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Anatomical Organization of Multiple Modulatory Inputs in a Rhythmic Motor System.

Swallie SE, Monti AM, Blitz DM - PLoS ONE (2015)

Bottom Line: The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1.The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations.Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, OH, United States of America.

ABSTRACT
In rhythmic motor systems, descending projection neuron inputs elicit distinct outputs from their target central pattern generator (CPG) circuits. Projection neuron activity is regulated by sensory inputs and inputs from other regions of the nervous system, relaying information about the current status of an organism. To gain insight into the organization of multiple inputs targeting a projection neuron, we used the identified neuron MCN1 in the stomatogastric nervous system of the crab, Cancer borealis. MCN1 originates in the commissural ganglion and projects to the stomatogastric ganglion (STG). MCN1 activity is differentially regulated by multiple inputs including neuroendocrine (POC) and proprioceptive (GPR) neurons, to elicit distinct outputs from CPG circuits in the STG. We asked whether these defined inputs are compact and spatially segregated or dispersed and overlapping relative to their target projection neuron. Immunocytochemical labeling, intracellular dye injection and three-dimensional (3D) confocal microscopy revealed overlap of MCN1 neurites and POC and GPR terminals. The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1. The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations. Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1. Consistent innervation patterns suggest that integration of the sensory GPR and peptidergic POC inputs occur through more distributed and more tightly constrained anatomical interactions with their common modulatory projection neuron target than anticipated.

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MCN1 neurites arborize in the anterior CoG.(A) A single optical slice includes a portion of MCN1 (intracellular fill with Alexa 568; green) and DIC optics to view the outline of the tissue. The MCN1 neurites are located in the anterior region of the CoG (bracket), while in this example, the soma is located more posteriorly (filled arrow) and the axon leaves the CoG through the ion (open arrow). (B) A higher magnification volume rendering of a z-stack (249 optical slices, 1.0 μm interval) of the MCN1 fill from (A) reveals the full extent of the MCN1 arborization within the anterior CoG. Arrows as in (A). (C) Average location of the MCN1 soma (open square) and MCN1 neurites (filled squares) in the x-y plane of the CoG are plotted. The soma was located in the posterior CoG while the neurites were in the anterior region. The neurite location is reported as the average (dark filled center box) and standard deviation (lighter center box) of the center of the arborization and the average (dark filled outer box) and standard deviation (lighter outer box) of the spread of the arborization as measured vertically and horizontally from center across preparations. Scale bars: 100 μm. coc, circumoesophageal connective; ion, inferior oesophageal nerve; son, superior oesophageal nerve.
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pone.0142956.g002: MCN1 neurites arborize in the anterior CoG.(A) A single optical slice includes a portion of MCN1 (intracellular fill with Alexa 568; green) and DIC optics to view the outline of the tissue. The MCN1 neurites are located in the anterior region of the CoG (bracket), while in this example, the soma is located more posteriorly (filled arrow) and the axon leaves the CoG through the ion (open arrow). (B) A higher magnification volume rendering of a z-stack (249 optical slices, 1.0 μm interval) of the MCN1 fill from (A) reveals the full extent of the MCN1 arborization within the anterior CoG. Arrows as in (A). (C) Average location of the MCN1 soma (open square) and MCN1 neurites (filled squares) in the x-y plane of the CoG are plotted. The soma was located in the posterior CoG while the neurites were in the anterior region. The neurite location is reported as the average (dark filled center box) and standard deviation (lighter center box) of the center of the arborization and the average (dark filled outer box) and standard deviation (lighter outer box) of the spread of the arborization as measured vertically and horizontally from center across preparations. Scale bars: 100 μm. coc, circumoesophageal connective; ion, inferior oesophageal nerve; son, superior oesophageal nerve.

Mentions: To quantify locations in the anteroposterior and mediolateral dimensions, both DIC and fluorescence signals were collected at multiple dorsoventral planes. For each ganglion, a single optical slice of the CoG at the depth at which the diameter of the ganglion was greatest was used to identify borders of the ganglion. Specifically, tangent lines along a standardized circular shape were aligned to the anterior, posterior, and medial edges of the CoG, with the circle oriented such that the lateral tangent line aligned with the axon tract of the coc (Fig 2, inset) (Corel Draw, Corel Corporation). In each preparation, this alignment was then maintained across single optical slices above and below the widest diameter slice to quantify structure locations in the anteroposterior and mediolateral axes. This allowed us to collapse the analysis into a two-dimensional plot despite differences in the diameter of the ganglion throughout the dorsoventral axis. To normalize the data and eliminate differences due to inter-preparation variability in CoG size, measurements were scaled such that medial was designated as 0, lateral as 100, posterior as 0, and anterior as 100 in the plane in which the CoG diameter was greatest (Fig 2). All analyses of structures throughout the depth of a ganglion were performed on a single z-stack from a continuous confocal session to ensure alignment of optical slices. Using a grid overlaid on the circle in each relevant image (VistaMetrix software; SkillCrest) locations of the MCN1 soma, MCN1 neurites, the ACO, and the GPR axon bundle were quantified. For larger structures such as the MCN1 neuropil and the ACO, both the center and the margins in the anteroposterior and mediolateral dimensions were determined. For the MCN1 soma and the GPR axon bundle, only the center of the structure was determined.


