PDF responses of type 1 pacemakers are mediated by cAMP-dependent pathways.

<p>In addition, PDF effects appear to depend on intracellular Ca²⁺ baseline levels and thus, most likely on the membrane potential. <b>A</b>. Adenylyl cyclase (AC) inhibitor SQ22536 (20 µM) blocks forskolin (AC activator, 10 µM) -dependent rise of the Ca²⁺ baseline and Ca²⁺ activity in this continuous recording of type 1 AMe cells. <b>B</b>. Also, SQ22536 (20 µM) reversibly blocks the PDF-induced increase of the Ca²⁺ baseline and Ca²⁺ activity in type 1 cells. <b>C</b>. Application of 8-bromo cAMP (10 µM) produces rapid and large increases in Ca²⁺ levels, which were not blocked by SQ22536 (20 µM). <b>D</b>. Incubation of the Ca²⁺ channel antagonist mibefradil (10 µM) inhibits spontaneous activity and decreases Ca²⁺ baseline levels apparently causing membrane potential hyperpolarizations. Mibefradil-dependent effects prevent PDF (100 µM) responses in type 1 AMe neurons.</p

Hypothetical model of PDF signaling in spontaneously active type 1 circadian pacemaker neurons.

<p><b>A</b>. We suggest that PDF signals via adenylyl cylcase activity in type 1 cells. The cAMP-dependent block of K⁺ channels depolarizes the cell and thereby opens voltage-gated Ca²⁺ channels. The resulting rise in intracellular Ca²⁺ together with the rise in cAMP concentrations then feeds back to the molecular clockwork (TTFL) via activation of PKA and PKC, thereby phase-advancing circadian rhythms of circadian pacemaker neurons. <b>B</b>. In contrast, PDF-dependent block of Na⁺ channels hyperpolarizes and thereby phase-delays pacemaker neurons only via PKA, but not concomitant PKC-dependent feedback to the TTFL. <b>C</b>. When PDF blocks both K⁺ and Na⁺ channels it promotes rhythmic membrane potential oscillations and causes bursting. <b>D</b>. Finally, PDF promotes fast synchronization between two pacemakers, which are coupled via their common PDF-sensitivity. If the PDF releasing pacemaker is also PDF-sensitive, because of autoreceptor expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108757#pone.0108757-Choi2" target="_blank">[48]</a> it will synchronize with the postsynaptic PDF-sensitive pacemaker neuron. PDF-dependent rhythmic bursting is suggested to promote fast synchronization.</p

Application of PDF blocks outward K⁺ and inward Na⁺ current components.

<p>In whole-cell patch clamp recordings AMe neurons in primary cell cultures were kept at a holding potential of −60 mV. Voltage-dependent currents were activated by series of depolarizing voltage steps from −140 mV to +80 mV with 10 mV increments. <b>A₁–A₂</b>. Current traces before and after application of 500 nM PDF (2 min) to the extracellular solution indicate that PDF inhibits part of a delayed rectifier type potassium (K⁺) outward current. <b>B</b>. The I-V plot of the same recording at the position indicated by the arrows in <b>A₁ and A₂</b> shows the decline of outward currents while sustained, small inward currents, which counteract outward K⁺ currents at hyperpolarized potentials, are not affected. <b>C</b>. In another recording PDF blocks Ca²⁺-dependent outward K⁺ currents, which cause the characteristic downward bend of the outward currents, while apparently not affecting delayed rectifier type K⁺ currents, or small sustained inward Ca²⁺ currents which counteract K⁺ outward currents at hyperpolarizing potentials. <b>D</b>. In another AMe neuron PDF inhibits voltage-gated fast Na⁺ inward currents that activate around −40 mV. Washing in of PDF-free saline containing the Na⁺ channel antagonist tetrodotoxin (TTX) almost competely blocks the residual inward current.</p

The spontaneous, regular activity of type 1 neurons depends on low voltage-activated (LVA) Ca²⁺ channels and on hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker currents.

<p><b>A</b>. Type 1 AMe neurons express regular Ca²⁺ transients, which can be blocked by the voltage-dependent Ca²⁺ channel antagonist mibefradil (10 µM), the HCN-channel antagonist DK-AH 269 (10 µM; which also reduces the baseline), and the Na⁺ channel antagonist TTX (100 nM). Consecutive recordings during constant perfusion of the same pacemaker neuron reveal that TEA-dependent block of K⁺ channels does not mimic all PDF effects in type 1 cells. <b>B</b>. Type 1 pacemakers increase baseline Ca²⁺ levels, amplitude, and frequency of Ca²⁺ transients with increasing concentrations (1–20 mM) of the K⁺ channel blocker TEA. Coapplication of the Na⁺ channel antagonist TTX (50 nM) increases the speed of TEA effects while truncating their durations, favoring burst-activity. <b>C</b>. Increasing the concentration of TTX (10–100 nM) finally blocks spontaneous activity and shortens TEA responses to a brief burst.</p

Application of PDF to accessory medulla (AMe) pacemakers in primary cell culture allows to distinguish 4 different response types 1–4 in Ca²⁺ imaging experiments.

<p><b>A</b>. Type 1 AMe neurons express regular Ca²⁺ transients. PDF increases the frequency of spontaneous Ca²⁺ transients and the Ca²⁺ baseline dose-dependently and reversibly. <b>B</b>. Type 2 AMe neurons are not spontaneously active and have low baseline Ca²⁺ levels. PDF slowly increases Ca²⁺ baseline levels. <b>C</b>. Type 3 cells are less regularly spontaneously active than type 1 cells. PDF application slightly increases baseline Ca²⁺ levels while suppressing high amplitude Ca²⁺ transients. <b>D</b>. Type 4 AMe neurons have a high Ca²⁺ baseline level which is decreased by PDF application.</p