[show abstract][hide abstract] ABSTRACT: 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 Ca(2+) 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-HT(3A) serotonin receptors and a chimera of human α7/mouse 5-HT(3A) 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.
PLoS ONE 01/2011; 6(1):e16519. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 G(q) protein-coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca(2+)] and report [Ca(2+)] with a genetically encoded fluorescent Ca(2+) 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.
[show abstract][hide abstract] ABSTRACT: A highly interconnected network of arterioles overlies mammalian cortex to route blood to the cortical mantle. Here we test if this angioarchitecture can ensure that the supply of blood is redistributed after vascular occlusion. We use rodent parietal cortex as a model system and image the flow of red blood cells in individual microvessels. Changes in flow are quantified in response to photothrombotic occlusions to individual pial arterioles as well as to physical occlusions of the middle cerebral artery (MCA), the primary source of blood to this network. We observe that perfusion is rapidly reestablished at the first branch downstream from a photothrombotic occlusion through a reversal in flow in one vessel. More distal downstream arterioles also show reversals in flow. Further, occlusion of the MCA leads to reversals in flow through approximately half of the downstream but distant arterioles. Thus the cortical arteriolar network supports collateral flow that may mitigate the effects of vessel obstruction, as may occur secondary to neurovascular pathology.
[show abstract][hide abstract] ABSTRACT: We tested the hypothesis that activation of nucleus basalis magnocellularis (NBM), which provides cholinergic input to cortex, facilitates motor control. Our measures of facilitation were changes in the direction and time-course of vibrissa movements that are elicited by microstimulation of vibrissa motor (M1) cortex. In particular, microstimulation led solely to a transient retraction of the vibrissae in the sessile animal but to a full motion sequence of protraction followed by retraction in the aroused animal. We observed that activation of NBM, as assayed by cortical desynchronization, induced a transition from microstimulation-evoked retraction to full sweep sequences. This dramatic change in the vibrissa response to microstimulation was blocked by systemic delivery of atropine and, in anesthetized animals, an analogous change was blocked by the topical administration of atropine to M1 cortex. We conclude that NBM significantly facilitates the ability of M1 cortex to control movements. Our results bear on the importance of cholinergic activation in schemes for neuroprosthetic control of movement.
Journal of Neurophysiology 08/2005; 94(1):699-711. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using two-photon microscopy, we quantify changes in blood flow after photothrombotic occlusion of individual blood vessels in rat neocortex, and find that flow reverses direction at the first branch that lies downstream from localized clots.
Orthopaedics & Traumatology-surgery & Research - ORTHOP TRAUMATOL-SURG RES. 05/2004;
[show abstract][hide abstract] ABSTRACT: 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.
Lasers and Electro-Optics Society, 2003. LEOS 2003. The 16th Annual Meeting of the IEEE; 11/2003
[show abstract][hide abstract] ABSTRACT: 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.