subcellular-imaging-of-neuronal-calcium-handling-in-vivo

Subcellular Imaging of Neuronal Calcium Handling <em>In Vivo</em>

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Colorado State University College of Veterinary Medicine and Biomedical Sciences

The AMPA subtype of synaptic glutamate receptors (AMPARs) plays an essential role in cognition. Their function, numbers, and change at synapses during synaptic plasticity are tightly regulated by neuronal activity. Although we know that long-distance transport of AMPARs is essential for this regulation, we do not understand the associated regulatory mechanisms of it. Neuronal transmission is a metabolically demanding process in which ATP consumption and production are tightly coupled and regulated. Aerobic ATP synthesis unavoidably produces reactive oxygen species (ROS), such as hydrogen peroxide, which are known modulators of calcium signaling. Although a role for calcium signaling in AMPAR transport has been described, there is little understanding of the mechanisms involved and no known link to physiological ROS signaling. Here, using real-time  imaging of AMPAR transport in the intact  nervous system, we demonstrate that long-distance synaptic AMPAR transport is bidirectionally regulated by calcium influx and activation of calcium/calmodulin-dependent protein kinase II. Quantification of  calcium dynamics revealed that modest, physiological increases in ROS decrease calcium transients in  glutamatergic neurons. By combining genetic and pharmacological manipulation of ROS levels and calcium influx, we reveal a mechanism in which physiological increases in ROS cause a decrease in synaptic AMPAR transport and delivery by modulating activity-dependent calcium signaling. Together, our results identify a novel role for oxidant signaling in the regulation of synaptic AMPAR transport and delivery, which in turn could be critical for coupling the metabolic demands of neuronal activity with excitatory neurotransmission. Synaptic AMPARs are critical for excitatory synaptic transmission. The disruption of their synaptic localization and numbers is associated with numerous psychiatric, neurologic, and neurodegenerative conditions. However, very little is known about the regulatory mechanisms controlling transport and delivery of AMPAR to synapses. Here, we describe a novel physiological signaling mechanism in which ROS, such as hydrogen peroxide, modulate AMPAR transport by modifying activity-dependent calcium signaling. Our findings provide the first evidence in support of a mechanistic link between physiological ROS signaling, AMPAR transport, localization, and excitatory transmission. This is of fundamental and clinical significance since dysregulation of intracellular calcium and ROS signaling is implicated in aging and the pathogenesis of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease.

Reactive Oxygen Species Modulate Activity-Dependent AMPA Receptor Transport in .

Synaptic plasticity depends on rapid experience-dependent changes in the number of neurotransmitter receptors. Previously, we demonstrated that motor-mediated transport of AMPA receptors (AMPARs) to and from synapses is a critical determinant of synaptic strength. Here, we describe two convergent signaling pathways that coordinate the loading of synaptic AMPARs onto scaffolds, and scaffolds onto motors, thus providing a mechanism for experience-dependent changes in synaptic strength. We find that an evolutionarily conserved JIP-protein scaffold complex and two classes of mitogen-activated protein kinase (MAPK) proteins mediate AMPAR transport by kinesin-1 motors. Genetic analysis combined with in vivo, real-time imaging in Caenorhabditis elegans revealed that CaMKII is required for loading AMPARs onto the scaffold, and MAPK signaling is required for loading the scaffold complex onto motors. Our data support a model where CaMKII signaling and a MAPK-signaling pathway cooperate to facilitate the rapid exchange of AMPARs required for early stages of synaptic plasticity.

MAPK signaling and a mobile scaffold complex regulate AMPA receptor transport to modulate synaptic strength.

 is an important genetic model for neuroscience studies, used for analyses of how genes control connectivity, neuronal function, and behavior. To date, however, most studies of neuronal function in  are incapable of obtaining microscopy imaging with subcellular resolution and behavior analysis in the same set of animals. This constraint stems from the immobilization requirement for high-resolution imaging that is incompatible with behavioral analysis using conventional immobilization techniques. Here, we present a novel microfluidic device that uses surface acoustic waves (SAW) as a non-contact method to temporarily immobilize worms for a short period (30 seconds). We optimize the SAW based protocol for rapid switching between free-swimming and immobilized states, facilitating non-invasive analysis of swimming behavior as well as high-resolution synaptic imaging in the same animal. We find that the coupling of heat and acoustic pressure play a key role in the immobilization process. We introduce a proof-of-concept longitudinal study, illustrating that the device enables repeated imaging of fluorescently tagged synaptic receptors in command interneurons and analysis of swimming behavior in the same animals for three days. This longitudinal approach provides the first correlative analysis of synaptic glutamatergic receptors and swimming behavior in aging animals. We anticipate that this device will enable further longitudinal analysis of animal motility and subcellular morphological changes during development and aging in .

Frederic Hoerndli

Department of Biomedical Sciences

Colorado State University College of Veterinary Medicine and Biomedical Sciences

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Frederic Hoerndli

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Sciences

Colorado State University College of Veterinary Medicine and Biomedical Sciences

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Department of Biomedical Sciences