Harold H. Zakon

The effects of steroid hormones on electrical activity of excitable cells

Steroid hormones influence the electrical activity of many neurons and effectors by regulating the transcription of their ion channels and neurotransmitter receptors, or by modulating the activity of their channels and receptors through second messenger-coupled membrane receptors, or both. In this article, four cell types with known functions and distinct electrical activities are focused on to illustrate how different steroids act synergistically with, or in opposition to, each other to modulate specific electrical phenomena such as spontaneous regular firing (GH3 cells, a pituitary cell line), action potential duration (electric organ cells), and intrinsic excitability and sensitivity to neurotransmitters (GnRH and opioidergic neurons).These examples illustrate how steroids might influence electrical activity in neurons involved in more complex central circuits.

Evolution of sodium channels predates the origin of nervous systems in animals.

Voltage-dependent sodium channels are believed to have evolved from calcium channels at the origin of the nervous system. A search of the genome of a single-celled choanoflagellate (the sister group of animals) identified a gene that is homologous to animal sodium channels and has a putative ion selectivity filter intermediate between calcium and sodium channels. Searches of a wide variety of animal genomes, including representatives of each basal lineage, revealed that similar homologs were retained in most lineages. One of these, the Placozoa, does not possess a nervous system. We cloned and sequenced the full choanoflagellate channel and parts of two placozoan channels from mRNA, showing that they are expressed. Phylogenetic analysis clusters the genes for these channels with other known sodium channels. From this phylogeny we infer ancestral states of the ion selectivity filter and show that this state has been retained in the choanoflagellate and placozoan channels. We also identify key gene duplications and losses and show convergent amino acid replacements at important points along the animal lineage.

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

Genomic basis for the convergent evolution of electric organs

Only one way to make an electric organ? Electric fish have independently evolved electric organs that help them to communicate, navigate, hunt, and defend themselves. Gallant et al. analyzed the genome of the electric eel and the genes expressed in two other distantly related electric fish. The same genes were recruited within the different species to make evolutionarily new structures that function similarly. Science, this issue p. 1522 Multiple divergent fish lineages have used the same evolutionary toolkit to produce electric organs. Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

Toxin-Resistant Sodium Channels: Parallel Adaptive Evolution across a Complete Gene Family

Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel α-subunit (SCNA) gene family and their encoded Nav1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, compared to ten in another vertebrate lineage, mammals. Prior phylogenetic analyses have indicated that the genomes of both teleosts and tetrapods contain four monophyletic groups of SCNA genes, and that tandem duplications expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods, suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analyses of other closely mapped genes in D. rerio as well as of SCNA genes from several teleost species all support the hypothesis that a whole-genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation.

Old gene duplication facilitates origin and diversification of an innovative communication system—twice

The genetic basis of parallel innovation remains poorly understood due to the rarity of independent origins of the same complex trait among model organisms. We focus on two groups of teleost fishes that independently gained myogenic electric organs underlying electrical communication. Earlier work suggested that a voltage-gated sodium channel gene (Scn4aa), which arose by whole-genome duplication, was neofunctionalized for expression in electric organ and subsequently experienced strong positive selection. However, it was not possible to determine if these changes were temporally linked to the independent origins of myogenic electric organs in both lineages. Here, we test predictions of such a relationship. We show that Scn4aa co-option and rapid sequence evolution were tightly coupled to the two origins of electric organ, providing strong evidence that Scn4aa contributed to parallel innovations underlying the evolutionary diversification of each electric fish group. Independent evolution of electric organs and Scn4aa co-option occurred more than 100 million years following the origin of Scn4aa by duplication. During subsequent diversification of the electrical communication channels, amino acid substitutions in both groups occurred in the same regions of the sodium channel that likely contribute to electric signal variation. Thus, the phenotypic similarities between independent electric fish groups are also associated with striking parallelism at genetic and molecular levels. Our results show that gene duplication can contribute to remarkably similar innovations in repeatable ways even after long waiting periods between gene duplication and the origins of novelty.

Diversity of sexual dimorphism in electrocommunication signals and its androgen regulation in a genus of electric fish, Apteronotus

Abstract Gymnotiform electric fish emit an electric organ discharge that, in several species, is sexually dimorphic and functions in gender recognition. In addition, some species produce frequency modulations of the electric organ discharge, known as chirps, that are displayed during aggression and courtship. We report that two congeneric species (Apteronotus leptorhynchus and A. albifrons) differ in the expression of sexual dimorphism in these signals. In A. leptorhynchus, males chirp more than females, but in A. albifrons chirping is monomorphic. The gonadosomatic index and plasma levels of 11-ketotestosterone were equivalent in both species, suggesting that they were in similar reproductive condition. Corresponding to this difference in dimorphism, A. leptorhynchus increases chirping in response to androgens, but chirping in A. albifrons is insensitive to implants of testosterone, dihydrotestosterone or 11-ketotestosterone. Species also differ in the sexual dimorphism and androgen sensitivity of electric organ discharge frequency. In A. leptorhynchus, males discharge at higher frequencies than females, and androgens increase electric organ discharge frequency. In A.␣albifrons, males discharge at lower frequencies than females, and androgens decrease electric organ discharge frequency. Thus, in both chirping and electric organ discharge frequency, evolutionary changes in the presence or direction of sexual dimorphism have been accompanied and perhaps caused by changes in the androgen regulation of the electric organ discharge.

Adaptive evolution of voltage-gated sodium channels: The first 800 million years

Voltage-gated Na+-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca2+ channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na+ and Ca2+. Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels “capitalized” on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (∼9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats).

Electric organ discharge frequency and plasma sex steroid levels during gonadal recrudescence in a natural population of the weakly electric fish Sternopygus macrurus

Summary1.Sternopygus macrurus were collected in Venezuela during the period of gonadal recrudescence in early or late dry season. Electric organ discharge (EOD) frequencies were recorded, blood samples were taken for analysis of steroid titers, and gonads were taken for determination of reproductive condition.2.Mean EOD frequencies were significantly lower in males than in females in all samples. EOD frequency was inversely correlated with body length in males in late, but not early, dry season, and these parameters were never correlated in females.3.Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT), but not estradiol-17β (E2), were inversely correlated with EOD frequency in males. No 11-KT was observed in plasma of females, and plasma levels of T and E2 in females were comparable to those of males. Neither T nor E2 were correlated with EOD frequency in females.4.Testes collected in late dry season were more mature than those from early dry season; androgen levels and EOD frequency were correlated with testicular maturity. Ovaries collected in early dry season were immature, while those from late dry season were more mature. There was no relationship between EOD frequency and stage of ovarian development.5.These results suggest that plasma androgens modulate EOD frequency in males during the reproductive season and that plasma E2 has little relationship to EOD frequency in either sex.