Currently available recording methods limit the study of fast neuronal signaling by forcing a tradeoff between spatial and temporal resolution. Fortunately, advanced optical imaging techniques can be used to overcome this limitation. Specifically, we are developing confocal microscopy schemes that allow multisite recordings of neuron function in live brain tissue with high spatial and temporal resolution. The first scheme that is currently being investigated involves the use of a digital micromirror device (DMD) to implement the light paths necessary for high-speed confocal imaging: addressable point illumination and spatial filtering via addressable point detection. The second scheme involves the use of acousto-optic deflectors (AODs) in the illumination path to increase the excitation intensity, along with the DMD or an addressable CMOS imager as the spatial filter in the detection path. Calculations of the signal-to-noise ratios and operating parameters of the three devices indicate that we will be able to study both calcium concentration and fast membrane potential changes at several sites within the dendritic tree of a neuron.
We have developed a fully automated procedure for extracting dendritic morphology from multiple three-dimensional image stacks produced by laser scanning microscopy. By eliminating human intervention, we ensure that the results are objective, quickly generated, and accurate. The software suite accounts for typical experimental conditions by reducing background noise, removing pipette artifacts, and aligning multiple overlapping image stacks. The output morphology is appropriate for simulation in compartmental simulation environments. In this report, we validate the utility of this procedure by comparing its performance on live neurons and test specimens with other fully and semiautomated reconstruction tools.
The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.
We studied the contribution of L-type Ca 2+ channels to action potential-evoked Ca 2+ influx in dendritic spines of CA1 pyramidal neurons and the modulation of these channels by the β 2 adrenergic receptor. Backpropagating action potentials (bAPs) (three at 50 Hz) were evoked by brief somatic current injections, and Ca 2+ transients were recorded in proximal basal dendrites and associated spines. The R- and T-type Ca 2+ channel blocker NiCl 2 (100 μ m ) significantly reduced Ca 2+ transients in both spines and their parent dendrites (∼50%), suggesting that these channels are the major source of bAP-evoked Ca 2+ influx in these structures. The L-type Ca 2+ channel blockers nimodipine and nifedipine (both 10 μ m ) reduced spine Ca 2+ transients by ∼10%, whereas the L-type Ca 2+ channel activators FPL 64176 (2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methylester) and Bay K 8644 ((±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester) (both 10 μ m ) significantly enhanced the spine Ca 2+ transients by 40-50%. Activation of β 2 adrenergic receptors with salbutamol (40 μ m ) or formoterol (5 μ m ) resulted in significant enhancements of the spine (40-50%) but not dendritic Ca 2+ transients. This increase was prevented when L-type Ca 2+ channels were blocked with nimodipine (10 μ m ) or when cAMP-dependent protein kinase A (PKA) was inhibited with KT5720 (3 μ m ), Rp-cAMPS (Rp-adenosine cyclic 3′,5′-phosphorothioate) (100 μ m ), or PKI (100 μ m ). The above data suggest that L-type Ca 2+ channels are functionally present in dendritic spines of CA1 pyramidal neurons, contribute to spine Ca 2+ influx, and can be modulated by the β 2 adrenergic receptor through PKA in a highly compartmentalized manner.
