Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull-push sensitivity modulation.

Olfactory receptor neurons (ORNs) of the hawkmoth Manduca sexta sensitize via cAMP- and adapt via cGMP-dependent mechanisms. Perforated patch clamp recordings distinguished 11 currents in these ORNs. Derivatives of cAMP and/or cGMP antagonistically affected three of five K+ currents and two non-specific cation currents. The Ca2+ -dependent K+ current IK(Ca2+) and the sensitive pheromone-dependent K+ current IK(cGMP-) , which both express fast kinetics, were inhibited by 8bcGMP, while a slow K+ current, IK(cGMP+) , was activated by 8bcGMP. Furthermore, application of 8bcAMP blocked slowly activating, zero mV-reversing, non-specific cation currents, ILL and Icat(PKC?) , which remained activated in the presence of 8bcGMP. Their activations pull the membrane potential towards their 0-mV reversal potentials, in addition to increasing intracellular Ca2+ levels voltage- and ILL -dependently. Twenty minutes after application, 8bcGMP blocked a TEA-independent K+ current, IK(noTEA) , and a fast cation current, Icat(nRP) , which both shift the membrane potential to negative values. We conclude that conditions of sensitization are maintained at high levels of cAMP, via specific opening/closure of ion channels that allow for fast kinetics, hyperpolarized membrane potentials, and low intracellular Ca2+ levels. In contrast, adaptation is supported via cGMP, which antagonizes cAMP, opening Ca2+ -permeable channels with slow kinetics that stabilize depolarized resting potentials. The antagonistic modulation of peripheral sensory neurons by cAMP or cGMP is reminiscent of pull-push mechanisms of neuromodulation at central synapses underlying metaplasticity.

Patch Clamp Technology in the Twenty-First Century.

In the almost four decades since its inception, the patch clamp technique has transitioned from a specialist skill to a method commonly used among many others in a lab. Development of patch clamp instrumentation has not been steady: A boost of product releases in rapid succession by multiple manufacturers in the 1990s had slowed to a trickle by the mid-2000s. In 2016, Sutter Instrument's entry into the market of turnkey patch clamp amplifier systems, defined as an amplifier with matching data acquisition hardware and software, caused a fresh breeze in a field in danger of going stale. Sutter has meanwhile completed the product line, culminating in the flagship dPatch® Ultra-fast, Low-noise Digital Amplifier. The dPatch System constitutes a contemporary, digital design that features many firsts, including digital signal compensation, an extremely high bandwidth and fully integrated dynamic clamp capability, paired with the increasingly popular SutterPatch® Software.This chapter compares feature sets of the new Sutter instrumentation with the established platforms by the other two providers of turnkey systems, Axon Instruments by Molecular Devices and HEKA Elektronik by Harvard Bioscience. A variety of products from other manufacturers, who rely on combination with components from other sources rather than offering turnkey systems, are listed, but for their conceptual diversity not compared at a great level of detail. The chapter further covers architectural considerations for patch clamp systems, headstage design, data acquisition strategies and efficient structuring of the recorded data, controlling and monitoring periphery, advanced technologies, such as software lock-in amplifier capability and dynamic clamp features, and application modules for efficient analysis of action potentials and postsynaptic events.

Pharmacological investigation of protein kinase C- and cGMP-dependent ion channels in cultured olfactory receptor neurons of the hawkmoth Manduca sexta.

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.

Perfusion with cGMP analogue adapts the action potential response of pheromone-sensitive sensilla trichoidea of the hawkmoth Manduca sexta in a daytime-dependent manner.

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.

Adaptation in pheromone-sensitive trichoid sensilla of the hawkmoth Manduca sexta.

In extracellular tip recordings from long trichoid sensilla of male Manduca sexta moths, we studied dose-response relationships in response to bombykal stimuli of two different durations in the adapted and the non-adapted state. Bombykal-responsive cells could be distinguished from non-bombykal-sensitive cells in each trichoid sensillum because the bombykal-responsive cell always generated the action potentials of larger initial amplitude. The bombykal cell, which was recorded at a defined location within a distal flagellar annulus, can resolve at least four log(10)-units of pheromone concentrations but is apparently unable to encode all stimulus durations tested. Parameters of the amplitude-modulated sensillar potential and the frequency-modulated action potential responses were examined in different states of adaptation. Evidence is presented for the existence of several mechanisms of adaptation, which affect distinct steps of the transduction cascade. After adapting pheromone stimuli, the sensillar potential rises to a lower amplitude and declines faster compared with the non-adapted response. In addition, the frequency of the adapted action potential response is reduced. Only the time of rise of the sensillar potential is differentially affected by adapting pheromone stimuli of different duration. The time of rise does not increase after short, but only after long, adapting stimuli. Both short and long adapting stimuli shift the dose-response curves of the sensillar potential amplitude, as well as the initial slope of its rising phase, to higher stimulus concentrations by approximately one log(10)-unit. The shift in the dose-response curve of the action potential response is larger than for the sensillar potential response, suggesting that an additional adaptation mechanism acts at the level of action potential generation. Furthermore, a faster decline of the sensillar potential after short and long adapting stimuli suggests that the resting potential of the olfactory receptor neuron is stabilized.