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Extraction of Total RNA from Single Neurons: Mauthner-cell Cytoplasm vs. Whole Brain

ABSTRACT: Astatotilapia burtoni males change their startle behavior in accordance with changes in a socially-mediated phenotypic state. When dominant (DOM), males are brightly colored, isolated from the school and startle readily in response to an auditory pulse. When subordinate (SUB), males are cryptically colored, school and startle less frequently. We hypothesize that the difference in startle responsiveness is governed by serotonergic modulation on the command-like Mauthner cells (M-cells). We used the selective serotonin receptor subtype 2 (5HTR2) antagonist ketanserin to show that serotonin receptors can modulate startle frequency and that the behavioral difference correlates with differences in electrophysiological recordings of M-cells. Specifically, SUB males have a higher serotonergic tone and demonstrate, by behavior and electrophysiological properties, increased sensitivity to 5-HT manipulations compared to DOMs. Immunohistochemistry for serotonin is present around the M-cells. Furthermore, we identified serotonin receptor transcripts in A. burtoni and used single-cell transcriptomics to determine the presence or absence of specific receptor subtypes in the M-cells. Microarray analysis of M-cell samples shows that the neuron’s transcriptome is relatively stable in individuals of different social status. These results are consistent with a role for serotonin in modulating the behavioral response to sensory stimuli in an ecologically-relevant manner through inhibitory interneurons, which establish the membrane resistance of the M-cell before the auditory stimulus.

ORGANISM(S): Haplochromis burtoni

PROVIDER: GSE26277 | GEO | 2010/12/23

SECONDARY ACCESSION(S): PRJNA135145

REPOSITORIES: GEO

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Project description:The presence of serotonergic system during early pre-neural development is conserved amongst all studied invertebrate and vertebrate animals. Previously, we discovered that early embryonic pre-neural serotonin relays physiological and behavioral information from mother to progeny in an invertebrate model system. However, the question of whether the similar logic might apply during vertebrate development remains unanswered. Thus, we took advantage of zebrafish model system to address this based on the systematic investigation of composition and function of early pre-neural serotonergic system in fish. As a result, we characterized the presence, distribution and activity of molecules involved in serotonin synthesis and transport at pre-neural and neural stages of development. Then, we experimentally revealed the existence of delayed developmental and behavioral effects resulting from the manipulations of pre-neural (zygote, blastula and gastrula) serotonergic system. In particular, the delayed effects included differences in the synthesis of serotonin in early serotonergic neurons in the central nervous system as well as in behavioral alterations in zebrafish larvae. At the same time, the effects resulting from manipulations of pre-neural serotonin appeared highly specific and did not coincide with any major abnormalities. Similar manipulations of the serotonergic system at later neural stages did not show similar effects, thus, demonstrating that transduction of a serotonergic signal is not based on the general level of retained serotonin but on its availability in the specific cells. Accordingly, gene expression analysis demonstrated specific changes in response to the elevation of early pre-neural serotonin, which included the delayed and pre-mature onsets of different gene expression programs. Taken together, our results introduce a novel function of early pre-neural serotonergic system in a vertebrate embryo – tuning and fine control of specific mechanisms at later neural developmental stages that result in a mild variation of an adaptive spectrum. We used microarrays to identify the aftermath of early pre-neural serotonin increase and decrease.

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Extraction of Total RNA from Single Neurons: Mauthner-cell Cytoplasm vs. Whole Brain

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