Elsevier

Neuroscience

Volume 155, Issue 2, 13 August 2008, Pages 374-386
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2008.05.052Get rights and content

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 (H-phenylalanine-methionine-arginine-phenylalanine-NH2) –gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (green fluorescent protein (GFP), red fluorescent protein from Discosoma sp (dsRed) or mutated form of red fluorescent protein from Discosoma sp (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 (APs). 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 RFamide-related peptide-1, which have amidated RF C-termini, did not affect HaFaNaC-expressing 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.

Section snippets

cDNA constructs

HaFaNaC constructs were contracted out to sequencing companies for subcloning into the mammalian expression vector pCMVTnT (Promega, Madison, WI, USA). HaFaNaC cDNA (EMBL accession number: X92113) was donated by Drs. Eric Lingueglia and Michel Lazdunski (CNRS-Université de Nice-Sophia, Antipolis, France). To generate pCMVTnT/HaFaNaC, the entire coding sequence of HaFaNaC was polymerase chain reaction (PCR) amplified and ligated between XbaI and SalI sites (Epoch Biolabs, Sugar Land, TX, USA).

Results

In this study we have examined the efficacy with which the invertebrate ligand-gated ion channel HaFaNaC can be expressed in mammalian neurons. In addition to its functional expression, we have investigated whether the expression of HaFaNaC or the application of FMRFamide would have any secondary confounding effects on mammalian neuronal function. Finally, we examined the subcellular localization of HaFaNaC in the somata, dendrites and axons of HaFaNaC-expressing mammalian neurons. We used

Discussion

We have shown that the invertebrate HaFaNaC can be functionally expressed in mammalian neurons in vitro. When HaFaNaC was expressed in CA3 pyramidal neurons of organotypic slice cultures, somatic application of FMRFamide was able to produce large depolarizations sufficient to elicit a burst of APs. The responses to FMRFamide were inhibited by amiloride and showed similar current–voltage relationships to those previously observed in cell lines and oocytes (Lingueglia et al., 1995). Importantly,

Conclusion

In conclusion, we have described the utility of heterologously expressing HaFaNaC in mammalian neurons as a potential genetically targeted method to excite specific subsets of neurons in intact mammalian CNS tissue. FMRFamide activation of heterologous expressed HaFaNaC could be used to study the effect of the excitation of specific subsets of neurons that are tonically active in the mammalian CNS. Strategies to improve the kinetics of activation of HaFaNaC and modify its subcellular

Acknowledgments

The authors would like to thank Drs. E. Lingueglia and M. Lazdunski for the donation of their FMRFamide-gated sodium channel, Dr. R. Y. Tsien for the tdTomato, Dr. E. Ruthazer for donating the GFP-tagged synaptophysin, and Dr. J. Dempster for the gift of his electrophysiological software. Microscopy was performed at the VCU-Dept. of Neurobiology and Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463. A. Rory McQuiston is supported by a

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