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Basic neuroscience research employs numerous forms of antibodies as key reagents in diverse applications. While the predominant use of antibodies is as immunolabeling reagents, neuroscientists are making increased use of intracellular antibodies or intrabodies. Intrabodies are recombinant antibodies genetically encoded for expression within neurons. These can be used to target various cargo (fluorescent proteins, reporters, enzymes, etc.) to specific molecules and subcellular domains to report on and manipulate neuronal function with high precision. Intrabodies have the advantages inherent in all genetically encoded recombinant antibodies but represent a distinct subclass in that their structure allows for their expression and function within cells. The high precision afforded by the ability to direct their expression to specific cell types, and the selective binding of intrabodies to targets within these allows intrabodies to offer unique advantages for neuroscience research, given the tremendous molecular, cellular and morphological complexity of brain neurons. Intrabodies expressed within neurons have been used for a variety of purposes in basic neuroscience research. Here I provide a general background to intrabodies and their development, and examples of their emerging utility as valuable basic neuroscience research tools.

Basic neuroscience research employs numerous forms of antibodies as key reagents in diverse applications. While the predominant use of antibodies is as immunolabeling reagents, neuroscientists are making increased use of intracellular antibodies or intrabodies. Intrabodies are recombinant antibodies genetically encoded for expression within neurons. These can be used to target various cargo (fluorescent proteins, reporters, enzymes, etc.) to specific molecules and subcellular domains to report on and manipulate neuronal function with high precision. Intrabodies have the advantages inherent in all genetically encoded recombinant antibodies but represent a distinct subclass in that their structure allows for their expression and function within cells. The high precision afforded by the ability to direct their expression to specific cell types, and the selective binding of intrabodies to targets within these allows intrabodies to offer unique advantages for neuroscience research, given the tremendous molecular, cellular and morphological complexity of brain neurons. Intrabodies expressed within neurons have been used for a variety of purposes in basic neuroscience research. Here I provide a general background to intrabodies and their development, and examples of their emerging utility as valuable basic neuroscience research tools. [ABSTRACT FROM AUTHOR]

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (DÖg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture. [ABSTRACT FROM AUTHOR]

The Ca2+/calmodulin-dependent protein kinase II (Ca MKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and in vitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca2+/CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation in vitro was reduced by the Ca MKII inhibitors tat CN21 and tat CN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block Ca MKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed that this is not the case. In vitro, staurosporine and H7 enabled Ca MKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous Ca MKII aggregation. This aggregation was blocked by tat CN21, but not by staurosporine, even though both effectively inhibit Ca MKII activity. These results support the mechanistic model for Ca MKII aggregation and show that kinase activity is not required. [ABSTRACT FROM AUTHOR]

Understanding behavior requires unraveling the mysteries of neurons, glia, and their extensive connectivity. Drosophila has emerged as an excellent organism for studying the neural basis of behavior. This can be largely attributed to the extensive effort of the fly community to develop numerous sophisticated genetic tools for visualizing, mapping, and manipulating behavioral circuits. Here, we attempt to highlight some of the new reagents, techniques and approaches available for dissecting behavioral circuits in Drosophila. We focus on detailing intersectional strategies such as the Flippase-induced intersectional Gal80/Gal4 repression (FINGR), because of the tremendous potential they possess for mapping the minimal number of cells required for a particular behavior. The logic and strategies outlined in this review should have broad applications for other genetic model organisms. [ABSTRACT FROM AUTHOR]

CaMKIIα is a central mediator of bidirectional synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). To study how CaMKIIα movement during plasticity is affected by soluble amyloid-β peptide oligomers (Aβ), we used FingR intrabodies to simultaneously image endogenous CaMKIIα and markers for excitatory versus inhibitory synapses in live neurons. Aβ blocks LTP-stimulus-induced CaMKIIα accumulation at excitatory synapses. This block requires CaMKII activity, is dose and time dependent, and also occurs at synapses without detectable Aβ; it is specific to LTP, as CaMKIIα accumulation at inhibitory synapses during LTD is not reduced. As CaMKII movement to excitatory synapses is required for normal LTP, its impairment can mechanistically explain Aβ-induced impairment of LTP. CaMKII movement during LTP requires binding to the NMDA receptor, and Aβ induces internalization of NMDA receptors. However, surprisingly, this internalization does not cause the block in CaMKIIα movement and is observed for extrasynaptic, but not synaptic, NMDA receptors. • FingR intrabodies enabled simultaneous live imaging of three endogenous proteins • Aβ inhibits CaMKII trafficking after LTP-, but not LTD-inducing, stimuli • The Aβ-induced block of CaMKII trafficking requires CaMKII activity • Aβ induced internalization of the extrasynaptic, but not synaptic, NMDA receptor Cook et al. used simultaneous live imaging of multiple endogenous proteins to probe LTP- versus LTD-induced CaMKII trafficking to excitatory versus inhibitory synapses. They show that Aβ inhibits LTP by blocking LTP-induced CaMKII trafficking via signaling mechanisms that require CaMKII activity. [ABSTRACT FROM AUTHOR]

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture., Competing Interests: The authors declare no conflict of interest.

The Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and in vitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca(2+) /CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation in vitro was reduced by the CaMKII inhibitors tatCN21 and tatCN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block CaMKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed that this is not the case. In vitro, staurosporine and H7 enabled CaMKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous CaMKII aggregation. This aggregation was blocked by tatCN21, but not by staurosporine, even though both effectively inhibit CaMKII activity. These results support the mechanistic model for CaMKII aggregation and show that kinase activity is not required. CaMKII aggregation is prevented by inhibiting kinase activity with mutations (red italics; shown previously) or inhibitors (red bold; shown here), indicating requirement of kinase activity. However, we show here that nucleotide-competitive inhibitors (green) allow CaMKII aggregation (including endogenous CaMKII within neurons), demonstrating that kinase activity is not required and supporting the current mechanistic model for CaMKII aggregation., (© 2015 International Society for Neurochemistry.)