In trans neuregulin3-Caspr3 interaction controls DA axonal bassoon cluster development
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Cited by 3 publications
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While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a presynaptic marker, bassoon, with two different postsynaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, postsynaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental neuron (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.
While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a presynaptic marker, bassoon, with two different postsynaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, postsynaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental neuron (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.
Investigation of the impact of the Caspr3 receptor in brain microvascular endothelial bEnd.3 cells on invasion function of Escherichia coli K1
Objective To investigate the role of the Caspr3 receptor in bEnd.3 cells on the invasion function of Escherichia coli K1 strain. Methods 1. Cell Culture and Bacterial Infection:bEnd.3 cells and E. coli K1 (E44) were cultured in vitro. Confocal laser microscopy was used to observe the changes in F-actin and Caspr3 in bEnd.3 cells following bacterial addition. 2. Caspr3 Knockdown: bEnd.3 cells were transfected with lentivirus to establish stable Caspr3 knockdown (Lenti-sh-Caspr3) and null-loaded (Lenti-sh-NC). Transfection efficiency was assessed via Western blot and confocal laser microscopy. 3. Bacterial Adhesion and Invasion Assays: Lenti-sh-NC and Lenti-sh-Caspr3 cells were infected with E. coli E44 to evaluate bacterial adhesion and invasion. 4. Changes in F-actin: Confocal laser microscopy was used to observe the changes in F-actin after E. coli E44 invaded Lenti-sh-Caspr3. Results 1. bEnd.3 cells expressed Caspr3. Compared with the control group, there was no significant change in the fluorescence intensity of Caspr3, F-actin aggregation occurred, and the co-localization with Caspr3 at the aggregation was more significant after E44 infection. 2. Western blot and confocal microscopy confirmed a significant reduction in Caspr3 protein expression in Lenti-sh-Caspr3 compared to bEnd.3 and Lenti-sh-NC. 3. No significant difference in E44 adhesion rate was observed between Lenti-sh-Caspr3 and the control group. However, the invasion rate of E44 in Lenti-sh-Caspr3 was significantly lower than in the control group. 4. F-actin aggregation was significantly reduced in Lenti-sh-Caspr3 after E44 infection. Conclusions bEnd.3 cells expresses Caspr3, which co-localizes with F-actin during E. coli meningitis invasion. Knockdown of Caspr3 did not affect the adhesion of E. coli E44 to host cells, but the number of bacterial invasions was significantly decreased, and F-actin aggregation of the host cells was reduced. Caspr3 may influence the invasion of host cells by E. coli meningitis by regulating F-actin rearrangement.
While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.
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