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N-cadherin relocalizes from the periphery to the center of the synapse after transient synaptic stimulation in hippocampal neurons.

Yam PT, Pincus Z, Gupta GD, Bashkurov M, Charron F, Pelletier L, Colman DR - PLoS ONE (2013)

Bottom Line: We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft.In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells.These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada ; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.

ABSTRACT
N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

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Structured illumination microscopy can resolve different synaptic domains.(A,B) 17-20 DIV hippocampal neurons immunostained for the active zone protein bassoon and the post-synaptic density protein PSD95, or (C) bassoon and the synaptic vesicle protein vGlut1. (A) Imaged with widefield fluorescence microscopy, bassoon and PSD95 puncta overlap. (B) Imaged with SIM, the increased resolution shows that bassoon and PSD95 occupy separate domains, consistent with their function in the pre- and post-synaptic compartments respectively. (C) Within the presynaptic bouton, the synaptic vesicle pool (represented by vGlut1) and the active zone (represented by bassoon) can be distinguished with SIM. (D) Manders’ coefficient measurement of the proportion of PSD95 signal which overlaps with the bassoon signal and vice versa. Unpaired t-test, 6 fields of view. (E) Manders’ coefficient measurement of the proportion of bassoon signal which overlaps with the vGlut1 signal and vice versa. Unpaired t-test, 6 fields of view. Graphs represent the mean ± s.e.m. (F,G) During depolarization of neurons with 50 mM KCl, the synaptic vesicle markers vGlut1 and synaptophysin become smaller and more numerous, and spread over larger area (brackets). (H) The vGlut1 puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, 7 fields of view, ≥980 vGlut1 puncta analyzed per condition. (I) The number of vGlut1 puncta per bassoon puncta increases during KCl depolarization. Graph represents the mean ± s.e.m. Unpaired t-test, 7 fields of view, ≥874 bassoon puncta analyzed per condition. (J) The synaptophysin puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, ≥10 fields of view per condition, ≥533 synaptophysin puncta analyzed per condition.
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pone-0079679-g001: Structured illumination microscopy can resolve different synaptic domains.(A,B) 17-20 DIV hippocampal neurons immunostained for the active zone protein bassoon and the post-synaptic density protein PSD95, or (C) bassoon and the synaptic vesicle protein vGlut1. (A) Imaged with widefield fluorescence microscopy, bassoon and PSD95 puncta overlap. (B) Imaged with SIM, the increased resolution shows that bassoon and PSD95 occupy separate domains, consistent with their function in the pre- and post-synaptic compartments respectively. (C) Within the presynaptic bouton, the synaptic vesicle pool (represented by vGlut1) and the active zone (represented by bassoon) can be distinguished with SIM. (D) Manders’ coefficient measurement of the proportion of PSD95 signal which overlaps with the bassoon signal and vice versa. Unpaired t-test, 6 fields of view. (E) Manders’ coefficient measurement of the proportion of bassoon signal which overlaps with the vGlut1 signal and vice versa. Unpaired t-test, 6 fields of view. Graphs represent the mean ± s.e.m. (F,G) During depolarization of neurons with 50 mM KCl, the synaptic vesicle markers vGlut1 and synaptophysin become smaller and more numerous, and spread over larger area (brackets). (H) The vGlut1 puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, 7 fields of view, ≥980 vGlut1 puncta analyzed per condition. (I) The number of vGlut1 puncta per bassoon puncta increases during KCl depolarization. Graph represents the mean ± s.e.m. Unpaired t-test, 7 fields of view, ≥874 bassoon puncta analyzed per condition. (J) The synaptophysin puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, ≥10 fields of view per condition, ≥533 synaptophysin puncta analyzed per condition.

Mentions: Conventional light microscopy has a resolution limit of ~200-250 nm, which is insufficient to resolve details within synapses which generally range in diameter from ~200-500 nm. Thus to visualize how N-cadherin localizes within synapses, we turned to SIM, a super-resolution microscopy technique with a two-fold greater resolution along the lateral and axial directions, resulting in an x-y resolution of ~100 nm [27–29]. We studied synapses in cultured rat hippocampal neurons at 17-20 DIV (days in vitro), by which time mature synapses were present. Immunostaining these neurons for bassoon, an active zone (presynaptic) protein, and PSD95, a postsynaptic density protein, revealed mostly overlapping puncta when imaged with conventional widefield fluorescence microscopy (Figure 1A). When imaged with SIM, the bassoon and PSD95 puncta were more clearly resolved and occupied adjacent domains apposed to each other, reflecting their localization to opposing sides of the synapse (Figure 1B). We measured the overlap between the bassoon and PSD95 signals with the Manders’ coefficient [30], which indicates the fraction of the bassoon signal coincident with the PSD95 signal and vice versa. This showed that only one-third of the signal between the bassoon and PSD95 stainings overlapped (Figure 1D), implying that most of the bassoon and PSD95 signals occupy different zones. Given that bassoon and PSD95 on either side of the synaptic cleft are separated by ~90 nm [31], which is less than the x-y resolution of SIM at ~100 nm, and that synapses are not always oriented with their pre- and post-synaptic domains aligned along the x-y plane, some colocalization between the bassoon and PSD95 signal would be expected. However, most of the bassoon and PSD95 signal are not colocalized, indicating that when imaged with SIM, the bassoon and PSD95 puncta are distinct, slightly overlapping domains.


N-cadherin relocalizes from the periphery to the center of the synapse after transient synaptic stimulation in hippocampal neurons.

