Abstract:Key points
CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held.
CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1.
The half‐life of ELKS is on the timescale of days and not weeks.
Synaptic transmission was not impacted by the loss of CAST/ELKS.
CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit… Show more
“…These values are similar to titers obtained using the loxP HV, as reported (NG163-R2). 17 , 27 , 28 , 29 , 30 Taken together, these data demonstrate that the vox sites have no effect on viral DNA replication, assembly, or infectivity of the HV. Figure 2 VikAD enables excision of VoxHV packaging signal and amplification of HdAd (A) Map of VoxHV genome with MfeI restriction sites.…”
Recombinant viral vectors have become integral tools for basic
in vivo
research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression
in vitro
and
in vivo
. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either
loxP
or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.
“…These values are similar to titers obtained using the loxP HV, as reported (NG163-R2). 17 , 27 , 28 , 29 , 30 Taken together, these data demonstrate that the vox sites have no effect on viral DNA replication, assembly, or infectivity of the HV. Figure 2 VikAD enables excision of VoxHV packaging signal and amplification of HdAd (A) Map of VoxHV genome with MfeI restriction sites.…”
Recombinant viral vectors have become integral tools for basic
in vivo
research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression
in vitro
and
in vivo
. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either
loxP
or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.
“…The clustering of membrane proteins is another parameter that is of interest for understanding protein organization ( Nakamura et al, 2015 ; Dong et al, 2018 ; Lübbert et al, 2019 ; Radulovic et al, 2020 ). By running the workflow of “Hierarchical Clustering of Particles,” gold particles are clustered into groups if they are within a certain distance of another particle (30 nm, or 27 pixels as the default; Figure 3A ).…”
Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses.
“…2016; Radulovic et al . 2020), to what extent postsynaptic activity plays a role in this selection process is still unclear. Without concurrent activity of other inputs, non‐calyceal inputs are unable to evoke postsynaptic APs, as they elicit postsynaptic firing almost exclusively by EPSP summation (Sierksma et al .…”
During development the giant, auditory calyx of Held forms a one-to-one connection with a principal neuron of the medial nucleus of the trapezoid body. r While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi-calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure-function discrepancy. r We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labelling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT). r During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma. r As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure-function discrepancy.
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