Several (five to eight) frames of baseline were recorded during focal application of control ACSF, and then drug-containing ACSF was applied for the remainder of the imaging series. Pharmacological agents were applied by a multi-valve, single-output focal drug application device (ALA Scientific, Westbury, NY). Slice viability was monitored by visualization of cellular integrity under infrared differential interference contrast videomicroscopy and/or by visualizing calcium responses to N-methyl-D-aspartate (NMDA 50 μM) or acetylcholine (ACh 100 μM). If saturation of responses was observed, the data were discarded and the experiment was repeated at nonsaturating gain levels. Gain, black level, and number of accumulated video frames per final frame were empirically adjusted to minimize background and maximize peak response intensity without causing saturation at either end of the signal. Each frame in a time-lapse sequence was captured every 3–4 s. Video-rate acquisition of frames minimized exposure (<500 ms) of the tissue at each timed shutter opening. Minimal exposure of the preparation to the excitation light was achieved by controlling a shutter in the excitation light path (Uniblitz vs.25 Vincent Associates, Rochester, NY) through digital outputs of the frame acquisition board.
Time-lapse sequences of fluo-3 fluorescence were acquired using custom macros within the public domain National Institutes of Health image program (available on the Internet at ) on a Macintosh computer equipped with a video frame acquisition board (Scion Corporation, Frederick, MD). Images were acquired using a 10× water-immersion objective. Fluorescence filters (Chroma Technology, Brattleboro, VT) for fluo-3 imaging were as follows: excitation filter, 480 ± 20 nm dichroic mirror, 505 nm longpass and emission filter, 535 ± 25 nm. Epifluorescence imaging of fluo-3 intensity was performed with a 100-W mercury light source and a low-light charge-coupled device video camera (Dage–MTI 300-T, Michigan City, IN). For imaging, slices were removed from the loading solution to standard oxygenated ACSF, transferred to a recording chamber on the stage of an upright compound microscope (Olympus BX50-WI, Tokyo, Japan), and perfused with oxygenated ACSF at a rate of 1–2 ml/min. Measurements of relative changes in i were made using vital epifluorescence microscopy of mouse neocortical slices loaded with fluo-3. Spontaneous oscillatory changes in i have also been observed in single developing neocortical neurons ( 8, 12, 16), but their mode of activation has not been characterized. Application of mGluR agonists has been observed to produce oscillations in i in neurons and glia in the developing hippocampus and neocortex ( 14, 15). Activation of ionotropic glutamate and γ-aminobutyric acid (GABA) receptors can cause elevations in i in developing neocortical neurons, and synaptic activation of these neurons can lead to monophasic i transients ( 12, 13).
Neocortical domains apparently can occur independently of action potential activity, but their frequency can be increased by activation of metabotropic glutamate receptors (mGluR) ( 11). These spontaneous events are mediated by gap junctional communication ( 10) and appear to involve cell-to-cell diffusion of the second messenger inositol trisphosphate ( 9, 11).
In the postnatal neocortex, coordinated i rises can occur in 3–100 adjacent neurons, termed neuronal domains ( 9). Proliferative neuroepithelial cells in the embryonic ventricular zone display several patterns of i dynamics, including isolated transients, coordinated i increases in local cell clusters, and synchronous transients in cell pairs undergoing mitotic division ( 8). In the developing neocortex, several patterns of calcium dynamics have been described.
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Spontaneous changes in the concentration of neuronal free calcium ( i) have been observed in a variety of developing systems, many of which exhibit distinct spatial and temporal patterns of i fluctuation ( 1– 7).