Yam PT, Pincus Z, Gupta GD, Bashkurov M, Charron F, Pelletier L, Colman DR - PLoS ONE (2013)

Structured illumination microscopy can resolve different synaptic domains.(A,B) 17-20 DIV hippocampal neurons immunostained for the active zone protein bassoon and the post-synaptic density protein PSD95, or (C) bassoon and the synaptic vesicle protein vGlut1. (A) Imaged with widefield fluorescence microscopy, bassoon and PSD95 puncta overlap. (B) Imaged with SIM, the increased resolution shows that bassoon and PSD95 occupy separate domains, consistent with their function in the pre- and post-synaptic compartments respectively. (C) Within the presynaptic bouton, the synaptic vesicle pool (represented by vGlut1) and the active zone (represented by bassoon) can be distinguished with SIM. (D) Manders’ coefficient measurement of the proportion of PSD95 signal which overlaps with the bassoon signal and vice versa. Unpaired t-test, 6 fields of view. (E) Manders’ coefficient measurement of the proportion of bassoon signal which overlaps with the vGlut1 signal and vice versa. Unpaired t-test, 6 fields of view. Graphs represent the mean ± s.e.m. (F,G) During depolarization of neurons with 50 mM KCl, the synaptic vesicle markers vGlut1 and synaptophysin become smaller and more numerous, and spread over larger area (brackets). (H) The vGlut1 puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, 7 fields of view, ≥980 vGlut1 puncta analyzed per condition. (I) The number of vGlut1 puncta per bassoon puncta increases during KCl depolarization. Graph represents the mean ± s.e.m. Unpaired t-test, 7 fields of view, ≥874 bassoon puncta analyzed per condition. (J) The synaptophysin puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, ≥10 fields of view per condition, ≥533 synaptophysin puncta analyzed per condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815108&req=5

pone-0079679-g001: Structured illumination microscopy can resolve different synaptic domains.(A,B) 17-20 DIV hippocampal neurons immunostained for the active zone protein bassoon and the post-synaptic density protein PSD95, or (C) bassoon and the synaptic vesicle protein vGlut1. (A) Imaged with widefield fluorescence microscopy, bassoon and PSD95 puncta overlap. (B) Imaged with SIM, the increased resolution shows that bassoon and PSD95 occupy separate domains, consistent with their function in the pre- and post-synaptic compartments respectively. (C) Within the presynaptic bouton, the synaptic vesicle pool (represented by vGlut1) and the active zone (represented by bassoon) can be distinguished with SIM. (D) Manders’ coefficient measurement of the proportion of PSD95 signal which overlaps with the bassoon signal and vice versa. Unpaired t-test, 6 fields of view. (E) Manders’ coefficient measurement of the proportion of bassoon signal which overlaps with the vGlut1 signal and vice versa. Unpaired t-test, 6 fields of view. Graphs represent the mean ± s.e.m. (F,G) During depolarization of neurons with 50 mM KCl, the synaptic vesicle markers vGlut1 and synaptophysin become smaller and more numerous, and spread over larger area (brackets). (H) The vGlut1 puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, 7 fields of view, ≥980 vGlut1 puncta analyzed per condition. (I) The number of vGlut1 puncta per bassoon puncta increases during KCl depolarization. Graph represents the mean ± s.e.m. Unpaired t-test, 7 fields of view, ≥874 bassoon puncta analyzed per condition. (J) The synaptophysin puncta volume decreases during KCl depolarization. Box and whiskers graph. Mann Whitney test, ≥10 fields of view per condition, ≥533 synaptophysin puncta analyzed per condition.
Mentions: Conventional light microscopy has a resolution limit of ~200-250 nm, which is insufficient to resolve details within synapses which generally range in diameter from ~200-500 nm. Thus to visualize how N-cadherin localizes within synapses, we turned to SIM, a super-resolution microscopy technique with a two-fold greater resolution along the lateral and axial directions, resulting in an x-y resolution of ~100 nm [27–29]. We studied synapses in cultured rat hippocampal neurons at 17-20 DIV (days in vitro), by which time mature synapses were present. Immunostaining these neurons for bassoon, an active zone (presynaptic) protein, and PSD95, a postsynaptic density protein, revealed mostly overlapping puncta when imaged with conventional widefield fluorescence microscopy (Figure 1A). When imaged with SIM, the bassoon and PSD95 puncta were more clearly resolved and occupied adjacent domains apposed to each other, reflecting their localization to opposing sides of the synapse (Figure 1B). We measured the overlap between the bassoon and PSD95 signals with the Manders’ coefficient [30], which indicates the fraction of the bassoon signal coincident with the PSD95 signal and vice versa. This showed that only one-third of the signal between the bassoon and PSD95 stainings overlapped (Figure 1D), implying that most of the bassoon and PSD95 signals occupy different zones. Given that bassoon and PSD95 on either side of the synaptic cleft are separated by ~90 nm [31], which is less than the x-y resolution of SIM at ~100 nm, and that synapses are not always oriented with their pre- and post-synaptic domains aligned along the x-y plane, some colocalization between the bassoon and PSD95 signal would be expected. However, most of the bassoon and PSD95 signal are not colocalized, indicating that when imaged with SIM, the bassoon and PSD95 puncta are distinct, slightly overlapping domains.

Bottom Line: We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft.In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells.These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University, Montreal, Quebec, Canada ; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.

ABSTRACT
N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

Show MeSH
Related in: MedlinePlus