Dentate Granule Cells Loaded with OGB-1, AM

Calcium imaging of hippocampal tissue slices has been ongoing for decades, with use of multiphoton microscopy in these studies reported since the 1990s. Conventional calcium imaging uses a calcium indicator dye loaded into cells. Calcium imaging is based on change in fluorescence emission spectra (e.g., OGB-1 and fluo3) or change in absorption spectra (e.g., fura-2) of calcium indicator dyes upon calcium ion binding. This process is reversible and follows the chemical equilibrium between cytosolic free Ca2+, the ion free-form, and the ion bound-form of the calcium indicator dye. There are three major routes of calcium influx into the cytosol in neurons. One is through voltage-gated calcium channels, which, due to the high depolarization threshold of these channels, usually only occurs when neurons fire APs. Another route is through NMDA receptor-mediated synaptic transmission, where activated receptors conduct large amounts of calcium. Lastly, activation of certain G-protein-coupled receptors may trigger the activation of phospholipase C and release of inositol trisphosphate (IP3), followed by activation of IP3 receptors in endoplasmic reticulum, causing Ca2+ release from internal calcium stores. Due to their high affinity for calcium, once Ca2+ binds to a calcium indicator dye, despite Ca2+ being pumped out of cells and sequestered in intracellular stores continuously, it takes several tens to several hundreds of milliseconds to unbind from the dye, and restore dye fluorescence to basal levels. Thus, due to the long duration response of calcium indicators, calcium imaging equipment normally does not require millisecond temporal resolution and conventional research CCD cameras, scanning confocal microscopy, and two photon microscopy can be used in calcium imaging experiments.

CA3 loaded with OGB-1,AM.

Monitoring neuronal activity from multiple neurons (multi-cellular imaging) can be accomplished by bulk-loading the acetoxymethyl (AM)-ester form of calcium indicator dyes. Fura-2 AM and OGB-1 AM are widely used. With this loading method, due to the relatively low cytoplasmic dye concentrations achieved (10 ┬ÁM or less), it is difficult to identify the dendrites and axon of a particular neuron, especially in pyramidal cells in CA regions or granule cells in the dentate gyrus of hippocampal slices. Instead, one can monitor signal changes from the soma of multiple neurons. While the temporal response of calcium imaging is slower than VSDI, changes in signal corresponding to cellular activity are very high, often 5-100% change in fluorescence during a response. This facilitates monitoring of cellular activity without event-averaging. Since fluorescence emission from bulk-loaded slices is weak, we typically use high numerical aperture water-immersion objective lenses with modest magnifications (16x, 20x, and 40x). With these magnifications, it is possible to monitor individual cellular behaviors of entire hippocampal sub-regions (CA1, CA3, DG, etc.). For reference, a 40x confocal image field could contain 100-300 cells while a 16x image field could contain 500-1000 neurons, all of which are accessible for imaging responses. Confocal microscopy or two photon microscopy is preferable for imaging slices at moderate to high spatial resolution since wide-field epi-fluorescence microscopy with a CCD camera collects fluorescence from the entire slice depth, confounding interpretation of the resulting responses. However, conventional confocal microscopy or two photon microscopy still suffers from slow image acquisition speeds (poor temporal resolution) because excitation spots must be scanned pixel by pixel, reducing typical imaging speed to 1 to 10 Hz frame rates. In our laboratory, we use an advanced form of confocal microscopy, known as swept-field microscopy, which incorporates multi-beam scanning to speed acquisition rates, coupled with an EM-CCD camera capable of recording at frame rates exceeding 1 kHz. Using this combination of technologies, one can achieve real experimental acquisition rate of 25 Hz for images with 512x512 pixels or 300 Hz at 128x128 pixels. Calcium indicators can also be internally loaded into neurons through patch pipettes using a conventional electrophysiology setup. With this loading method, one can study dynamic properties of fine processes, or even activity in individual synaptic spines. In addition, there are many genetically encoded calcium indicator probes available that can be used to study single cell activity, multi-cellular activity and even network properties.