FMRFamide modulates potassium currents in circadian pacemaker neurons of Bulla gouldiana

doi:10.1016/S0306-4522(01)00566-8

Neuroscience

Volume 110, Issue 1, 1 March 2002, Pages 181-190

https://doi.org/10.1016/S0306-4522(01)00566-8 Get rights and content

Abstract

The peptide FMRFamide (Phe-Met-Arg-Phe-NH₂) is known to modulate the circadian pacemaker found in the eye of the marine snail Bulla gouldiana. In the present study, we investigated the cellular mechanisms underlying this modulation by examining the effects of FMRFamide on the membrane properties of the circadian pacemaker cells, known as basal retinal neurons in this preparation. Bath application of FMRFamide (0.1–1 μM) increased the membrane conductance, and hyperpolarized the membrane potential of these neurons. Next, perforated-patch recordings were used to demonstrate that FMRFamide reversibly increased the outward current amplitude due to an augmentation of a non-inactivating calcium-independent current. Reversal potential of the tail currents and its dependence on extracellular potassium concentration suggested potassium ions as the charge carrier for this current. The peptide-modulated outward current was blocked by 54% after bath application of the potassium channel blocker tetraethylammonium chloride and completely blocked by substituting cesium for intracellular potassium. Voltage dependence, activation kinetics and tail current kinetics of the FMRFamide-modulated current were consistent with values found for the delayed rectifier current.

Overall, our data suggest that FMRFamide modulates a delayed rectifier potassium current and at least one other, less voltage-dependent conductance. This provides a mechanistic explanation for FMRFamide’s ability to both shift the phase and attenuate light-induced phase shifts of the circadian pacemaker in B. gouldiana.

Section snippets

Experimental procedures

B. gouldiana were obtained from a commercial supplier (Marinus Inc., Long Beach, USA) and maintained in seawater tanks (15°C, light 12 h:dark 12 h) for at least 7 days prior to experiments. During the subjective day animals were immobilized by injecting 10 ml isotonic MgCl₂ and then dissected on ice. All recordings were performed under dark-room red lighting.

Intracellular recordings of pacemaker cells in the semi-intact retina preparation were performed as previously described (McMahon and

Modulation of passive membrane properties

During the subjective day the membrane potential of BRNs in the semi-intact retina is compared to the subjective night relatively depolarized and these neurons generate spontaneous action potentials (McMahon and Block, 1984). Bath application of the peptide FMRFamide (0.1–1 μM) led to a hyperpolarization of the BRN membrane potential (V_m) from −54 mV to −66 mV (mean ΔV_m=12.8±5 mV; n=6) and a subsequent termination of their spontaneous activity (Fig. 1A). After washout of the peptide, the

Discussion

The peptide FMRFamide is known to modulate the circadian pacemaker found in the eye of the marine snail B. gouldiana (Colwell et al., 1992a). In the current study, we investigated the mechanisms underlying this modulation by examining the effects of FMRFamide on membrane properties of circadian pacemaker cells (BRNs) in the retina and in culture. We found that in both, the retina and cultured BRNs, the peptide increased a conductance, which led to a hyperpolarization of the cells’ resting

Acknowledgements

We thank Dr. C.S. Colwell for helpful comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG-Mi 328/2-2).

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Citation Excerpt :
Application of FMRF-amide to the eye results in a decrease of CAP activity, induces phase shifts in the CAP rhythm and modulates light-induced phase shifts (Jacklet et al., 1987; Colwell et al., 1992). FMRF-amide affects the ocular circadian system by modulation of a delayed rectifier potassium current and a second, yet unidentified, conductance, thereby regulating the excitability of the basal retinal neurons (Michel et al., 2002). These data indicate that efferent modulation of the Bulla pacemaker may involve the action of FMRF-amide.
In most animal species, a circadian timing system has evolved as a strategy to cope with 24-hour rhythms in the environment. Circadian pacemakers are essential elements of the timing system and have been identified in anatomically discrete locations in animals ranging from insects to mammals. Rhythm generation occurs in single pacemaker neurons and is based on the interacting negative and positive molecular feedback loops. Rhythmicity in behavior and physiology is regulated by neuronal networks in which synchronization or coupling is required to produce coherent output signals. Coupling occurs among individual clock cells within an oscillating tissue, among functionally distinct subregions within the pacemaker, and between central pacemakers and the periphery. Recent evidence indicates that peripheral tissues can influence central pacemakers and contain autonomous circadian oscillators that contribute to the regulation of overt rhythmicity. The data discussed in this review describe coupling and synchronization mechanisms at the cell and tissue levels. By comparing the pacemaker systems of several multicellular animal species (Drosophila, cockroaches, crickets, snails, zebrafish and mammals), we will explore general organizational principles by which the circadian system regulates a 24-hour rhythmicity.
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FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure–function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K⁺ channels in the same tissue. The use of K⁺ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.
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FMRFamide modulates potassium currents in circadian pacemaker neurons of Bulla gouldiana

Abstract

Section snippets

Experimental procedures

Modulation of passive membrane properties

Discussion

Acknowledgements

Neurosci. Lett.

Neurosci. Biobehav. Rev.

Brain Res.

Brain Res.

Brain Res.

Ionic currents of isolated retinal pacemaker neurons: projected daily phase differences and selective enhancement by a phase-shifting neurotransmitter

J. Neurophys.

Neuronal inhibition by the peptide FMRFamide involves opening of S-K+ channels

Nature

FMRFamide produces biphasic modulation of the LFS motor neurons in the neural circuit of the siphon withdrawal reflex of Aplysia by activating Na+ and K+ currents

J. Neurosci.

Unwinding the snails clock: Cellular analysis of a retinal circadian pacemaker

Neth. J. Zool.

Localization of a circadian pacemaker in the eye of a mollusc, Bulla

Science

Suppression of calcium current by an endogenous neuropeptide in neurones of Aplysia californica

J. Physiol.

The neuropeptide FMRFamide decreases both the Ca2+ conductance and a cyclic 3′,5′-adenosine monophosphate-dependent K+ conductance in identified molluscan neurons

J. Neurosci.

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

J. Comp. Physiol. A

Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia

J. Comp. Physiol. A

Modulatory effects of serotonin, FMRFamide, and myomodulin on the duration of action potentials, excitability, and membrane currents in tail sensory neurons of Aplysia

J. Neurophys.

Neuronal inhibition by the peptide FMRFamide involves opening of S-K⁺ channels

FMRFamide produces biphasic modulation of the LFS motor neurons in the neural circuit of the siphon withdrawal reflex of Aplysia by activating Na⁺ and K⁺ currents

The neuropeptide FMRFamide decreases both the Ca²⁺ conductance and a cyclic 3′,5′-adenosine monophosphate-dependent K⁺ conductance in identified molluscan neurons