Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: High content monitoring of SV recycling. Hippocampal neurons were transfected with synaptophysin-pHluorin (sypHy) on day in vitro ( DIV) 7 and imaged at DIV 13–15. Transfected neurons were challenged with a pulse of impermeant acidic buffer for 1 min (to reveal the surface fraction of sypHy, shaded pink). After returning to imaging buffer for 2 min, neurons were stimulated with a train of 300 action potentials (10 Hz) followed by a 5 min rest period. After this rest period neurons were stimulated with a further train of 400 action potentials (40 Hz). Finally, after a further 3 min neurons were exposed to an alkaline buffer (NH 4 Cl, to reveal the total SV pool, blue shaded region). Stimulation is indicated by bars. Mean trace displays the average sypHy fluorescent response of wild-type neurons ± SEM. Traces are ΔF/F 0 and are presented as a fraction of the total SV pool

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Transfection, In Vitro, Imaging

Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: Nrxn1 +/− neurons display depressed ADBE. A-C ) Hippocampal synaptosome lysates from either wild-type (WT) or Nrxn1 +/− rats were probed for the presence of Neurexin-1 (Nrxn1, A ) and total protein ( B ). ( C ) Quantification of Nrxn1 levels in both are displayed, normalised to total protein ± SEM, n = 3 independent synaptosome preparations for both WT and Nrxn1 +/− , * p = 0.0365, unpaired t test. D-J ) Hippocampal neurons derived from either WT or Nrxn1 +/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. D , H ) Mean sypHy fluorescence traces of WT and Nrxn1 +/− hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( D ) or peak fluorescence during electrical stimulation ( H ) ± SEM. E ) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F , G ) Mean peak sypHy response in response to either 10 Hz ( F ) or 40 Hz ( G ) action potential trains ± SEM. ( I, J ) Mean sypHy retrieval time constants (t) in response to either 10 Hz ( I ) or 40 Hz ( J ) action potential trains ± SEM. For D-J n = 9 coverslips for both WT and Nrxn1 +/− from 3 independent cultures. ( K , L ) Primary hippocampal cultures derived either WT or Nrxn1 +/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. ( K ) Representative images of TMR-dextran uptake in WT and Nrxn1 +/− cultures. Scale bar = 50 μm. L ) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM ( n = 13 coverslips from 3 independent cultures for WT and Nrxn1 +/− ). ( M ) Primary hippocampal cultures derived from either WT or Nrxn1 +/− rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. ( N ) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 12 coverslips, Nrxn1 +/− n = 15 coverslips from 3 independent cultures). In all cases an unpaired two-sided students t test was performed, ** p = 0.0037, unpaired t test

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Derivative Assay, Transfection, In Vitro, Fluorescence, Staining

Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: Nlgn3 −/y neurons display depressed ADBE. A-C ) Hippocampal synaptosome lysates from either wild-type (WT) or Nlgn3 −/y rats were probed for the presence of Neuroligin-3 (Nlgn3, A ) and total protein ( B ). ( C ) Quantification of Nlgn3 levels in both are displayed, normalised to total protein ± SEM, n = 4 independent synaptosome preparations for both WT and Nlgn3 −/y , *** p < 0.0001, unpaired t test. ( D-J ) Hippocampal neurons derived from either WT or Nlgn3 −/y rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. ( D, H ) Mean sypHy fluorescence traces of WT and Nlgn3 −/y hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( D ) or peak fluorescence during electrical stimulation ( H ) ± SEM. ( E ) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F , G ) Mean peak sypHy response in response to either 10 Hz ( F ) or 40 Hz ( G ) action potential trains ± SEM. ( I , J ) Mean sypHy retrieval time constants (τ) in response to either 10 Hz ( I ) or 40 Hz ( J ) action potential trains ± SEM. For D-J WT n = 11 coverslips, Nlgn3 −/y n = 14 from 3 independent cultures. ( K , L ) Primary hippocampal cultures derived either WT or Nlgn3 −/y rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. ( K ) Representative images of TMR-dextran uptake in WT and Nlgn3 −/y cultures. Scale bar = 50 μm. ( L ) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 11 coverslips, Nlgn3 −/y n = 12 from 3 independent cultures). ( M ) Primary hippocampal cultures derived from either WT or Nlgn3 −/y rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. ( N ) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 14 coverslips, Nlgn3 −/y n = 15 coverslips from 3 independent cultures). In all cases an unpaired two-sided students t test was performed, except E , F and J , * p = 0.0112, unpaired t test

