antennal lobe PNs Drosophila calcium currents that regulate repetitive firing in 1T calcium channels mediate transient a 3-type v Ca

: Projection neurons (PNs), located in the antennal lobe region of the insect brain, play a key role in processing olfactory information. To explore how activity is regulated at the level of single PNs within this central circuit we have recorded from these neurons in adult Drosophila melanogaster brains. Our previous study demonstrated that PNs express voltage-gated calcium currents with a transient and sustained component. We found that the sustained component is mediated by cac gene-encoded Cav2-type channels involved in regulating action potential-independent release of neurotransmitter at excitatory cholinergic synapses. The function of the transient calcium current and the gene encoding the underlying channels, however, were unknown. Here we report that the transient current blocked by prepulse inactivation is sensitive to amiloride, a vertebrate Cav3-type channel blocker. In addition PN-specific RNAi knockdown of α1T, the Drosophila Cav3-type gene, caused a dramatic reduction in the transient current without altering the sustained component. These data demonstrate that the α1T gene encodes voltage-gated calcium channels underlying the amiloride-sensitive transient current. Alterations in evoked firing and spontaneous burst firing in the α1T knockdowns demonstrate that the Cav3-type calcium channels are important in regulating excitability in adult PNs. Copyright Ca v 3-type (cid:1) 1T calcium channels mediate transient calcium currents that regulate repetitive ﬁring in Drosophila antennal lobe PNs. J 1490–1496, 2013. neurons (PNs), located in the antennal lobe region of the insect brain, play a key role in processing olfactory information. To explore how activity is regulated at the level of single PNs within this central circuit we have recorded from these neurons in adult Drosophila melanogaster brains. Our previous study demonstrated that PNs express voltage-gated calcium currents with a transient and sustained component. We found that the sustained component is mediated by cac gene-encoded Ca v 2-type channels involved in regulating action potential-independent release of neurotransmitter at excitatory cholinergic synapses. The function of the transient calcium current and the gene encoding the underlying channels, however, were unknown. Here we report that the transient current blocked by prepulse inacti- vation is sensitive to amiloride, a vertebrate Ca v 3-type channel blocker. In addition PN-speciﬁc RNAi knockdown of (cid:1) 1T, the Drosophila Ca v 3-type gene, caused a dramatic reduction in the transient current without altering the sustained component. These data demonstrate that the (cid:1) 1T gene encodes voltage-gated calcium channels underlying the amiloride-sensitive transient current. Alterations in evoked ﬁring and spontaneous burst ﬁring in the (cid:1) 1T knockdowns demonstrate that the Ca v 3-type calcium channels are , 5.4 mM 5 mM 1.25 mM NaH 2 PO 4 , and 20.7 mM NaHCO 3 . were added to block synaptic transmission, sodium current, and potassium currents: 20 (cid:2) M D -tubocurarine (Sigma), 10 (cid:2) M picrotoxin (Sigma), 1 (cid:2) M tetrodotoxin (Alomone), 2.5 mM TEA (Sigma), and 1.0 mM 4-aminopyridine (Sigma). The osmolarity was adjusted to 250 mosM, and the solution was saturated with 95% oxygen-5% CO 2 . For experiments evaluating the effect of amiloride (Sigma), the drug was made up fresh each day by dissolving in H 2 O. This was added to the extracellular solution for a ﬁnal concentration of 1 mM. Firing properties were recorded in current-clamp mode with internal solution as described above except that cesium gluconate was replaced by potassium gluconate. Intrinsic ﬁring properties, both evoked and spontaneous, were evaluated in external solution that contained picrotoxin and D -tubocurarine to block synaptic transmission. All recordings were performed during continuous perfusion with oxygenated saline at room temperature.