Behavioral Comorbidities and Drug Treatments in a Zebrafish scn1lab Model of Dravet Syndrome.

Loss-of-function mutations in SCN1A cause Dravet syndrome (DS), a catastrophic childhood epilepsy in which patients experience comorbid behavioral conditions, including movement disorders, sleep abnormalities, anxiety, and intellectual disability. To study the functional consequences of voltage-gated sodium channel mutations, we use zebrafish with a loss-of-function mutation in scn1lab, a zebrafish homolog of human SCN1A. Homozygous scn1labs552/s552 mutants exhibit early-life seizures, metabolic deficits, and early death. Here, we developed in vivo assays using scn1labs552 mutants between 3 and 6 d postfertilization (dpf). To evaluate sleep disturbances, we monitored larvae for 24 h with locomotion tracking software. Locomotor activity during dark (night phase) was significantly higher in mutants than in controls. Among anticonvulsant drugs, clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved at night for scn1labs552 mutant larvae. To monitor exploratory behavior in an open field, we tracked larvae in a novel arena. Mutant larvae exhibited impaired exploratory behavior, with increased time spent near the edge of the arena and decreased mobility, suggesting greater anxiety. Both clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved and increased time spent in the center of the arena. Counting inhibitory neurons in vivo revealed no differences between scn1labs552 mutants and siblings. Taken together, our results demonstrate conserved features of sleep, anxiety, and movement disorders in scn1lab mutant zebrafish, and provide evidence that a zebrafish model allows effective tests of treatments for behavioral comorbidities associated with DS

A second corticotropin-releasing hormone gene (CRH2) is conserved across vertebrate classes and expressed in the hindbrain of a basal Neopterygian fish, the spotted gar (Lepisosteus oculatus)

© 2015 Wiley Periodicals, Inc. To investigate the origins of the vertebrate stress-response system, we searched sequenced vertebrate genomes for genes resembling corticotropin-releasing hormone (CRH). We found that vertebrate genomes possess, in addition to CRH, another gene that resembles CRH in sequence and syntenic environment. This paralogous gene was previously identified only in the elephant shark (a holocephalan), but we find it also in marsupials, monotremes, lizards, turtles, birds, and fishes. We examined the relationship of this second vertebrate CRH gene, which we name CRH2, to CRH1 (previously known as CRH) and urocortin1/urotensin1 (UCN1/UTS1) in primitive fishes, teleosts, and tetrapods. The paralogs CRH1 and CRH2 likely evolved via duplication of CRH during a whole-genome duplication early in the vertebrate lineage. CRH2 was subsequently lost in both teleost fishes and eutherian mammals but retained in other lineages. To determine where CRH2 is expressed relative to CRH1 and UTS1, we used in situ hybridization on brain tissue from spotted gar (Lepisosteus oculatus), a neopterygian fish closely related to teleosts. In situ hybridization revealed widespread distribution of both crh1 and uts1 in the brain. Expression of crh2 was restricted to the putative secondary gustatory/secondary visceral nucleus, which also expressed calcitonin-related polypeptide alpha (calca), a marker of parabrachial nucleus in mammals. Thus, the evolutionary history of CRH2 includes restricted expression in the brain, sequence changes, and gene loss, likely reflecting release of selective constraints following whole-genome duplication. The discovery of CRH2 opens many new possibilities for understanding the diverse functions of the CRH family of peptides across vertebrates

Three Distinct Glutamate Decarboxylase Genes in Vertebrates

Gamma-aminobutyric acid (GABA) is a widely conserved signaling molecule that in animals has been adapted as a neurotransmitter. GABA is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). Two vertebrate genes, GAD1 and GAD2, encode distinct GAD proteins: GAD67 and GAD65, respectively. We have identified a third vertebrate GAD gene, GAD3. This gene is conserved in fishes as well as tetrapods. We analyzed protein sequence, gene structure, synteny, and phylogenetics to identify GAD3 as a homolog of GAD1 and GAD2. Interestingly, we found that GAD3 was lost in the hominid lineage. Because of the importance of GABA as a neurotransmitter, GAD3 may play important roles in vertebrate nervous systems

Divergent evolution of two corticotropin-releasing hormone (CRH) genes in teleost fishes

© 2015 Grone and Maruska. Genome duplication, thought to have happened twice early in vertebrate evolution and a third time in teleost fishes, gives rise to gene paralogs that can evolve subfunctions or neofunctions via sequence and regulatory changes. To explore the evolution and functions of corticotropin-releasing hormone (CRH), we searched sequenced teleost genomes for CRH paralogs. Our phylogenetic and synteny analyses indicate that two CRH genes, crha and crhb, evolved via duplication of crh1 early in the teleost lineage. We examined the expression of crha and crhb in two teleost species from different orders: an African cichlid, Burton\u27s mouthbrooder, (Astatotilapia burtoni; Order Perciformes) and zebrafish (Danio rerio; Order Cypriniformes). Furthermore, we compared expression of the teleost crha and crhb genes with the crh1 gene of an outgroup to the teleost clade: the spotted gar (Lepisosteus oculatus). In situ hybridization for crha and crhb mRNA in brains and eyes revealed distinct expression patterns for crha in different teleost species. In the cichlid, crha mRNA was found in the retina but not in the brain. In zebrafish, however, crha mRNA was not found in the retina, but was detected in the brain, restricted to the ventral hypothalamus. Spotted gar crh1 was found in the retina as well as the brain, suggesting that the ancestor of teleost fishes likely had a crh1 gene expressed in both retina and brain. Thus, genome duplication may have freed crha from constraints, allowing it to evolve distinct sequences, expression patterns, and likely unique functions in different lineages

Social status regulates kisspeptin receptor mRNA in the brain of Astatotilapia burtoni

The brain controls reproduction in response to relevant external and internal cues. Central to this process in vertebrates is gonadotropin-releasing hormone (GnRH1) produced in neurons of the hypothalamic-preoptic area (POA). GnRH1 released from the POA stimulates pituitary release of gonadotropins, which in males causes sperm production and concomitant steroid hormone release from the testes. Kisspeptin, a neuropeptide acting via the kisspeptin receptor (Kiss1r), increases GnRH1 release and is linked to development of the reproductive system in mammals and other vertebrates. In both fish and mammals, kiss1r mRNA levels increase in the brain around the time of puberty but the environmental and other stimuli regulating kisspeptin signaling to GnRH1 neurons remain unknown. To understand where kiss1r is expressed and how it is regulated in the brain of a cichlid fish, Astatotilapia burtoni, we measured expression of a kiss1r homolog mRNA by in situ hybridization and quantitative reverse transcription-PCR (qRT-PCR). We found kiss1r mRNA localized in the olfactory bulb, specific nuclei in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, as well as in GnRH1 and GnRH3 neurons. Since males\u27 sexual physiology and behavior depend on social status in A. burtoni, we also tested how status influenced kiss1r mRNA levels. We found higher kiss1r mRNA levels in whole brains of high status territorial males and lower levels in low status non-territorial males. Our results are consistent with the hypothesis that Kiss1r regulates many functions in the brain, making it a strong candidate for mediating differences in reproductive physiology between territorial and non-territorial phenotypes. © 2010 Elsevier Inc

Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parent\u27s mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation. © 2012 Elsevier Inc

Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations