A molecularly defined cardiorespiratory interneuron expressing SDPFLRFamide/GDPFLRFamide in the snail Lymnaea: monosynaptic connections and pharmacology

Abstract

1. Neuron-specific expression of alternately spliced exons of the gene encoding the Phe-Met-Arg-Phe-NH2 (FMRFamide) family of neuropeptides and the role of encoded peptides in synaptic transmission were examined in an identified cardiorespiratory interneuron, the visceral white interneuron (VWI), in the snail Lymnaea. 2. In situ hybridization using exon-specific probes showed VWI cytoplasmic expression of the exon encoding the Lymnaea heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRF amide) Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRF amide) but not the exon encoding the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide). 3. The absence of the tetrapeptides (FMRFamide and FLRFamide) in the VWI was indirectly confirmed by the lack of immunoreactivity to a specific antibody raised against the sequence Leu-Tyr. This sequence is present in the Lymnaea tetrapeptide precursor, but not the heptapeptide precursor. 4. The VWI has monosynaptic connections with many identifiable neurons in the CNS. These were excitatory on three clusters of identified neurons [B group (Bgp), E group (Egp), and F group (Fgp)], inhibitory on another cluster [A group (Agp)] or biphasic (excitation followed by inhibition) on a single giant neuron [right pedal dorsal 1 (RPeD1)]. 5. The role of GDPFLRFamide/SDPFLRFamide as putative neurotransmitters was examined by comparing neuronally evoked postsynaptic responses with the effects of focal peptide application. 6. The heptapeptides closely mimicked the inhibitory responses (threshold pressure pipette concentration 10(-9) M) on the Agp cells and RPeD1, including an increase in membrane conductance. FMRFamide was 1 order of magnitude less potent. GDPFLRFamide/SDPFLRFamide, applied either alone or in "cocktails" (combinations of GDPFLRFamide, SDPFLRFamide, FMRFamide, and FLRFamide), did not reproduce the excitatory effect of the VWI on the Bgp, Egp, and Fgp cells. These peptides, applied either together or separately, inhibited the cells. 7. FMRFamide or FLRFamide, but not GDPFLRFamide or SDPFLRFamide, could reproduce the initial depolarizing component of the biphasic response on RPeD1. This only occurred at concentrations of > or = 10(-4) M (10(-3) M was necessary to get spikes on RPeD1) and may not be physiologically significant. 8. We conclude that at least one so far unidentified co-transmitter must be present in the VWI to account for its full range of synaptic responses.

Genomic organization of the FMRFamide gene in Lymnaea: multiple exons encoding novel neuropeptides

“…The characterization of these peptides by protein sequencing (Ebberink et al, 1987;de With et al, 1992) used, as these are the targets of the N-terminally extended peptides (heptapeptides, hexapeptides) encoded on exons III-V of the FMRFamide gene Skingsley et al, 1993). As was found previously, SDPFLRFamide inhibited neuronal firing (e.g., Egp cell of Fig.…”

“…Neuropeptides SDPFLRFamide and SKPYMRFamide were applied to the surface of identified neurons via pressure pipettes while recording the responses using intracellular microelectrodes. Methods were the same as Skingsley et al (1993). Isolated Lymnaea brains were continuously perfused with HEPES-buffered saline (Benjamin and Winlow,198 1) during pharmacological application of neuropeptides.…”

“…To some extent, peptides encoded by this exon can mimic aspects of the action ofthe VW1 but cannot explain all its actions. For instance, the inhibitory effects of the VW1 on its postsynaptic cells can be satisfactorily mimicked by GDPFLRFamide, but this does not explain the excitatory responses on other neurons (Skingsley et al, 1993). The possibility arises that the VW1 therefore contains other peptides (or other transmitters) that may be encoded on separate exons within the same gene.…”

“…This is expected, as the peptides probably act as transmitters in this system (Buckett et al, 1990;Brezden et al, 1991). As a consequence of alternative RNA splicing, a second exon encodes a class of N-terminally extended peptides, predominantly GDP/ SDPFLRFamide, which are expressed in approximately 57 cells ) including a cardiorespiratory cell, the visceral white interneuron (VWI) (Skingsley et al, 1993). To some extent, peptides encoded by this exon can mimic aspects of the action ofthe VW1 but cannot explain all its actions.…”

Comparative Physiology/Comparative & Invertebrate Neuroscience

“…1B In Lymnaea, the large interneurons RPeD1 and VWI are located in the pedal and visceral ganglia respectively. These cells have wide-ranging effects on a large number of respiratory and cardiac interneurons and motoneurons (Buckett et al 1990;Skingsley et al 1993). The RPeD1 interneuron inhibits the VWI while the VWI has a dual excitatory-inhibitory connection to the RPeD1.…”