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Kohn, A. B.

Publications and source records attributed to Kohn, A. B..

9 recordsLinked to original sources
bioRxiv2020

ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of one of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica - the prominent model in cellular and system neuroscience. These functional P2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP (EC50=306 M) with two-phased kinetics as well as Na+-dependence. The ATP analog, Bz-ATP, was a less effective agonist (~20%) than ATP, and PPADS was a more potent inhibitor of the P2X receptors than the suramin. We showed that P2X receptors are uniquely expressed within Aplysias cerebral bioenergetic center (also known as F-cluster). Using RNA-seq, we found that the F-cluster contains more than a dozen unique secretory peptides, including three insulins, interleukins, and potential toxins, as well as ecdysone-type receptors and a district subset of ion channels. This structure is one of the most prominent integrative centers in the entire CNS and remarkably different from the morphologically similar neurosecretory center (bag cluster) involved in egg-laying behavior. Using RNA-seq, we also characterized the expression of P2X receptors across more than a dozen Aplysia peripheral tissues and developmental stages. We showed that P2X receptors are predominantly expressed in chemosensory structures and during early cleavage stages. The localization and pharmacology of P2X receptors in Aplysia highlight the evolutionary conservation of bioenergetic sensors and chemosensory purinergic transmission across animals. This study also provides a foundation to decipher homeostatic mechanisms in development and neuroendocrine systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/392001v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ea3ab5org.highwire.dtl.DTLVardef@b45aadorg.highwire.dtl.DTLVardef@ad1851org.highwire.dtl.DTLVardef@17abbc_HPS_FORMAT_FIGEXP M_FIG We show that ATP and its ligand-gated P2X receptors are essential signaling components within both the chemosensory systems and the unique integrative neurosecretory center, present in the CNS of the sea slug Aplysia - a prominent model in neuroscience. Expression and pharmacology of P2X receptors in Aplysia confirms the preservation of evolutionary conserved bioenergetic sensors across animals and provide new tools to decipher homeostatic mechanisms in neuro-endocrine systems in general. C_FIG

neuroscience↗
bioRxiv2020

Action Potentials and Na+ voltage-gated ion channels in Placozoa

Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched animal lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals. HighlightsO_LIPlacozoans are the simplest known animals without recognized neurons and muscles C_LIO_LIWith only six morphological cell types, placozoans showed complex & rapid behaviors C_LIO_LISodium-dependent action potentials have been discovered in intact animals C_LIO_LIVoltage-gated sodium channels (Nav) in Placozoa support a rapid behavioral integration C_LIO_LIPlacozoans have more Nav channels that any studied invertebrate animal so far C_LIO_LIDiversification of Nav-channels highlight the unique evolution of these nerveless animals C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/243113v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@4d9c5forg.highwire.dtl.DTLVardef@15539bforg.highwire.dtl.DTLVardef@4246faorg.highwire.dtl.DTLVardef@141e5fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗
bioRxiv2020

P2X receptors in Aplysia californica: Chemosensory systems, bio-energetic and development

ATP and its ionotropic P2X receptors are components of one of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica - a prominent model in cellular and system neuroscience. We showed that ATP and P2X receptors are essential signaling components within the unique bioenergetic center located in the CNS of Aplysia, also known as the cerebral F-cluster of insulin-containing neurons. Functional P2X receptors were successfully expressed in Xenopus oocytes to characterize their ATP-dependence (EC50=306M), two-phased kinetics, ion selectivity (Na+-dependence), sensitivity to the ATP analog Bz-ATP (~20% compare to ATP) and antagonists (with PPADS as a more potent inhibitor compared to suramin). Next, using RNA-seq, we characterized the expression of P2X receptors across more than a dozen Aplysia peripheral tissues and developmental stages. We showed that P2X receptors are predominantly expressed in chemosensory structures and during early cleavage stages. The localization and pharmacology of P2X receptors in Aplysia highlight the evolutionary conservation of bioenergetic sensors and chemosensory purinergic transmission across animals. This study also provides a foundation to decipher homeostatic mechanisms in development and neuroendocrine systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/243196v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@d5c2faorg.highwire.dtl.DTLVardef@1f3dab3org.highwire.dtl.DTLVardef@9b49daorg.highwire.dtl.DTLVardef@10dc71f_HPS_FORMAT_FIGEXP M_FIG C_FIG We show that ATP and its ligand-gated P2X receptors are essential signaling components within both the chemosensory systems and the unique bioenergetic center, present in the CNS of the sea slug Aplysia californica - a prominent model in neuroscience. Expression and pharmacology of P2X receptors in Aplysia confirms the preservation of evolutionary conserved bioenergetic sensors across animals and provide new tools to decipher homeostatic mechanisms in neuro-endocrine systems in general.

