High-speed Two-photon Imaging Research Articles

Functional Microstructure of CaV-Mediated Calcium Signaling in the Axon Initial Segment.

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (CaV). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of CaVs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: CaV3 generates hotspot regions of calcium influx coupled to calcium stores, whereas CaV2 channels underlie diffuse calcium influx between hotspots. Therefore, different CaV classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.

Optogenetic Stimulation of Cholinergic Amacrine Cells Improves Capillary Blood Flow in Diabetic Retinopathy.

PurposeDisruption in blood supply to active retinal circuits is the earliest hallmark of diabetic retinopathy (DR) and has been primarily attributed to vascular deficiency. However, accumulating evidence supports an early role for a disrupted neuronal function in blood flow impairment. Here, we tested the hypothesis that selectively stimulating cholinergic neurons could restore neurovascular signaling to preserve the capillary circulation in DR.MethodsWe used wild type (wt) and choline acetyltransferase promoter (ChAT)-channelrhodopsin-2 (ChR2) mice expressing ChR2 exclusively in cholinergic cells. Mice were made diabetic by streptozotocin (STZ) injections. Two to 3 months after the last STZ injection, the rate of capillary blood flow was measured in vivo within each retinal vascular layer using high speed two-photon imaging. Measurements were done at baseline and following ChR2-driven activation of retinal cholinergic interneurons, the sole source of the vasodilating neurotransmitter acetylcholine. After recordings, retinas were collected and assessed for physiological and structural features.ResultsIn retinal explants from ChAT-ChR2 mice, we found that channelrhodopsin2 was selectively expressed in all cholinergic amacrine cells. Its direct activation by blue light led to dilation of adjacent retinal capillaries. In living diabetic ChAT-ChR2 animals, basal capillary blood flow was significantly higher than in diabetic mice without channelrhodopsin. However, optogenetic stimulation with blue light did not result in flickering light-induced functional hyperemia, suggesting a necessity for a concerted neurovascular interaction.ConclusionsThese findings provide direct support to the utility and efficacy of an optogenetic approach for targeting selective retinal circuits to treat DR and its complications.

High-Speed Two-Photon Imaging

The small size of neuronal dendrites and spines combined with the high speed of neurophysiological signals, such as transients in membrane potential or ion concentration, necessitates that any functional study of these structures uses recording methods with both high spatial and high temporal resolutions. In this regard, conventional two-photon microscopy, in combination with fluorescent indicators sensitive to physiological parameters, has proved to be only a partial solution by providing near-diffraction-limited spatial resolution even when imaging structures deep inside light-scattering tissue. This is because the relatively slow beam-scanning methods used in most conventional two-photon microscopes severely limit the extent to which functional data can be recorded. Here, we detail developments to create high-speed two-photon imaging systems that overcome this limitation and discuss important considerations that must be taken into account when attempting to construct such systems.

LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

Pyramidal cells accumulate chloride at seizure onset

Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity.

Dendritic organization of sensory input to cortical neurons in vivo

In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.

Dendritic organization of sensory input to cortical neurons in vivo

In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.