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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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The ACO and MCN1 morphologies are similar within preparations despite variability across preparations.Volume renderings of z-stacks demonstrate the arborizations of the ACO (A, D) and MCN1 (B, E) and their organization relative to each other (C, F) in two preparations. Both in an example of a round-shaped ACO (A-C) and an elongated ACO (D-F), the MCN1 and ACO arborizations share a similar morphology. Images consist of CabTRP Ia-IR (ACO: red) and intracellular fills of MCN1 with Alexa 568 (green). A-C: 231 slices, 1.0 μm interval; D-F: 246 slices, 1.0 μm interval. Open arrows indicate the MCN1 axon (B, E) and POC (A, D) axon bundle while the filled arrows point to the MCN1 primary neurite (B, E) and the ACO (A, D). Scale bars: 50 μm.
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pone.0142956.g006: The ACO and MCN1 morphologies are similar within preparations despite variability across preparations.Volume renderings of z-stacks demonstrate the arborizations of the ACO (A, D) and MCN1 (B, E) and their organization relative to each other (C, F) in two preparations. Both in an example of a round-shaped ACO (A-C) and an elongated ACO (D-F), the MCN1 and ACO arborizations share a similar morphology. Images consist of CabTRP Ia-IR (ACO: red) and intracellular fills of MCN1 with Alexa 568 (green). A-C: 231 slices, 1.0 μm interval; D-F: 246 slices, 1.0 μm interval. Open arrows indicate the MCN1 axon (B, E) and POC (A, D) axon bundle while the filled arrows point to the MCN1 primary neurite (B, E) and the ACO (A, D). Scale bars: 50 μm.

Mentions: A monoclonal antibody generated against substance P (Accurate Chemical and Scientific Corporation; 1:300 for 72 hours) (Table 1) was used with goat anti-rat Alexa Fluor 488 or 568 (dilution of 1:300 for 16–18 hours; Life Technologies) secondary antibody to label the POC axons and their terminals (ACO). The substance P antibody specifically recognizes the conserved C-terminal of substance P and related peptides [42–44]. This includes specificity for the endogenous crab peptide, Cancer borealis tachykinin related peptide Ia (CabTRP Ia). Labeling in the STNS is blocked by preabsorption with substance P (10−7 M) and with native CabTRP Ia (10−4 M) [43,44]. The distribution of CabTRP Ia immunoreactivity in this study was similar to previous studies [28,34,43]. As the native C. borealis peptide recognized by the monoclonal antibody generated against substance P has been identified, we will refer to labeling with this antibody as CabTRP Ia-immunoreactivity (CabTRP Ia-IR). MCN1 also contains CabTRP Ia and is labeled with the Substance P antibody. However, the MCN1 label is typically weak while the ACO label is intense [28, 34]. In order to prevent saturation of the ACO label, it was necessary to decrease the illumination intensity much below that necessary to visualize CabTRP-IR in MCN1. In preparations in which MCN1 was labeled with Alexa dye the Alexa labeled MCN1 soma, axon, and neurites which did not overlap with the ACO structure were not visible in the CabTRP Ia only channel (e.g., Fig 6A and 6D). This was verified in each preparation in which double labeling was performed. Additionally, the ACO is a flocculent structure [34] (e.g., Figs 5–7), distinct from the more typical neuronal branch structure of MCN1 neurites. Thus, the distinct morphologies and intensity of CabTPR Ia-IR of MCN1 and the ACO enabled identification of CabTRP Ia-IR as ACO processes, distinct from MCN1 neurites, using appropriate confocal microscope settings.


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

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

The ACO and MCN1 morphologies are similar within preparations despite variability across preparations.Volume renderings of z-stacks demonstrate the arborizations of the ACO (A, D) and MCN1 (B, E) and their organization relative to each other (C, F) in two preparations. Both in an example of a round-shaped ACO (A-C) and an elongated ACO (D-F), the MCN1 and ACO arborizations share a similar morphology. Images consist of CabTRP Ia-IR (ACO: red) and intracellular fills of MCN1 with Alexa 568 (green). A-C: 231 slices, 1.0 μm interval; D-F: 246 slices, 1.0 μm interval. Open arrows indicate the MCN1 axon (B, E) and POC (A, D) axon bundle while the filled arrows point to the MCN1 primary neurite (B, E) and the ACO (A, D). Scale bars: 50 μm.
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Related In: Results  -  Collection

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pone.0142956.g006: The ACO and MCN1 morphologies are similar within preparations despite variability across preparations.Volume renderings of z-stacks demonstrate the arborizations of the ACO (A, D) and MCN1 (B, E) and their organization relative to each other (C, F) in two preparations. Both in an example of a round-shaped ACO (A-C) and an elongated ACO (D-F), the MCN1 and ACO arborizations share a similar morphology. Images consist of CabTRP Ia-IR (ACO: red) and intracellular fills of MCN1 with Alexa 568 (green). A-C: 231 slices, 1.0 μm interval; D-F: 246 slices, 1.0 μm interval. Open arrows indicate the MCN1 axon (B, E) and POC (A, D) axon bundle while the filled arrows point to the MCN1 primary neurite (B, E) and the ACO (A, D). Scale bars: 50 μm.
Mentions: A monoclonal antibody generated against substance P (Accurate Chemical and Scientific Corporation; 1:300 for 72 hours) (Table 1) was used with goat anti-rat Alexa Fluor 488 or 568 (dilution of 1:300 for 16–18 hours; Life Technologies) secondary antibody to label the POC axons and their terminals (ACO). The substance P antibody specifically recognizes the conserved C-terminal of substance P and related peptides [42–44]. This includes specificity for the endogenous crab peptide, Cancer borealis tachykinin related peptide Ia (CabTRP Ia). Labeling in the STNS is blocked by preabsorption with substance P (10−7 M) and with native CabTRP Ia (10−4 M) [43,44]. The distribution of CabTRP Ia immunoreactivity in this study was similar to previous studies [28,34,43]. As the native C. borealis peptide recognized by the monoclonal antibody generated against substance P has been identified, we will refer to labeling with this antibody as CabTRP Ia-immunoreactivity (CabTRP Ia-IR). MCN1 also contains CabTRP Ia and is labeled with the Substance P antibody. However, the MCN1 label is typically weak while the ACO label is intense [28, 34]. In order to prevent saturation of the ACO label, it was necessary to decrease the illumination intensity much below that necessary to visualize CabTRP-IR in MCN1. In preparations in which MCN1 was labeled with Alexa dye the Alexa labeled MCN1 soma, axon, and neurites which did not overlap with the ACO structure were not visible in the CabTRP Ia only channel (e.g., Fig 6A and 6D). This was verified in each preparation in which double labeling was performed. Additionally, the ACO is a flocculent structure [34] (e.g., Figs 5–7), distinct from the more typical neuronal branch structure of MCN1 neurites. Thus, the distinct morphologies and intensity of CabTPR Ia-IR of MCN1 and the ACO enabled identification of CabTRP Ia-IR as ACO processes, distinct from MCN1 neurites, using appropriate confocal microscope settings.

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