Lee F. Schroeder

An in vivo biosensor for neurotransmitter release and in situ receptor activity

Tools from molecular biology, combined with in vivo optical imaging techniques, provide new mechanisms for noninvasively observing brain processes. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. We devised cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, use the Gq protein–coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca2+] and report [Ca2+] with a genetically encoded fluorescent Ca2+ sensor. The initial realization of CNiFERs detected acetylcholine release via activation of M1 muscarinic receptors. We used chronic implantation of M1-CNiFERs in frontal cortex of the adult rat to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We found that these drugs potently inhibited in situ muscarinic receptor activity.

Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

All-optical thrombotic stroke model for near-surface blood vessels in rat: focal illumination of exogeneous photosensitizers combined with real-time two-photon imaging

Photothrombotic microstrokes are produced in rat cortex by 532-nm single-photon optical excitation of an intravenously injected photosensitizer, rose bengal. The dynamics of blood flow and clot formation in the cortical vasculature are observed using two-photon laser scanning microscopy of an intravenously injected fluorescent dye. Flowing and clotted vessels are clearly distinguishable in both large and small vessels, down to individual capillaries, using this technique. We find that by tightly focusing the laser light used to excite the photosensitizer, clots can be formed in individual blood vessels without affecting neighboring vessels tens of micrometers away. We observe many changes in blood flow as a result of localized clot formation, including upstream vascular dilation, clot clearing, i.e. recanalization, and complete reversal of blood flow direction downstream.

Real-time two-photon fluorescence microscopy of blood flow dynamics following photothrombotic stroke in rat neocortex

We use two-photon microscopy to observe changes in blood flow after localized photothrombotic clotting of individual blood vessels in rat neocortex. We find that flow reverses direction downstream from clotted arterioles, thereby maintaining tissue perfusion.