Biomedical subjects
Nima Marandi
Publications and source records attributed to Nima Marandi.
Optical monitoring of brain function in vivo: from neurons to networks.
The precise understanding of the cellular and molecular basis of brain function requires the direct assessment of the activity of defined cells in vivo. A promising approach for such analyses is two-photon microscopy in combination with appropriate cell labeling techniques. Here, we review the multi-cell bolus loading (MCBL) method that involves the use of membrane-permeant fluorescent indicator dyes. We show that this approach is useful for the functional analysis of clusters of neurons and glial cells in vivo. Work from our and other laboratories shows that the techniques that were previously feasible only in brain slices, like targeted patch clamp recordings from identified cells or pharmacological manipulations in confined brain regions, can now be used also in vivo. We also show that MCBL and two-photon imaging can be easily combined with other labeling techniques, particularly with those involving the use of genetically encoded, green-fluorescent-protein-based indicators. Finally, we examine recent applications of MCBL/two-photon imaging for the analysis of various brain regions, including the somatosensory and the visual cortex.
Quantitative single-cell RT-PCR and Ca2+ imaging in brain slices.
We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in individual cells of living brain slices. Quantification is achieved by using rapid-cycle, real-time PCR protocols and high-resolution external cDNA standard curves for the gene of interest. The method consists of several procedures, including cell soma harvest, reverse transcription, and an optimized cDNA purification step, which allowed us to quantify transcripts in small types of neurons, like cerebellar granule cells. Thus, we detected in single granule cells an average of 20 transcript copies of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon calcium imaging and quantitative RT-PCR in single Purkinje and granule cells, respectively, and identified distinct glutamate receptor-dependent Ca2+ responses in these two cell types. The approach was further tested by profiling the expression of the ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum. Our study revealed a developmental switch from an average of 15 NR2B copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell after P26. Taken together, our results demonstrate that the new method is rapid, highly sensitive, provides reliable results in neurons of various sizes, and can be used in combination with Ca2+ imaging.
Two-photon chloride imaging in neurons of brain slices.
Two-photon laser scanning microscopy has been used successfully for imaging activity-dependent changes of intracellular calcium and sodium levels. Here we introduce a simple technique for two-photon chloride imaging in intact neurons. It involves the use of the membrane-permeable Cl(-) indicator dye MQAE [N-(6-methoxyquinolyl) acetoethyl ester]. Brief incubation with MQAE produced a robust loading of cells in slices from various brain regions including hippocampus, cortex and cerebellum. In contrast to conventional fluorescence measurements using MQAE, two-photon imaging was not affected in a major way by dye bleaching and phototoxic damage. Instead, it allowed prolonged recordings of time-resolved changes in intracellular chloride concentration in somata and dendrites. As an example of an application we imaged GABA-mediated Cl(-) transients in pyramidal cells of cortical and hippocampal slices as well as in cerebellar Purkinje neurons. By combining Cl(-) imaging with the gramicidin-based perforated-patch-clamp technique we showed that changes in MQAE fluorescence are proportional to the magnitudes of GABA-evoked transmembrane Cl(-) fluxes. Thus, MQAE-based two-photon microscopy promises to be a valuable technique for many applications requiring chloride imaging in single cells.