Cultured Insect Mushroom Body Neurons Express Functional Receptors for Acetylcholine, GABA, Glutamate, Octopamine, and Dopamine

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Abstract
Cayre, M., S. D. Buckingham, S. Yagodin, and D. B. Sattelle. Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine. J. Neurophysiol. 81: 1–14, 1999. Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 μM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 μM nicotine. The ACh response was insensitive to atropine (50 μM) but was reduced by mecamylamine (50 μM) and α-bungarotoxin (α-bgt, 10 μM). ACh-induced inward ion currents (n = 28, EACh ∼0 mV) were also blocked by 1 μM mecamylamine and by 1 μM α-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional α-bgt–sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. γ-Aminobutyric acid (GABA, 100 μM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 μM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30–100 μM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to l-glutamate (100 μM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 μM) l-glutamate with rapidly desensitizing, inward currents that reversed at approximately −30 mV. Dopamine (100 μM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 μM) in ∼54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).