Anatomical Organization of Multiple Modulatory Inputs in a Rhythmic Motor System.

Swallie SE, Monti AM, Blitz DM - PLoS ONE (2015)

MCN1 neurites arborize in the anterior CoG.(A) A single optical slice includes a portion of MCN1 (intracellular fill with Alexa 568; green) and DIC optics to view the outline of the tissue. The MCN1 neurites are located in the anterior region of the CoG (bracket), while in this example, the soma is located more posteriorly (filled arrow) and the axon leaves the CoG through the ion (open arrow). (B) A higher magnification volume rendering of a z-stack (249 optical slices, 1.0 μm interval) of the MCN1 fill from (A) reveals the full extent of the MCN1 arborization within the anterior CoG. Arrows as in (A). (C) Average location of the MCN1 soma (open square) and MCN1 neurites (filled squares) in the x-y plane of the CoG are plotted. The soma was located in the posterior CoG while the neurites were in the anterior region. The neurite location is reported as the average (dark filled center box) and standard deviation (lighter center box) of the center of the arborization and the average (dark filled outer box) and standard deviation (lighter outer box) of the spread of the arborization as measured vertically and horizontally from center across preparations. Scale bars: 100 μm. coc, circumoesophageal connective; ion, inferior oesophageal nerve; son, superior oesophageal nerve.
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Related In: Results  -  Collection

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pone.0142956.g002: MCN1 neurites arborize in the anterior CoG.(A) A single optical slice includes a portion of MCN1 (intracellular fill with Alexa 568; green) and DIC optics to view the outline of the tissue. The MCN1 neurites are located in the anterior region of the CoG (bracket), while in this example, the soma is located more posteriorly (filled arrow) and the axon leaves the CoG through the ion (open arrow). (B) A higher magnification volume rendering of a z-stack (249 optical slices, 1.0 μm interval) of the MCN1 fill from (A) reveals the full extent of the MCN1 arborization within the anterior CoG. Arrows as in (A). (C) Average location of the MCN1 soma (open square) and MCN1 neurites (filled squares) in the x-y plane of the CoG are plotted. The soma was located in the posterior CoG while the neurites were in the anterior region. The neurite location is reported as the average (dark filled center box) and standard deviation (lighter center box) of the center of the arborization and the average (dark filled outer box) and standard deviation (lighter outer box) of the spread of the arborization as measured vertically and horizontally from center across preparations. Scale bars: 100 μm. coc, circumoesophageal connective; ion, inferior oesophageal nerve; son, superior oesophageal nerve.
Mentions: To quantify locations in the anteroposterior and mediolateral dimensions, both DIC and fluorescence signals were collected at multiple dorsoventral planes. For each ganglion, a single optical slice of the CoG at the depth at which the diameter of the ganglion was greatest was used to identify borders of the ganglion. Specifically, tangent lines along a standardized circular shape were aligned to the anterior, posterior, and medial edges of the CoG, with the circle oriented such that the lateral tangent line aligned with the axon tract of the coc (Fig 2, inset) (Corel Draw, Corel Corporation). In each preparation, this alignment was then maintained across single optical slices above and below the widest diameter slice to quantify structure locations in the anteroposterior and mediolateral axes. This allowed us to collapse the analysis into a two-dimensional plot despite differences in the diameter of the ganglion throughout the dorsoventral axis. To normalize the data and eliminate differences due to inter-preparation variability in CoG size, measurements were scaled such that medial was designated as 0, lateral as 100, posterior as 0, and anterior as 100 in the plane in which the CoG diameter was greatest (Fig 2). All analyses of structures throughout the depth of a ganglion were performed on a single z-stack from a continuous confocal session to ensure alignment of optical slices. Using a grid overlaid on the circle in each relevant image (VistaMetrix software; SkillCrest) locations of the MCN1 soma, MCN1 neurites, the ACO, and the GPR axon bundle were quantified. For larger structures such as the MCN1 neuropil and the ACO, both the center and the margins in the anteroposterior and mediolateral dimensions were determined. For the MCN1 soma and the GPR axon bundle, only the center of the structure was determined.

Bottom Line: The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1.The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations.Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, OH, United States of America.

ABSTRACT
In rhythmic motor systems, descending projection neuron inputs elicit distinct outputs from their target central pattern generator (CPG) circuits. Projection neuron activity is regulated by sensory inputs and inputs from other regions of the nervous system, relaying information about the current status of an organism. To gain insight into the organization of multiple inputs targeting a projection neuron, we used the identified neuron MCN1 in the stomatogastric nervous system of the crab, Cancer borealis. MCN1 originates in the commissural ganglion and projects to the stomatogastric ganglion (STG). MCN1 activity is differentially regulated by multiple inputs including neuroendocrine (POC) and proprioceptive (GPR) neurons, to elicit distinct outputs from CPG circuits in the STG. We asked whether these defined inputs are compact and spatially segregated or dispersed and overlapping relative to their target projection neuron. Immunocytochemical labeling, intracellular dye injection and three-dimensional (3D) confocal microscopy revealed overlap of MCN1 neurites and POC and GPR terminals. The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1. The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations. Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1. Consistent innervation patterns suggest that integration of the sensory GPR and peptidergic POC inputs occur through more distributed and more tightly constrained anatomical interactions with their common modulatory projection neuron target than anticipated.

Show MeSH
Related in: MedlinePlus