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Synapse clusters are preferentially formed by synapses with large recycling pool sizes.

Welzel O, Tischbirek CH, Jung J, Kohler EM, Svetlitchny A, Henkel AW, Kornhuber J, Groemer TW - PLoS ONE (2010)

Bottom Line: Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes.Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43.Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany.

ABSTRACT
Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1-43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.

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Distribution of the number of neighbouring synapses in cultured hippocampal neurons.A, Difference-image of images before and after electrical stimulation of FM 1–43 labelled neurons in a representative experiment. B, Analytical image calculated from A. Positions of automatically detected synapses are represented by dots and colour-coded according to their number of neighbouring synapses in a 50 µm2 environment. Environments of two representative synapses with two and eleven neighbours are highlighted, respectively. C, Distribution of the number of synapse neighbours (N = 5, n = 3149). (Scale bars 4 µm).
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pone-0013514-g001: Distribution of the number of neighbouring synapses in cultured hippocampal neurons.A, Difference-image of images before and after electrical stimulation of FM 1–43 labelled neurons in a representative experiment. B, Analytical image calculated from A. Positions of automatically detected synapses are represented by dots and colour-coded according to their number of neighbouring synapses in a 50 µm2 environment. Environments of two representative synapses with two and eleven neighbours are highlighted, respectively. C, Distribution of the number of synapse neighbours (N = 5, n = 3149). (Scale bars 4 µm).

Mentions: All image and data analysis was performed using custom-written routines in MATLAB (The MathWorks, Inc., Natick). The mean background determined from the intensity histogram of recorded image stacks was subtracted [36] and the resulting image stacks were used to automatically define peak regions of interest of synaptic bouton size [37], where AP-evoked fluorescence decrease (FM 1–43 and anti-synaptotagmin-1-cypHer) or increase (synaptopHluorin) occurred in difference images. The average for all regions of interest was calculated for each image to obtain fluorescence intensity profiles. For the determination of the total pool size ΔFtot the mean of five values before the onset of the stimulus was subtracted from the mean of five values after the total destain period. The absolute fluorescence decrease ΔF (FM 1–43 and anti-synaptotagmin-1-cypHer) or increase (synaptopHluorin) was calculated as the difference of the mean of three (anti-synaptotagmin-1-cypHer) or five (FM 1–43 and synaptopHluorin) values before the first stimulation and the mean of three or five values after the end of the first stimulus with 600 APs or 1200 APs, respectively. The number of neighbours was determined by counting the detected spots in a circular region with a diameter of 30 pixels (pixel size ps = 267 nm at binning 2×2), which correspond to a diameter of 8 µm, around each spot (see Figure 1B) or approximately 50 µm2. For the determination of the synapse diameter each spot was fitted by a rotation symmetric gaussian profile: , where x and y are the image coordinates, μx and μy are the center coordinates of each spot and σ is the standard deviation. The area around each spot chosen for the fit was 10×10 pixels. The tolerance for the used unconstrained nonlinear optimization was and results of the fit with a coefficient of determination below 0.8 were discarded. The diameter of each synapse was calculated as the full width at half maximum, using the following equation: , with ps as the pixel size obtained from physical camera pixel size and magnification of the used objective.


Synapse clusters are preferentially formed by synapses with large recycling pool sizes.

Welzel O, Tischbirek CH, Jung J, Kohler EM, Svetlitchny A, Henkel AW, Kornhuber J, Groemer TW - PLoS ONE (2010)

Distribution of the number of neighbouring synapses in cultured hippocampal neurons.A, Difference-image of images before and after electrical stimulation of FM 1–43 labelled neurons in a representative experiment. B, Analytical image calculated from A. Positions of automatically detected synapses are represented by dots and colour-coded according to their number of neighbouring synapses in a 50 µm2 environment. Environments of two representative synapses with two and eleven neighbours are highlighted, respectively. C, Distribution of the number of synapse neighbours (N = 5, n = 3149). (Scale bars 4 µm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958124&req=5

pone-0013514-g001: Distribution of the number of neighbouring synapses in cultured hippocampal neurons.A, Difference-image of images before and after electrical stimulation of FM 1–43 labelled neurons in a representative experiment. B, Analytical image calculated from A. Positions of automatically detected synapses are represented by dots and colour-coded according to their number of neighbouring synapses in a 50 µm2 environment. Environments of two representative synapses with two and eleven neighbours are highlighted, respectively. C, Distribution of the number of synapse neighbours (N = 5, n = 3149). (Scale bars 4 µm).
Mentions: All image and data analysis was performed using custom-written routines in MATLAB (The MathWorks, Inc., Natick). The mean background determined from the intensity histogram of recorded image stacks was subtracted [36] and the resulting image stacks were used to automatically define peak regions of interest of synaptic bouton size [37], where AP-evoked fluorescence decrease (FM 1–43 and anti-synaptotagmin-1-cypHer) or increase (synaptopHluorin) occurred in difference images. The average for all regions of interest was calculated for each image to obtain fluorescence intensity profiles. For the determination of the total pool size ΔFtot the mean of five values before the onset of the stimulus was subtracted from the mean of five values after the total destain period. The absolute fluorescence decrease ΔF (FM 1–43 and anti-synaptotagmin-1-cypHer) or increase (synaptopHluorin) was calculated as the difference of the mean of three (anti-synaptotagmin-1-cypHer) or five (FM 1–43 and synaptopHluorin) values before the first stimulation and the mean of three or five values after the end of the first stimulus with 600 APs or 1200 APs, respectively. The number of neighbours was determined by counting the detected spots in a circular region with a diameter of 30 pixels (pixel size ps = 267 nm at binning 2×2), which correspond to a diameter of 8 µm, around each spot (see Figure 1B) or approximately 50 µm2. For the determination of the synapse diameter each spot was fitted by a rotation symmetric gaussian profile: , where x and y are the image coordinates, μx and μy are the center coordinates of each spot and σ is the standard deviation. The area around each spot chosen for the fit was 10×10 pixels. The tolerance for the used unconstrained nonlinear optimization was and results of the fit with a coefficient of determination below 0.8 were discarded. The diameter of each synapse was calculated as the full width at half maximum, using the following equation: , with ps as the pixel size obtained from physical camera pixel size and magnification of the used objective.

Bottom Line: Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes.Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43.Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany.

ABSTRACT
Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1-43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.

Show MeSH
Related in: MedlinePlus