neuroscience↗
bioRxiv2020

Lineage-specific diversification in the usage of D-glutamate and D-aspartate in early-branching metazoans

D-amino acids are unique and essential signaling molecules in neural, hormonal, and immune systems. However, the presence of D-amino acids and their recruitment in early animals is mostly unknown due to limited information about prebilaterian metazoans. Here, we performed the comparative survey of L-/D-aspartate and L-/D-glutamate in representatives of four phyla of basal Metazoa: cnidarians (Aglantha); placozoans (Trichoplax), sponges (Sycon) and ctenophores (Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe), which are descendants of ancestral animal lineages distinct from Bilateria. Specifically, we used high-performance capillary electrophoresis for microchemical assays and quantification of the enantiomers. L-glutamate and L-aspartate were abundant analytes in all species studied. However, we showed that the placozoans, cnidarians, and sponges had high micromolar concentrations of D-aspartate, whereas D-glutamate was not detectable. In contrast, we found that in ctenophores, D-glutamate was the dominant enantiomer with no or trace amounts of D-aspartate. This situation illuminates prominent lineage-specific diversifications in the recruitment of D-amino acids and suggests distinct signaling functions of these molecules early in the animal evolution. We also hypothesize that a deep ancestry of such recruitment events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa. HighlightsO_LID-amino acids are essential for intercellular signaling and evolution C_LIO_LIEnantiomers have been quantified in early-branching animals C_LIO_LILineage-specific recruitment of D-glutamate could occur in ctenophores C_LIO_LID-aspartate is one of the primary enantiomers in other metazoans C_LIO_LIDeep ancestry of such events could provide constraints in the evolution of signaling C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY D-amino acids are essential for intercellular signaling. Direct microchemical quantification of enantiomers in representatives of early-branching animals suggests lineage-specific recruitments of D-glutamate and D-aspartate. Deep ancestry of such events might provide some constraints underlying the evolution of neural and other signaling systems in Metazoa. C_FIG_DISPLAY

evolutionary biology↗
bioRxiv2020

Nitric oxide (NO) signaling in Trichoplax and related species: Microchemical characterization and the lineage-specific diversification

Nitric oxide (NO) is a free radical gaseous messenger with a broad distribution across the animal kingdom. However, the early evolution of nitric oxide-mediated signaling in animals is unclear due to limited information about prebilaterian metazoans such as placozoans. Here, we analyzed NO synthases (NOS) in four different species of placozoans (haplotypes H1, H2, H4, H13). In contrast to all other invertebrates studied, Hoilungia and Trichoplax have three distinct NOS genes, including PDZ domain-containing NOS. To characterize NOS activity in Trichoplax adhaerens, we used capillary electrophoresis for microchemical assays of NO-related metabolites. Specifically, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional NOS. Next, using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest the compartmentalized release of this messenger and a greater diversity of cell types in placozoans than anticipated. We also revealed a dramatic diversification of NO receptor machinery, including identification of both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax is considered to be one of the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa. Short AbstractNitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans - one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa.

evolutionary biology↗
bioRxiv2019

Comparative single-cell transcriptomics of complete insect nervous systems

Molecular profiles of neurons influence information processing, but bridging the gap between genes, circuits, and behavior has been very difficult. Furthermore, the behavioral state of an animal continuously changes across development and as a result of sensory experience. How behavioral state influences molecular cell state is poorly understood. Here we present a complete atlas of the Drosophila larval central nervous system composed of over 200,000 single cells across four developmental stages. We develop polyseq, a python package, to perform cell-type analyses. We use single-molecule RNA-FISH to validate our scRNAseq findings. To investigate how internal state affects cell state, we optogentically altered internal state with high-throughput behavior protocols designed to mimic wasp sting and over activation of the memory system. We found nervous system-wide and neuron-specific gene expression changes. This resource is valuable for developmental biology and neuroscience, and it advances our understanding of how genes, neurons, and circuits generate behavior.

neuroscience↗
bioRxiv2019

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover presynaptic genes in an amoeboid-neuroid cell type, and postsynaptic genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.

developmental biology↗
bioRxiv2018

Polymorphism within the mitochondrial genome of the ctenophore, Pleurobrachia bachei, and its ongoing rapid evolution

The mitochondrial genomes in ctenophores are among the most compact in the animal kingdom with multiple rearrangements and examples of gene loss. Here, by resequencing of the Pleurobrachia bachei mitochondrial genome, we show that the high level of polymorphism (>10%) in Pleurobrachia might contribute to the ongoing fast evolution of ctenophores including the presence of truncated versions of apparently canonical genes such as cox1. Second, the codon interpretations in ctenophores, without robust proteomic data related to mitochondrial genes, is still a challenging issue, which is open for future experimental analyses.

evolutionary biology↗