Biomedical subjects

Peter Saggau

Publications and source records attributed to Peter Saggau.

11 recordsLinked to original sources

PubMed2006

New methods and uses for fast optical scanning.

Advanced optical imaging techniques used in neurobiology commonly employ fluorescent molecules for studying the structure and function of neural tissue. To obtain adequate spatio-temporal resolution, sophisticated scanning schemes are used to manage the excitation light going to and emission light coming from objects under observation. Although the fundamental principles of these techniques remain the same, such as scanning point illumination and point detection for confocal imaging, their physical implementation is the subject of technological advance, for example, the advent of inertia-free discontinuous scanning schemes. In general, the aims of these technological advances are to improve the spatio-temporal resolution of and/or reduce potential photodamage caused by optical imaging in live neural tissue. The number of recent advances in scanning methods indicates their increasing importance in imaging techniques.

Animals↗

PubMed2006

Photometric recording of transmembrane potential in outer hair cells.

Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 microm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

Animals↗

PubMed2005

Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy.

The successful study of dendritic signaling and computation requires the ability to simultaneously monitor neuronal activity at multiple cellular sites. While the difficulties of accessing dendritic submicron structures with conventional micropipette approaches are generally overcome by optical recording techniques, their spatio-temporal resolution has limited such studies to few sites or slow signals. Here we present a novel approach to functional imaging, termed random-access multiphoton (RAMP) microscopy, which combines multiphoton excitation with an inertia-free scanning mechanism. RAMP microscopy employs two-dimensional acousto-optic deflection to rapidly position a focused near-infrared ultrafast laser beam between dwell periods at multiple user-selected sites. Because neuronal structures are generally sparse, activity located throughout various compartments, including thin dendritic branches and spines, can be mapped at high frame rates while maintaining the signal-to-noise ratio of conventional scanning microscopy. Moreover, RAMP microscopy maintains the excellent structural imaging capability of multiphoton excitation, i.e., intrinsic optical sectioning and high lateral resolution from within highly light-scattering brain tissue. RAMP microscopy thus comprises a versatile tool for investigating correlations of dendritic structure and function with significantly enhanced experimental throughput.

Action Potentials↗

PubMed2004

Facilitation of L-type Ca2+ channels in dendritic spines by activation of beta2 adrenergic receptors.

We studied the contribution of L-type Ca2+ channels to action potential-evoked Ca2+ influx in dendritic spines of CA1 pyramidal neurons and the modulation of these channels by the beta2 adrenergic receptor. Backpropagating action potentials (bAPs) (three at 50 Hz) were evoked by brief somatic current injections, and Ca2+ transients were recorded in proximal basal dendrites and associated spines. The R- and T-type Ca2+ channel blocker NiCl2 (100 microm) significantly reduced Ca2+ transients in both spines and their parent dendrites (approximately 50%), suggesting that these channels are the major source of bAP-evoked Ca2+ influx in these structures. The L-type Ca2+ channel blockers nimodipine and nifedipine (both 10 microm) reduced spine Ca2+ transients by approximately 10%, whereas the L-type Ca2+ 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 microm) significantly enhanced the spine Ca2+ transients by 40-50%. Activation of beta2 adrenergic receptors with salbutamol (40 microm) or formoterol (5 microm) resulted in significant enhancements of the spine (40-50%) but not dendritic Ca2+ transients. This increase was prevented when L-type Ca2+ channels were blocked with nimodipine (10 microm) or when cAMP-dependent protein kinase A (PKA) was inhibited with KT5720 (3 microm), Rp-cAMPS (Rp-adenosine cyclic 3',5'-phosphorothioate) (100 microm), or PKI (100 microm). The above data suggest that L-type Ca2+ channels are functionally present in dendritic spines of CA1 pyramidal neurons, contribute to spine Ca2+ influx, and can be modulated by the beta2 adrenergic receptor through PKA in a highly compartmentalized manner.

Action Potentials↗

PubMed2003

Mu-opioid receptors facilitate the propagation of excitatory activity in rat hippocampal area CA1 by disinhibition of all anatomical layers.

Hippocampal mu-opioid receptors (MORs) have been implicated in memory formation associated with opiate drug abuse. MORs modulate hippocampal synaptic plasticity acutely, when chronically activated, and during drug withdrawal. At the network level, MORs increase excitability in area CA1 by disinhibiting pyramidal cells. The precise inhibitory interneuron subtypes affected by MOR activation are unknown; however, not all subtypes are inhibited, and specific interneuron subtypes have been shown to preferentially express MORs. Here we investigate, using voltage-sensitive dye imaging in brain slices, the effect of MOR activation on the patterns of inhibition and on the propagation of excitatory activity in rat hippocampal CA1. MOR activation augments excitatory activity evoked by stimulating inputs in stratum oriens [i.e., Schaffer collateral and commissural pathway (SCC) and antidromic], stratum radiatum (i.e., SCC), and stratum lacunosum-moleculare (SLM; i.e., perforant path and thalamus). The augmented excitatory activity is further facilitated as it propagates through the CA1 network. This was observed as a proportionately larger increase in amplitudes of excitatory activity at sites distal from where the activity was evoked. This facilitation was observed for excitatory activity propagating from all three stimulation sites. The augmentation and facilitation were prevented by GABAA receptor antagonists (bicuculline, 30 microM), but not by GABAB receptor antagonists (CGP 55845, 10 microM). Furthermore, MOR activation inhibited IPSPs in all layers of area CA1. These findings suggest that MOR-induced suppression of GABA release onto GABAA receptors augments all inputs to CA1 pyramidal cells and facilitates the propagation of excitatory activity through the network of area CA1.