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Derivative Assay, Transfection, In Vitro, Fluorescence, Staining

Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: Syngap −/− neurons display depressed ADBE. Hippocampal neurons derived from either wild-type (WT), Syngap +/− or Syngap −/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. A , E ) Mean sypHy fluorescence traces of WT, Syngap +/− or Syngap −/− hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM. B ) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. C , D ) Mean peak sypHy response in response to either 10 Hz ( C ) or 40 Hz ( D ) action potential trains ± SEM. F, G ) Mean sypHy retrieval time constants (τ) in response to either 10 Hz ( F ) or 40 Hz ( G ) action potential trains ± SEM. For A-G , WT n = 12 coverslips, Syngap +/− n = 11 and Syngap −/− n = 12 from 3 independent cultures. H-I ) Primary hippocampal cultures derived either wild-type (WT), Syngap +/− or Syngap −/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. H ) Representative images of TMR-dextran uptake in WT, Syngap +/− or Syngap −/− cultures. Scale bar = 50 μm. I ) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 15 coverslips, Syngap +/− and Syngap −/− n = 16 from 3 independent cultures). J ) Primary hippocampal cultures derived from either WT, Syngap +/− or Syngap −/− rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. K ) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT and Syngap +/− n = 15 coverslips, Syngap −/− n = 13 from 3 independent cultures). In all cases a one-way ANOVA was performed, ** p = 0.01

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Derivative Assay, Transfection, In Vitro, Fluorescence, Staining

Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: Syngap Δ − GAP /Δ−GAP neurons display depressed ADBE. Hippocampal neurons derived from either wild-type (WT), Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM. B ) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. C , D ) Mean peak sypHy response in response to either 10 Hz ( C ) or 40 Hz ( D ) action potential trains ± SEM. F , G ) Mean sypHy retrieval time constants (τ) in response to either 10 Hz ( F ) or 40 Hz ( G ) action potential trains ± SEM. For A-G , WT n = 14 coverslips, Syngap +/Δ−GAP n = 12 and Syngap Δ − GAP /Δ−GAP n = 10 from 3 independent cultures. H-I ) Primary hippocampal cultures derived either wild-type (WT), Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. H ) Representative images of TMR-dextran uptake in WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP cultures. Scale bar = 50 μm. I ) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 8 coverslips, Syngap +/Δ−GAP and Syngap Δ − GAP /Δ−GAP n = 9 from 3 independent cultures). J ) Primary hippocampal cultures derived from either WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. K ) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 10 coverslips, Syngap +/Δ−GAP n = 11 and Syngap Δ − GAP /Δ−GAP n = 13 from 3 independent cultures). In all cases a one-way ANOVA was performed, * p = 0.017

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Derivative Assay, Transfection, In Vitro, Fluorescence, Staining

Journal: Molecular Autism

Article Title: Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

doi: 10.1186/s13229-025-00660-6

Figure Lengend Snippet: Pten +/− neurons display depressed ADBE. A-C ) Hippocampal synaptosome lysates from either wild-type (WT) or Pten +/− rats were probed for the presence of PTEN ( A ) and total protein ( B ). ( C ) Quantification of PTEN levels in both are displayed, normalised to total protein ± SEM, n = 5 independent synaptosome preparations for both WT and Pten +/− , * p = 0.0178, unpaired t test. D-J ) Hippocampal neurons derived from either WT or Pten +/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. D , H ) Mean sypHy fluorescence traces of WT and Pten +/− hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( D ) or peak fluorescence during electrical stimulation ( H ) ± SEM. E ) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F , G ) Mean peak sypHy response in response to either 10 Hz ( F ) or 40 Hz ( G ) action potential trains ± SEM. I , J ) Mean sypHy retrieval time constants (τ) in response to either 10 Hz ( I ) or 40 Hz ( J ) action potential trains ± SEM. For D-J , WT n = 15 coverslips, Pten +/− n = 19 from 3 independent cultures. K , L ) Primary hippocampal cultures derived either wild-type (WT) or Pten +/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. K ) Representative images of TMR-dextran uptake in WT and Pten +/− cultures. Scale bar = 50 μm. L ) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 24 coverslips, Pten +/− n = 20 from 3 independent cultures)

Article Snippet: A , E ) Mean sypHy fluorescence traces of WT, Syngap +/Δ−GAP or Syngap Δ − GAP /Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH 4 Cl ( A ) or peak fluorescence during electrical stimulation ( E ) ± SEM.

Techniques: Derivative Assay, Transfection, In Vitro, Fluorescence