotonin [5‐hydroxytryptamine (5‐HT)] on the morphological development of antennal lobe (AL) neurons in the brain of the sphinx moth, Manduca sexta. The majority of cells used in this study were from animals at stage 5 of the 18 stages of metamorphic adult development. 5‐HT did not affect the survival of M. sexta AL neurons in culture, but did increase the numbers of cells displaying features characteristic of certain cell types. Three morphologically distinct cell types were examined in detail. The principal effect of 5‐HT on these neurons was enhancement of cell growth. The magnitude of responses to this amine was cell‐type specific. Site‐specific responses to 5‐HT were apparent also in one cell type. Our results suggest that the effects of 5‐HT can change during the course of metamorphic development. These changes coincide temporally with the development of fast, sodium‐based action potentials. © 1996 John Wiley & Sons, Inc.
Voltage-activated currents from adult honey bee antennal motor neurons were characterized with in vitro studies in parallel with recordings taken from cells in situ. Two methods were used to ensure unequivocal identification of cells as antennal motor neurons: 1) selective backfilling of the neurons with fluorescent markers before dissociation for cell culture or before recording from cells in intact brains, semiintact brains, or in brain slices or 2) staining with a fluorescent marker via the patch pipette during recordings and identifying antennal motor neurons in situ on the basis of their characteristic morphology. Four voltage-activated currents were isolated in these antennal motor neurons with pharmacological, voltage, and ion substitution protocols. The neurons expressed at least two distinct K+ currents, a transient current (IA) that was blocked by 4-aminopyridine (4-5 x 10(-3) M), and a sustained current (IK(V)) that was partially blocked by tetraethylammonium (2-3 x 10(-2) M) and quinidine (5 x 10(-5) M). IA activated above -40 to -30 mV and the half-maximal voltages for steady-state activation and inactivation were -8.8 and -43.2 mV, respectively. IK(V) activated above -50 to -40 mV and the midpoint of the steady-state activation curve was +11.2 mV. IK(V) did not show steady-state inactivation. Additionally, two inward currents were isolated: a tetrodotoxin (10(-7) M)-sensitive, transient Na+ current (INa) that activated above -35 mV, with a maximum around -5 mV and a half-maximal voltage for inactivation of -72.6 mV, and a CdCl2 (5 x 10(-5) M)-sensitive Ca2+ current that activated above -45 to -40 mV, with a maximum around -15 mV. This study represents the first step in our effort to analyze the cellular and ionic mechanisms underlying the intrinsic properties and plasticity of antennal motor neurons.
We developed a cell culture system for thoracic neurons of fifth instar or adult locusts (Locusta migratoria) in order to obtain maximum visualization of cellular morphology and direct access to the neurons for electrophysiological analysis. The dissociated neurons regenerated new neurites in a serum-free defined culture medium, in which they remained viable for up to 3 weeks. Viability of the cells was confirmed by intracellular recordings demonstrating active membrane properties and action potentials. While the morphology of the cultured neurons is distinct from their in vivo counterparts, they retained some cellular surface properties and markers related to transmitter metabolism. Two factors influencing cellular morphology in vitro were identified in Locusta: 1) the presence of a primary neurite stump, and 2) membrane contacts between cells. Dissociated neurons of the locust species Schistocerca gregaria grown in a hemolymph-enriched medium showed a marked reduction in branching patterns and a tenfold increase in neurite length compared to neurons growing in a medium without hemolymph. This culture system could prove useful for identifying the action of hemolymph-derived growth factors.
We have used the D2-specific dopamine receptor ligand spiperone [N-(p-aminophenethyl) spiperone; NAPS] coupled to the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) to visualize dopamine receptors expressed in vitro by neurons of the primary antennosensory centers (antennal lobes) of the brain of the honey bee, Apis mellifera. Changes in the percentage of antennal lobe neurons exhibiting spiperone binding sites over time in culture and at different stages of metamorphic adult development have been investigated. Neurons obtained from animals at all stages of development exhibited spiperone binding sites, but only after 2 days or more in vitro. The percentage of antennal lobe neurons in vitro expressing spiperone binding sites increased significantly with the development of the antennal lobe neuropil. Fluorescently labelled spiperone (120 nM) could be displaced effectively by 1 mM dopamine but not by the same concentration of tyramine, octopamine, or serotonin. In addition, the D2 antagonist spiperone and the D2/D1 antagonist fluphenazine were more effective at displacing the fluorescent ligand than the D1-specific antagonist SCH23390. Our results indicate that Apis antennal lobe neurons in culture express a dopamine receptor and that this receptor is more likely to be D2-like than D1-like in nature. The receptor is expressed early in the metamorphic adult development of the antennal lobe neuropil of the brain.
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