FMRFamide-related peptide expression in the vestibular-afferent neurons.

Vestibular-afferent neurons innervate hair cells from the sensory epithelia of vestibular end-organs and their action-potential discharge dynamics are driven by linear and angular accelerations of the head. The electrical activity of the vestibular-afferent neurons depends on their intrinsic properties and on the synaptic input from hair cells and from the terminals of the efferent system. Here we report that vestibular-afferent neurons of the rat are immunoreactive to RFamide-related peptides, and that the stronger signal comes from calyx-shaped neuron dendrites, with no signal detected in hair cells or supporting cells. The whole-cell voltage clamp recording of isolated afferent neurons showed that they express robust acid-sensing ionic currents (ASICs). Extracellular multiunit recordings of the vestibular nerve in a preparation in vitro of the rat inner ear showed that the perfusion of FMRFamide (a snail ortholog of this family of neuropeptides) exerts an excitatory effect on the afferent-neurons spike-discharge rate. Because the FMRFamide cannot activate the ASIC but reduces its desensitization generating a more robust current, its effect indicates that the ASIC are tonically active in the vestibular-afferent neurons and modulated by RFamide-like peptides.

Neuronal phenotype in the mature nervous system is maintained by persistent retrograde bone morphogenetic protein signaling.

The terminal differentiation of many developing neurons occurs after they innervate their target cells and is triggered by secreted target-derived signals that are transduced by presynaptic cognate receptors. Such retrograde signaling induces the expression of genes that are often distinctive markers of neuronal phenotype and function. However, whether long-term maintenance of neuronal phenotype requires persistent retrograde signaling remains poorly understood. Previously, we demonstrated that retrograde bone morphogenetic protein (BMP) signaling induces expression of a phenotypic marker of Drosophila Tv neurons, the neuropeptide FMRFamide (FMRFa). Here, we used a genetic technique that spatiotemporally targets transgene expression in Drosophila to test the role of persistent BMP signaling in the maintenance of Tv phenotype. We show that expression of dominant blockers of BMP signaling selectively in adult Tv neurons dramatically downregulated FMRFa expression. Moreover, adult-onset expression of mutant Glued, which blocks dynein/dynactin-mediated retrograde axonal transport, eliminated retrograde BMP signaling and dramatically downregulated FMRFa expression. Finally, we found that BMP deprivation did not affect Tv neuron survival and that FMRFa expression fully recovered to control levels after the termination of BMP blockade or Glued expression. Our results show that persistent retrograde BMP signaling is required to induce and to subsequently maintain the expression of a stably expressed phenotypic marker in a subset of mature Drosophila neurons. We postulate that retrograde maintenance of neuronal phenotype is conserved in vertebrates, and as a consequence, neuronal phenotype is likely vulnerable to neurodegenerative disease pathologies that disrupt neuronal connectivity or axonal transport.

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

Presumptive FMRF-amide-like immunoreactive retinopetal fibres in Crocodylus niloticus.

A small contingent of 30-50 of centrifugal visual fibres, showing FMRF-amide-like immunoreactivity, has been identified in C. niloticus; these fibres extend from the chiasmatic region into the retina. They do not take the marginal optic tract, but pass medially to the chiasmatic fascicles, from the preoptic region. The cells of origin of these fibres have not been identified. However, none of the retinopetal neurons of the brainstem [M. Medina, J. Reperant, R. Ward, D. Miceli, Centrifugal visual system of Crocodylus niloticus : a hodological, histochemical and immunocytochemical study, J. Comp. Neurol. 468 (2004) 65-85], labelled by retrograde transport of rhodamine beta-isothiocyanate after intraocular injection of this tracer, show FMRF-amide-like immunoreactivity; neither are any of the FMRF-amide-like immunopositive neurons in the crocodile brain, particularly those of the complex involving the terminal nerve and the septo-preoptic region, labelled by rhodamine after its intraocular injection.

Measuring peptide secretion using the FMRFamide tagging technique.

