Neurogranin enhances synaptic strength through its interaction with calmodulin

Zhong, Ling; Cherry, Tiffani; Bies, Christine E; Florence, Matthew A.; Gerges, Nashaat Z.

doi:10.1038/emboj.2009.236

Cited by 121 publications

(181 citation statements)

References 84 publications

(113 reference statements)

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“…Here, we labeled Ng with 10 nm colloidal gold. We then quantified the gold to generate labeling pattern of Ng that helped to understand its role in synaptic function ( Figure 2B) 8 .…”

Section: Discussionmentioning

confidence: 99%

“…Ng molecules within the spine (but not at the PSD) exhibit preferential localization to the plasma membrane. This histogram was originally published in the EMBO Journal 8 . Figure 1.…”

Section: Notementioning

confidence: 99%

“…We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) 10 . As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine 8 . Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.…”

mentioning

confidence: 98%

“…These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously 8 , and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function 8,9 .…”

mentioning

confidence: 99%

“…Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously 8 , and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function 8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) 10 .…”

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confidence: 99%

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Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Zhong

Brown

Wells

et al. 2013

JoVE

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Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues 1 . The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback 2 . The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure [3][4][5] .Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO 4 ), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO 4 is highly reactive and has been shown to mask antigens 6 , resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO 4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues 7 . We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously 8 , and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function 8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca 2+ /CaM-dependent ...

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“…Here, we labeled Ng with 10 nm colloidal gold. We then quantified the gold to generate labeling pattern of Ng that helped to understand its role in synaptic function ( Figure 2B) 8 .…”

Section: Discussionmentioning

confidence: 99%

“…Ng molecules within the spine (but not at the PSD) exhibit preferential localization to the plasma membrane. This histogram was originally published in the EMBO Journal 8 . Figure 1.…”

Section: Notementioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

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