Animals↗

PubMed2003

Mitochondrial regulation of synaptic plasticity in the hippocampus.

Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used an in vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity.

Animals↗

PubMed2003

Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy.

We describe novel approaches for compensating dispersion effects that arise when acousto-optic (AO) beam deflection of ultrafast laser pluses is used for multiphoton laser-scanning microscopy (MPLSM). AO deflection supports quick positioning of a laser beam to random locations, allowing high frame-rate imaging of user-selected sites of interest, in addition to conventional raster scanning. Compared to standard line-scan approaches, this results in improved signal strength (and thus increased signal-to-noise) as well as reduced photobleaching and photodamage. However, 2-D AO scanning has not yet been applied for multiphoton microscopy, largely because ultrafast laser pulses experience significant spatial and temporal dispersion while propagating through AO materials. We describe and quantify spatial dispersion, demonstrating it to be a significant barrier to achieving maximal spatial resolution. We also address temporal dispersion, which is a well-documented effect that limits multiphoton excitation efficacy, and is particularly severe for AO devices. To address both problems, we have developed a single diffraction grating scheme that reduces spatial dispersion more than three-fold throughout the field of view, and a novel four-pass stacked-prism prechirper that fully compensates for temporal dispersion while reducing by two-fold the required physical length relative to commonly employed designs. These developments enable the construction of a 2-D acousto-optic multiphoton laser-scanning microscope system.

Acoustics↗

PubMed2002

Modulation of presynaptic calcium transients by metabotropic glutamate receptor activation: a differential role in acute depression of synaptic transmission and long-term depression.

Activation of group I metabotropic glutamate receptors (mGluRs) can induce acute depression of excitatory synaptic transmission and long-term depression (LTD) in area CA1 of the rat hippocampus. The underlying mechanisms for both forms of depression are unknown. By measuring presynaptic calcium transients, we show that a reduction in the stimulation-induced presynaptic calcium rise that triggers vesicular release causes the acute depression of transmission by group I mGluRs. In contrast, the mechanism underlying mGluR-induced LTD does not involve a persistent change in stimulation-induced calcium influx. However, analysis of paired-pulse facilitation experiments suggests a presynaptic location for expression of this form of LTD. Furthermore, we show that mGluR-induced LTD can be completely blocked by a specific mGluR5 antagonist, whereas mGluR1 antagonists strongly attenuate the acute depression of transmission. These results support the hypothesis that the acute depression of transmission caused by activation of group I mGluRs involves regulation of stimulation-induced presynaptic calcium transients, whereas mGluR-induced LTD involves a distinct presynaptic modulation downstream of calcium influx.

Animals↗

PubMedDate not supplied

Fast three-dimensional laser scanning scheme using acousto-optic deflectors.

A scheme for fast 3-D laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counterpropagating acoustic waves that are both chirped and offset in their frequencies, we show that it is possible to simultaneously scan both axially and laterally with frame rates on the order of tens of kilohertz. This scheme was specifically designed for application with multiphoton imaging, particularly of neurons, where it will enable the concurrent monitoring of physiological signals at multiple locations within a microscopic 3-D volume (350 x 350 x 200 microm). When used for this purpose, we demonstrate how this scheme would also inherently compensate for spatial dispersion when ultrafast laser pulses are used in acousto-optic multiphoton microscopy.

Acoustics↗

PubMedDate not supplied

High-speed addressable confocal microscopy for functional imaging of cellular activity.

Due to cellular complexity, studying fast signaling in neurons is often limited by: 1. the number of sites that can be simultaneously probed with conventional tools, such as patch pipettes, and 2. the recording speed of imaging tools, such as confocal or multiphoton microscopy. To overcome these spatiotemporal limitations, we develop an addressable confocal microscope that permits concurrent optical recordings from multiple user-selected sites of interest at high frame rates. Our system utilizes acousto-optic deflectors (AODs) for rapid positioning of a focused laser beam and a digital micromirror device (DMD) for addressable spatial filtering to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current system has an adjustable spatial resolution of approximately 0.5 to 1 microm. Furthermore, we show that recordings can be made at an aggregate frame rate of approximately 40 kHz. The system is capable of optical sectioning; this property is used to create 3-D reconstructions of fluorescently labeled test specimens and visualize neurons in brain slices. Additionally, we use the system to record intracellular calcium transients at several sites in hippocampal neurons using the fluorescent calcium indicator Oregon Green BAPTA-1.

Animals↗

PubMedDate not supplied

Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells.

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Equipment Design↗