Peptides are transmitters produced by a wide variety of neurons and neuroendocrine cells. They mediate a remarkable range of physiological processes. To better understand the roles played by peptides, a number of methods have been developed that can monitor their secretion. Although each has particular strengths, they cannot rapidly detect the secretion of chemically defined peptides. However, a recently developed approach termed "FMRFamide-tagging" may be useful in this regard. A genetically encoded electrophysiological tag is attached to the peptide prohormone of interest. The "tagged" prohormone together with an ionotropic receptor that binds the tag are expressed in the cell type under investigation. Secretion of the tag (and the co-secreted peptide of interest) are revealed by rapid inward membrane currents that are due to the activation of the tag receptor. In this manner, peptide secretion can be followed on a millisecond time scale. This protocol gives the details of the approach and its potential application to a range of cell types.

Peripheral nerve connections influence the appearance of neurosecretary material in neural sheath of ventral ganglion of the fly Sarcophaga bullata: an immunocytochemical study.

This study examined the role of the brain and peripheral connections with the target organs in the appearance of neurosecretary material within the dorsal neural sheath of the ventral ganglion of the fly S. bullata. Specifically, the accumulation of the neuropeptide FMRFamide (the neurosecretary material) was examined by immunocytochemistry. Immunoreactions were performed on: (1) a normal intact ventral ganglion, (2) an isolated ventral ganglion that was cultured in vivo, and (3) a ventral ganglion that was isolated by transection from the brain, but retained its peripheral nerve connections. The results demonstrate that (a) the neurons of the ganglia survive and exhibit FMRFamide immune reaction independent of their peripheral connections, and (b) the accumulation of neuropeptide in the dorsal neural sheath is controlled by intact peripheral nerve connections with the ganglion. It is suggested that in the absence of their peripheral connections, the axons of FMRFamide immunoreactive neurons fail to invade the neural sheath resulting in the accumulation of neurosecretary material.

The brain of the Nemertodermatida (Platyhelminthes) as revealed by anti-5HT and anti-FMRFamide immunostainings.

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.

Cell type-specific regulatory sequences control expression of the Drosophila FMRF-NH2 neuropeptide gene.

The FMRFamide (dFMRFa) neuropeptide gene is expressed in about 17 diverse cell types in the Drosophila central nervous system. This expression pattern is generated by transcriptional control elements that are distributed over 8 kilobases of dFMRFa DNA. Previous studies identified one enhancer within the dFMRFa 5' region that is both necessary and sufficient to drive reporter transgene expression in one of the 17 dFMRFa cell types, the OL2 neurons. We now report the presence of two additional, non-overlapping enhancers within the gene: One drives expression by the six Tv neuroendocrine cells, and another in the four X and X2 interneurons. We also show that the Tv neuron-specific enhancer itself has complex organization, with several positively and negatively acting cis elements. Together, these results describe the organization of what is likely to be a prototypic neuronal gene promoter: an assemblage of multiple, independent, cell type-specific enhancers, each consisting of multiple quantitative elements.

Cell type-specific regulation of the Drosophila FMRF-NH2 neuropeptide gene by Apterous, a LIM homeodomain transcription factor.

We describe the direct and cell-specific regulation of the Drosophila FMRFa neuropeptide gene by Apterous, a LIM homeodomain transcription factor. dFMRFa and Apterous are expressed in partially overlapping subsets of neurons, including two of the seventeen dFMRFa cell types, the Tv neuroendocrine cells and the SP2 interneurons. Apterous contributes to the initiation of dFMRFa expression in Tv neurons, but not in those dFMRFa neurons that do not express Apterous. Apterous is not required for Tv neuron survival or morphological differentiation. Apterous contributes to the maintenance of dFMRFa expression by postembryonic Tv neurons, although the strength of its regulation is diminished. Apterous regulation of dFMRFa expression includes direct mechanisms, although ectopic Apterous does not induce ectopic dFMRFa. These findings show that, for a subset of neurons that share a common neurotransmitter phenotype, the Apterous LIM homeoprotein helps define neurotransmitter expression with very limited effects on other aspects of differentiation.