Biomedical subjects

Thomas A Pologruto

Publications and source records attributed to Thomas A Pologruto.

5 recordsLinked to original sources

PubMed2004

Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators.

Genetically encoded Ca2+ indicators (GECIs) based on fluorescent proteins (XFPs) and Ca2+-binding proteins [like calmodulin (CaM)] have great potential for the study of subcellular Ca2+ signaling and for monitoring activity in populations of neurons. However, interpreting GECI fluorescence in terms of neural activity and cytoplasmic-free Ca2+ concentration ([Ca2+]) is complicated by the nonlinear interactions between Ca2+ binding and GECI fluorescence. We have characterized GECIs in pyramidal neurons in cultured hippocampal brain slices, focusing on indicators based on circularly permuted XFPs [GCaMP (Nakai et al., 2001), Camgaroo2 (Griesbeck et al., 2001), and Inverse Pericam (Nagai et al., 2001)]. Measurements of fluorescence changes evoked by trains of action potentials revealed that GECIs have little sensitivity at low action potential frequencies compared with synthetic [Ca2+] indicators with similar affinities for Ca2+. The sensitivity of GECIs improved for high-frequency trains of action potentials, indicating that GECIs are supralinear indicators of neural activity. Simultaneous measurement of GECI fluorescence and [Ca2+] revealed supralinear relationships. We compared GECI fluorescence saturation with CaM Ca2+-dependent structural transitions. Our data suggest that GCaMP and Camgaroo2 report CaM structural transitions in the presence and absence of CaM-binding peptide, respectively.

Action Potentials↗

PubMed2004

Stereotyped odor-evoked activity in the mushroom body of Drosophila revealed by green fluorescent protein-based Ca2+ imaging.

To study the representation of olfactory information in higher brain centers, we expressed a green fluorescent protein-based Ca2+ sensor, G-CaMP, in the Drosophila mushroom body (MB). Using two-photon microscopy, we imaged odor-evoked G-CaMP fluorescence transients in MB neurons [Kenyon cells (KCs)] with single-cell resolution. Odors produced large fluorescence transients in a subset of KC somata and in restricted regions of the calyx, the neuropil of the MB. In different KCs, odor-evoked fluorescence transients showed diverse changes with odor concentration: in some KCs, fluorescence transients were evoked by an odor at concentrations spanning several orders of magnitude, whereas in others only at a narrow concentration range. Different odors produced fluorescence transients in different subsets of KCs. The spatial distributions of KCs showing fluorescence transients evoked by a given odor were similar across individuals. For some odors, individual KCs with fluorescence transients evoked by a particular odor could be found in similar locations in different flies with spatial precisions on the order of the size of KC somata. These results indicate that odor-evoked activity can have remarkable spatial specificity in the MB.

Animals↗

PubMed2004

Imaging calcium concentration dynamics in small neuronal compartments.

Calcium and its regulation play central roles diverse physiologic processes. Quantification of calcium concentrations ([Ca2+]) in small neuronal compartments is crucial to understanding Ca2+-dependent signaling. Here, we describe techniques that are optimized for 2-photon imaging of [Ca2+] dynamics in small compartments such as dendrites and dendritic spines.

Animals↗

PubMed2003

ScanImage: flexible software for operating laser scanning microscopes.

BACKGROUND: Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. RESULTS: We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. CONCLUSIONS: We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.

Equipment Design↗

PubMed2003

Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex.

Sensory deprivation during a critical period reduces spine motility and disrupts receptive field structure of layer 2/3 neurons in rat barrel cortex. To determine the locus of plasticity, we used laser scanning photostimulation, allowing us to rapidly map intracortical synaptic connectivity in brain slices. Layer 2/3 neurons differed in their spatial distributions of presynaptic partners: neurons directly above barrels received, on average, significantly more layer 4 input than those above the septa separating barrels. Complementary connectivity was found in deprived cortex: neurons above septa were now strongly coupled to septal regions, while connectivity between barrel regions and layer 2/3 was reduced. These results reveal competitive interactions between barrel and septal circuits in the establishment of precise intracortical circuits.

Afferent Pathways↗