Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

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The striatum plays a central role in sensorimotor learning and action selection. Tonically active cholinergic interneurons in the striatum give rise to dense axonal arborizations and significantly shape striatal output. However, it is not clear how the activity of these neurons is regulated within the striatal microcircuitry. In this study, using rat brain slices, we find that stimulation of intrastriatal cholinergic fibers evokes polysynaptic GABA A IPSCs in cholinergic interneurons. These polysynaptic GABA A IPSCs were abolished by general nicotinic acetylcholine receptor antagonists and also by a specific antagonist of nicotinic receptors containing ␤2 subunits. Dopamine receptor antagonists or dopamine depletion failed to block polysynaptic IPSCs, indicating that phasic dopamine release does not directly mediate the polysynaptic transmission. Dual recording from pairs of cholinergic interneurons revealed that activation of a single cholinergic interneuron is capable of eliciting polysynaptic GABA A IPSCs both in itself and in nearby cholinergic interneurons. Although polysynaptic transmission arising from a single cholinergic interneuron was depressed during repetitive 2 Hz firing, intrastriatal stimulation reliably evoked large polysynaptic IPSCs by recruiting many cholinergic fibers. We also show that polysynaptic GABAergic inhibition leads to a transient suppression of tonic cholinergic interneuron firing. We propose a novel microcircuit in the striatum, in which cholinergic interneurons are connected to one another through GABAergic interneurons. This may provide a mechanism to convert activation of cholinergic interneurons into widespread recurrent inhibition of these neurons via nicotinic excitation of striatal GABAergic neurons.

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8 -22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca 2ϩ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca 2ϩ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K ϩ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8 -22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to G q/11 -proteins as well as PTX-sensitive G i/o -proteins, and modulates neuronal Ca 2ϩ channels, K ϩ channels, and synaptic transmission.