Heterologous expression of the invertebrate FMRFamide–gated sodium channel as a mechanism to selectively activate mammalian neurons

Abstract

Considerable effort has been directed toward the development of methods to selectively activate specific subtypes of neurons. Focus has been placed on the heterologous expression of proteins that are capable of exciting neurons in which they are expressed. Here we describe the heterologous expression of the invertebrate FMRFamide-gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (GFP, dsRed or tdTomato) in CA3 pyramidal neurons of rat hippocampal slice cultures using single cell electroporation. Pressure application of the agonist FMRFamide to HaFaNaC-expressing neuronal somata produced large prolonged depolarizations and bursts of action potentials (AP). FMRFamide responses were inhibited by amiloride (100 µM). In contrast, pressure application of FMRFamide to the axons of neurons expressing HaFaNaC produced no response. Fusion of GFP to the N-terminus of HaFaNaC showed that GFP-HaFaNaC was absent from axons. Bath application of FMRFamide produced persistent AP firing in HaFaNaC-expressing neurons. This FMRFamide-induced increase in the frequency of APs was dose-dependent. The concentrations of FMRFamide required to activate HaFaNaC-expressing neurons were below that required to activate the homologous acid sensing ion channel normally found in mammalian neurons. Furthermore, the mammalian neuropeptides neuropeptide FF and RFRP-1, which have amidated RF C-termini, did not affect HaFaNaCexpressing neurons. Antagonists of NPFF receptors (BIBP3226) also had no effect on HaFaNaC. Therefore, we suggest that heterologous-expression of HaFaNaC in mammalian neurons could be a useful method to selectively and persistently excite specific subtypes of neurons in intact nervous tissue.

A review of FMRFamide- and RFamide-like peptides in metazoa

“…These data are informative as they provide an insight regarding when other aromatic amino acids can replace phenylalanine and the neuropeptide can still be considered a FLP in terms of FLP-like activity. HaFaNaC has been expressed in CA3 pyramidal neurones from rat hippocampal slice cultures (Schanuel et al 2008). FMRFa depolarizes and excites these neurones inducing bursts of activity, and this effect is blocked by amiloride.…”

An organotypic hippocampal slice culture model of excitotoxic injury induced spontaneous recurrent epileptiform discharges

“…Slice cultures were prepared using the method of Stoppini et al (Stoppini et al, 1991), as previously reported by (Schanuel et al, 2008). All animal use protocols are in strict accordance with the National Institute of Health guidelines and are approved by the International Animal Care and Use Committee of Virginia Commonwealth University.…”

New techniques, applications and perspectives in neuropeptide research

“…In another example, it has been shown that heterologous expression of FaNaC in mammalian hippocampal neurons provides a means to depolarize the neurons and induce bursts of action potentials upon focal application of FMRFa (Schanuel et al, 2008). FaNaC has a somato-dendritic localization and is absent from axons (Schanuel et al, 2008).…”

“…In another example, it has been shown that heterologous expression of FaNaC in mammalian hippocampal neurons provides a means to depolarize the neurons and induce bursts of action potentials upon focal application of FMRFa (Schanuel et al, 2008). FaNaC has a somato-dendritic localization and is absent from axons (Schanuel et al, 2008). As FMRFa is not present in the mammalian nervous system, and endogenous RFamides apparently do not activate FaNaC (Schanuel et al, 2008), it is in principle possible to activate specific subsets of neurons selectively in intact nervous tissue.…”

“…FaNaC has a somato-dendritic localization and is absent from axons (Schanuel et al, 2008). As FMRFa is not present in the mammalian nervous system, and endogenous RFamides apparently do not activate FaNaC (Schanuel et al, 2008), it is in principle possible to activate specific subsets of neurons selectively in intact nervous tissue. Transgenic expression of FaNaC under the control of specific promoters would enable driving of its expression only in specific subsets of neurons in living animals.…”