In VivoImaging of Intersynaptic Vesicle Exchange Using VGLUT1^VenusKnock-In Mice

Abstract

The vesicular glutamate transporter VGLUT1 loads synaptic vesicles with the neurotransmitter glutamate and thereby determines glutamate release at many synapses in the mammalian brain. Due to its function and selective localization, VGLUT1 is one of the most specific markers for glutamatergic synaptic vesicles. It has been used widely to identify glutamatergic synapses, and its expression levels are tightly correlated with changes in quantal size, modulations of synaptic plasticity, and corresponding behaviors. We generated a fluorescent VGLUT1Venus knock-in mouse for the analysis of VGLUT1 and glutamatergic synaptic vesicle trafficking. The mutation does not affect glutamatergic synapse function, and thus the new mouse model represents a universal tool for the analysis of glutamatergic transmitter systems in the forebrain. Previous studies demonstrated synaptic vesicle exchange between terminals in vitro. Using the VGLUT1 Venus knock-in, we show that synaptic vesicles are dynamically shared among boutons in the cortex of mice in vivo. We provide a detailed analysis of synaptic vesicle sharing in vitro, and show that network homeostasis leads to dynamic scaling of synaptic VGLUT1 levels.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…Malatesta et al, 2003;Zhu et al, 2008;Platel et al, 2009;Huang et al, 2014), visualization of astrocytes and astrogliosis in living brain tissue (Nolte et al, 2001;Weimer et al, 2008;Tang et al, 2009) and other dynamic interactions which are hard to study immunocytochemically (e.g. Reichenbach et al, 2010;Young et al, 2010;Herzog et al, 2011). There are, however, issues with reporter mice also as the transcriptional activity can be influenced by multiple factors.…”

Imaging Synaptic Vesicles Using VGLUT1-Venus Knock-In Mice: Insights into the Dynamic Nature of Intersynaptic Vesicle Exchange: Figure 1.

“…This observation gave rise to the concept of the synaptic vesicle "superpool," a shared pool of vesicles from several adjacent terminals that can be rapidly and directly accessed by individual synapses (Staras et al, 2010). Recently, a paper by Herzog et al (2011) in The Journal of Neuroscience examined intersynaptic vesicle exchange both in vitro and in vivo, using mice expressing a knock-in vesicular glutamate transporter (VGLUT1) fused to the YFP variant Venus.…”

“…VGLUT1 localizes to synaptic vesicles, and its expression level within axonal boutons has been shown to accurately report synaptic vesicle number and influence presynaptic strength (see Herzog et al, 2006Herzog et al, , 2011. To enable live imaging of synaptic vesicles, Herzog et al (2011) generated a mouse in which the endogenous VGLUT1 locus was modified to encode a VGLUT1-Venus fusion protein.…”

“…To enable live imaging of synaptic vesicles, Herzog et al (2011) generated a mouse in which the endogenous VGLUT1 locus was modified to encode a VGLUT1-Venus fusion protein. Because the Venus tag does not impair VGLUT1 function and endogenous levels of VGLUT1 expression are maintained by the knock-in approach, mice exhibited normal vesicular glutamate uptake and neurotransmitter release.…”

“…Thus, the authors could study synaptic vesicle trafficking and function under physiological conditions without the potential confounding effects associated with either the overexpression of genetic reporters or the administration of exogenous labels such as FM dyes. Herzog et al (2011) examined intersynaptic vesicle trafficking by monitoring fluorescence recovery after photobleaching (FRAP) at individual terminals in VGLUT1-Venus mice in hippocampal neuronal cultures. Consistent with previous studies, fluorescence recovery was best described as a biexponential function with two time constants: an early, fast component ( F ϭ 44.0 s), presumably reflecting rapid axonal transport of unbleached vesicles to the terminal, and a later component ( S ϭ 28.5 min), likely mediated by the slower exchange of vesicles with neighboring, unbleached boutons (Darcy et al, 2006;Staras et al, 2010).…”