Imaging membrane potential in dendritic spines

Dendritic spines mediate most excitatory inputs in the brain. Although it is clear that spines compartmentalize calcium, it is still unknown what role, if any, they play in integrating synaptic inputs. To investigate the electrical function of spines directly, we used second harmonic generation (SHG) imaging of membrane potential in pyramidal neurons from hippocampal cultures and neocortical brain slices. With FM 4-64 as an intracellular SHG chromophore, we imaged membrane potential in the soma, dendritic branches, and spines. The SHG response to voltage was linear and seemed based on an electro-optic mechanism. The SHG sensitivity of the chromophore in spines was similar to that of the parent dendritic shaft and the soma. Backpropagation of somatic action potentials generated SHG signals at spines with similar amplitude and kinetics to somatic ones. Our optical measurements of membrane potential from spines demonstrate directly that backpropagating action potentials invade the spines

Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner

High-Resolution Plasma Membrane-Selective Imaging by Second Harmonic Generation

Summary: The plasma membrane is the site of intercellular communication and subsequent intracellular signal transduction. The specific visualization of the plasma membrane in living cells, however, is difficult using fluorescence-based techniques owing to the high background signals from intracellular organelles. In this study, we show that second harmonic generation (SHG) is a high-resolution plasma membrane-selective imaging technique that enables multifaceted investigations of the plasma membrane. In contrast to fluorescence imaging, SHG specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows high-resolution imaging because of its subresolution nature. These properties were exploited to measure the distances from the plasma membrane to subcortical actin and tubulin fibers, revealing the precise cytoskeletal organization beneath the plasma membrane. Thus, SHG imaging enables the specific visualization of phenomena at the plasma membrane with unprecedented precision and versatility and should facilitate cell biology research focused on the plasma membrane. : Biological Sciences; Biophysics; Membrane Architecture Subject Areas: Biological Sciences, Biophysics, Membrane Architectur

Protocol to image deuterated propofol in living rat neurons using multimodal stimulated Raman scattering microscopy

Summary: Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics.For complete details on the use and execution of this protocol, please refer to Oda et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression

Glutamate receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. It has been reported that protein kinase A (PKA) can phosphorylate the 03B1-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR4 on Ser842, both in vitro and in vivo. Here, we studied the regulation of GluR4 phosphorylation and intracellular trafficking by PKA and by metabotropic receptors coupled to adenylyl cyclase (AC), in cultured chick retinal amacrine-like neurones, which are enriched in GluR4. The regulation of AMPA receptor activity by PKA and by metabotropic AC-coupled receptors was also investigated by measuring the [Ca2+]i response to kainate in Na+-free medium. Stimulation of AC with forskolin (FSK), or using the selective agonist of dopamine D1 receptors (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF38393), increased the [Ca2+]i response to kainate, GluR4 phosphorylation at Ser842 and GluR4 surface expression. Pre-incubation of the cells with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist of group II metabotropic glutamate receptors (mGluR), which are coupled to inhibition of AC, inhibited the effect of FSK and of SKF38393 on AMPA receptor activity, GluR4 phosphorylation and expression at the plasma membrane. These results indicate that there is a functional cross-talk between dopamine D1 receptors and group II mGluR in the regulation of GluR4 phosphorylation and AMPA receptor activity. Our data show that GluR4 phosphorylation at Ser842 by PKA, and its recruitment to the plasma membrane upon phosphorylation, is regulated by metabotropic receptors