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By combining patch clamping with injection of selective dyes, the dynamics of calcium can be imaged in real time within isolated neurons. (A) Using infrared differential interference contrast (IR-DIC) microscopy, the image of a patch pipette can be observed attached to a neuron during a whole-cell electrophysiology experiment. Note the relatively large size of the pipette tip (coming from the leftside of the image). Calibration bar = 2 Urn. (B) After filling the neuron with a calcium-sensitive dye, bis-Fura 2, the live neuron can be imaged with a fluorescence microscope. The dye takes about 10 minutes to fill the neuron after rupturing the patch membrane. Calibration bar = 10 I'm. (C) A unique property of the dye bis-Fura 2 is that it changes its fluorescence properties as it binds calcium. This can be observed by the changes in the fluorescence signal in response to a single (top) or five (bottom) action potentials. The fluorescence traces correspond to two regions, one close to the cell body (red box and red trace) and one farther out in the apical dendrite (orange box, orange trace). In this way, one can observe changes in calcium dynamics and how they correspond to activity states within single neurons.
When penetrating at the level of the soma, the somatic current clamp and voltage clamp recordings are capable of measuring the voltage and conductance changes that occur at the soma or proximal dendrites. As a result, the complex membrane dynamics and synaptic events that occur along the dendritic tree cannot be resolved by somatic penetration in neurons with a complex morphology. One powerful application of patch recording techniques is targeted recordings of dendrites and presynaptic terminals, structures previously not accessible to direct electrophysiological measurement. Recordings at dendritic sites have demonstrated dendritic membrane properties that often are quite distinct from properties determined from somatic recordings. From these findings, it can be inferred that dendrites are not just extensions of the soma but also play an active role in the propagation of postsynaptic potentials from the synapse to the soma.
However, many structures, such as finer dendrites and smaller axonals, are still resistant to direct electrophysiological measurement with patch electrodes. Another technique that allows for examination of minute structures is the use of fluorescent dyes. Two basic classes of dyes are commonly used: 1) dyes sensitive to changes in ion concentration, which indirectly reflect the membrane potential, and 2) dyes sensitive to voltage changes, with fluorescence directly responsive to changes of membrane potential. Because these dyes readily diffuse throughout the neuron, real-time imaging of changes in dye fluorescence can be performed in live cells (see Figure 5-6). Although the temporal resolutions and signal-to-noise ratios of imaging technologies do not rival those achievable with direct electrical recordings, imaging allows examination of minute structures and wide spatial regions. Major drawbacks of this technique include the toxicity of some dyes and the phototoxicity induced by light sources used to excite the dyes. More recently, this technique has been applied in vivo, providing new avenues for study of neuronal function.
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