SUMMARY Pheromone-dependent mate search is under strict circadian control in different moth species. But it remains unknown whether daytime-dependent changes in pheromone sensitivity already occur at the periphery in male moths. Because adapting pheromone stimuli cause rises of cyclic guanosine monophosphate (cGMP) in pheromone-sensitive trichoid sensilla of the night-active hawkmoth Manduca sexta, we wanted to determine whether cGMP decreases pheromone-sensitivity of olfactory receptor neurons in a daytime-dependent manner. Long-term tip recordings from trichoid sensilla were performed at the early day (ZT 1-4), when many moths are still active, and at the middle of the day (ZT 8-11), when moths are resting. A non-adapting pheromone-stimulation protocol combined with perfusion of the sensillum lymph with the membrane-permeable cGMP analogue 8bcGMP adapted the action potential response but not the sensillar potential. Perfusion with 8bcGMP decreased the initial action potential frequency, decreased the numbers of action potentials elicited in the first 100 ms of the pheromone response and attenuated the reduction of action potential amplitude. Furthermore, the decrease in 8bcGMP-dependent action potential frequency was stronger in recordings made at ZT 8-11 than at ZT 1-4. In the control recordings during the course of the day the pheromone responses became increasingly tonic and less phasic. At ZT 8-11 only, this daytime-dependent effect was further enhanced by 8bcGMP application. Thus we hypothesize that during the moths' resting phase,elevated cGMP levels underlie a daytime-dependent decrease in pheromone sensitivity and a decline in the temporal resolution of pheromone pulses.

In the hawkmoth Manduca sexta, pheromone stimuli of different strength and duration rise the intracellular Ca2+ concentration in olfactory receptor neurons (ORNs). While second-long pheromone stimuli activate protein kinase C (PKC), which apparently underlies processes of short-term adaptation, minute-long pheromone stimuli elevate cyclic guanosine monophosphate (cGMP) concentrations, which correlates with time courses of long-term adaptation. To identify ion channels involved in the sliding adjustment of olfactory sensitivity, inside-out patch clamp recordings on cultured ORNs of M. sexta were performed to characterize Ca2+-, PKC-, and cGMP-dependent ion channels. Stepping to positive holding potentials in high intracellular Ca2+ elicits different Ca2+-dependent ion channels, namely small-conductance channels (2–20 ps), medium-conductance channels (20–100 ps), and large-conductance channels (>100 ps). Ion channels of 40, 60, and 70 ps opened after PKC activation, whereas 10- and >100-ps channels were observed less frequently. Application of 8-bromo cyclic guanosine monophosphate opened 55- and 70-ps channels and increased the open probability of >100-ps channels, whereas even in the presence of phorbol ester 40-ps channels were inhibited. Thus, cGMP elevations activate a different set of ion channels as compared with PKC and suppress at least one PKC-dependent ion channel.

Krannich S, Stengl M. Cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the hawkmoth Manduca sexta. J Neurophysiol 100: 2866 -2877, 2008. First published August 6, 2008 doi:10.1152/jn.01400.2007. Moth pheromones cause rises in intracellular Ca 2ϩ concentrations that activate Ca 2ϩ -dependent cation channels in antennal olfactory receptor neurons. In addition, mechanisms of adaptation and sensitization depend on changes in cyclic nucleotide concentrations. Here, cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the moth Manduca sexta are described, which share properties with currents through vertebrate cyclic nucleotide-gated channels. The cyclic nucleotide-activated currents of M. sexta carried Ca 2ϩ and monovalent cations. They were directly activated by cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), modulated by Ca 2ϩ /calmodulin, and inhibited by lanthanum. M. sexta cyclic nucleotide-activated currents developed in an all-or-none manner, which suggests that the underlying channels are coupled and act coordinately. At least one cAMP-and two cGMP-activated nonselective cation currents could be distinguished. Compared with the cAMP-activated current, both cGMP-activated currents appeared to conduct more Ca 2ϩ and showed a stronger down-regulation by Ca 2ϩ /calmodulin-dependent negative feedback. Furthermore, both cGMP-activated currents differed in their Ca 2ϩ -dependent inhibition. Thus M. sexta olfactory receptor neurons, like vertebrate sensory neurons, appear to express nonselective cyclic nucleotide-activated cation channels with different subunit compositions. Besides the nonselective cyclic nucleotide-activated cation currents, olfactory receptor neurons express a cAMP-dependent current. This current resembled a protein kinase-modulated low-voltageactivated Ca 2ϩ current.

SUMMARY The biogenic amine octopamine is known to enhance the sensitivity of male moths to their species-specific pheromones in flight-tunnel experiments. This sensitization of pheromone-guided upwind flight is at least partly due to octopamine-dependent increases in the peak nerve impulse frequency of the pheromone response of olfactory receptor neurons. It is not known, however,whether octopamine exerts its effects directly on the electrical properties of the olfactory receptor neurons or indirectly, via modulation of the accessory cells of the sensillum. In extracellular tip recordings of pheromone-dependent trichoid sensilla on the antennae of male Manduca sexta moths, we investigated the effects of octopamine and serotonin on the transepithelial potential, which is generated by the activity of V-ATPases in sensillar accessory cells. In addition, the action potential activity of unstimulated olfactory receptor neurons was examined in the presence of biogenic amines. Under constant environmental conditions, the transepithelial potential oscillated regularly with periods of 2-8 min and with a 1-25 mV peak-to-peak amplitude over periods of several hours. These oscillatory intervals were interrupted by periods of relatively stable transepithelial potential, correlated with flight activity by the moth. Octopamine reduced the amplitude of the transepithelial potential oscillation and decreased the resistance of the sensillum preparation in a dose-dependent manner. Serotonin altered the waveform of the transepithelial potential, but did not change the resistance of the preparation. Thus, both amines affect the accessory cells, but have different targets in the regulation of the transepithelial potential. Neither amine significantly influenced the spontaneous action potential activity of the olfactory receptor neurons.