Channelrhodopsins are used to optogenetically depolarize neurons. We engineered a variant of channelrhodopsin, denoted Red-activatable Channelrhodopsin (ReaChR), that is optimally excited with orange to red light (λ ~ 590 to 630 nm) and offers improved membrane trafficking, higher photocurrents, and faster kinetics compared with existing red-shifted channelrhodopsins. Red light is more weakly scattered by tissue and absorbed less by blood than the blue to green wavelengths required by other channelrhodopsin variants. ReaChR expressed in vibrissa motor cortex was used to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illuminating with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window, or implant optical fibers.

Neuronal coding of stimulus-to-action sequences are believed to involve the release of dopamine (DA) and norepinephrine (NE). The electrochemical similarity of these monoamines, however, confounds real-time measurements of their release. Here we report the creation of cell-based neurotransmitter fluorescent-engineered reporters (CNiFERs) that utilize the specificity of G-protein coupled receptors (GPCRs) to discriminate nanomolar concentrations of DA and NE. CNiFERs were implanted into frontal cortex of mice to measure the timing of neurotransmitter release during classical conditioning using two-photon microscopy. The onset of DA release correlated with that of licking and monotonically shifted from the time of the reward toward that of the cue. In contrast, concurrent release of NE did not correlate with licking or the cue. This new generation of CNiFERs provides unique tools to assess the release of monoamines. The molecular design of these CNiFERs may be generalized to realize CNiFERs for any molecule that activates a GPCR.

Tools from molecular biology, in combination with in vivo optical imaging techniques, provide new mechanisms to noninvasively observe brain processing. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. Here we introduce CNiFERs, cell-based neurotransmitter fluorescent engineered reporters, to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, make use of 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 detects acetylcholine release via activation of M1 muscarinic receptors. Chronic implantation of M1-CNiFERs in frontal cortex of the adult rat is used to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We show that these drugs potently inhibit in situ muscarinic receptor activity.

The neurovascular system may be viewed as a distributed nervous system within the brain. It transforms local neuronal activity into a change in the tone of smooth muscle that lines the walls of arterioles and microvessels. We review the current state of neurovascular coupling, with an emphasis on signaling molecules that convey information from neurons to neighboring vessels. At the level of neocortex, this coupling is mediated by: (i) a likely direct interaction with inhibitory neurons, (ii) indirect interaction, via astrocytes, with excitatory neurons, and (iii) fiber tracts from subcortical layers. Substantial evidence shows that control involves competition between signals that promote vasoconstriction versus vasodilation. Consistent with this picture is evidence that, under certain circumstances, increased neuronal activity can lead to vasoconstriction rather than vasodilation. This confounds naïve interpretations of functional brain images. We discuss experimental approaches to detect signaling molecules in vivo with the goal of formulating an empirical basis for the observed logic of neurovascular control.

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.