Identification of Neural Circuits by Imaging Coherent Electrical Activity with FRET-Based Dyes

AbstractWe show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons

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

Oleate but not stearate induces the regulatory phenotype of myeloid suppressor cells

Tumor infiltrating myeloid cells play contradictory roles in the tumor development. Dendritic cells and classical activated macrophages support anti- tumor immune activity via antigen presentation and induction of pro- inflammatory immune responses. Myeloid suppressor cells (MSCs), for instance myeloid derived suppressor cells (MDSCs) or tumor associated macrophages play a critical role in tumor growth. Here, treatment with sodium oleate, an unsaturated fatty acid, induced a regulatory phenotype in the myeloid suppressor cell line MSC-2 and resulted in an increased suppression of activated T cells, paralleled by increased intracellular lipid droplets formation. Furthermore, sodium oleate potentiated nitric oxide (NO) production in MSC-2, thereby increasing their suppressive capacity. In primary polarized bone marrow cells, sodium oleate (C18:1) and linoleate (C18:2), but not stearate (C18:0) were identified as potent FFA to induce a regulatory phenotype. This effect was abrogated in MSC-2 as well as primary cells by specific inhibition of droplets formation while the inhibition of de novo FFA synthesis proved ineffective, suggesting a critical role for exogenous FFA in the functional induction of MSCs. Taken together our data introduce a new unsaturated fatty acid-dependent pathway shaping the functional phenotype of MSCs, facilitating the tumor escape from the immune system

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

iGluSnFR variants with improved signal-to-noise ratios and targeting to postsynaptic sites have been developed, enabling the analysis of glutamatergic neurotransmission in vivo as illustrated in the mouse visual and somatosensory cortex. The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Non-Linear Population Firing Rates and Voltage Sensitive Dye Signals in Visual Areas 17 and 18 to Short Duration Stimuli

Visual stimuli of short duration seem to persist longer after the stimulus offset than stimuli of longer duration. This visual persistence must have a physiological explanation. In ferrets exposed to stimuli of different durations we measured the relative changes in the membrane potentials with a voltage sensitive dye and the action potentials of populations of neurons in the upper layers of areas 17 and 18. For durations less than 100 ms, the timing and amplitude of the firing and membrane potentials showed several non-linear effects. The ON response became truncated, the OFF response progressively reduced, and the timing of the OFF responses progressively delayed the shorter the stimulus duration. The offset of the stimulus elicited a sudden and strong negativity in the time derivative of the dye signal. All these non-linearities could be explained by the stimulus offset inducing a sudden inhibition in layers II–III as indicated by the strongly negative time derivative of the dye signal. Despite the non-linear behavior of the layer II–III neurons the sum of the action potentials, integrated from the peak of the ON response to the peak of the OFF response, was almost linearly related to the stimulus duration

On the mechanism of ubiquinone mediated photocurrent generation by a reaction center based photocathode

Upon photoexcitation, the reaction center (RC) pigment-proteins that facilitate natural photosynthesis achieve a metastable separation of electrical charge among the embedded cofactors. Because of the high quantum efficiency of this process, there is a growing interest in their incorporation into biohybrid materials for solar energy conversion, bioelectronics and biosensing. Multiple bioelectrochemical studies have shown that reaction centers from various photosynthetic organisms can be interfaced with diverse electrode materials for the generation of photocurrents, but many mechanistic aspects of native protein functionality in a non-native environment is unknown. In vivo, RC's catalyse ubiquinone-10 reduction, protonation and exchange with other lipid phase ubiquinone-10s via protein-controlled spatial orientation and protein rearrangement. In contrast, the mechanism of ubiquinone-0 reduction, used to facilitate fast RC turnover in an aqueous photoelectrochemical cell (PEC), may not proceed via the same pathway as the native cofactor. In this report we show truncation of the native isoprene tail results in larger RC turnover rates in a PEC despite the removal of the tail's purported role of ubiquinone headgroup orientation and binding. Through the use of reaction centers with single or double mutations, we also show the extent to which two-electron/two-proton ubiquinone chemistry that operates in vivo also underpins the ubiquinone-0 reduction by surface-adsorbed RCs in a PEC. This reveals that only the ubiquinone headgroup is critical to the fast turnover of the RC in a PEC and provides insight into design principles for the development of new biophotovoltaic cells and biosensors

Mechanisms underlying a thalamocortical transformation during active tactile sensation

During active somatosensation, neural signals expected from movement of the sensors are suppressed in the cortex, whereas information related to touch is enhanced. This tactile suppression underlies low-noise encoding of relevant tactile features and the brain’s ability to make fine tactile discriminations. Layer (L) 4 excitatory neurons in the barrel cortex, the major target of the somatosensory thalamus (VPM), respond to touch, but have low spike rates and low sensitivity to the movement of whiskers. Most neurons in VPM respond to touch and also show an increase in spike rate with whisker movement. Therefore, signals related to self-movement are suppressed in L4. Fast-spiking (FS) interneurons in L4 show similar dynamics to VPM neurons. Stimulation of halorhodopsin in FS interneurons causes a reduction in FS neuron activity and an increase in L4 excitatory neuron activity. This decrease of activity of L4 FS neurons contradicts the "paradoxical effect" predicted in networks stabilized by inhibition and in strongly-coupled networks. To explain these observations, we constructed a model of the L4 circuit, with connectivity constrained by in vitro measurements. The model explores the various synaptic conductance strengths for which L4 FS neurons actively suppress baseline and movement-related activity in layer 4 excitatory neurons. Feedforward inhibition, in concert with recurrent intracortical circuitry, produces tactile suppression. Synaptic delays in feedforward inhibition allow transmission of temporally brief volleys of activity associated with touch. Our model provides a mechanistic explanation of a behavior-related computation implemented by the thalamocortical circuit

Determinants of Outdoor Time in Children and Youth: A Systematic Review of Longitudinal and Intervention Studies

Spending more time outdoors can improve children’s social and cognitive development, physical activity, and vision. Our systematic review summarized the determinants of outdoor time (OT) based on the social-ecological model. We searched nine databases: MEDLINE, APA PsycINFO, Web of Science, Cochrane Central Register of Controlled Trials (CENTRAL), CINAHL, SPORTDiscus, ERIC, SocINDEX, and ProQuest Dissertations and Theses. To be included, studies needed to be quantitative and longitudinal, include ≥1 potential determinant of OT among 0- to 17-year-olds, and be published in English, French, Japanese, or Spanish. We extracted the authors, publication year, country, design, sample size, OT measures, follow-up period, potential determinants, main results, and potential moderators or mediators. Fifty-five studies examining 119 potential determinants met the inclusion criteria. OT was consistently higher in warmer seasons and among participants reporting more OT at baseline. All three interventions that included both parent sessions and additional resources to promote OT (e.g., specific advice and community guides) were effective. COVID-19 restrictions and sun safety interventions discouraging midday outdoor activities led to less OT. The quality of evidence was rated as weak for 46 studies. Most potential determinants were examined in ≤3 studies; thus, more longitudinal studies are needed to enable stronger conclusions about the consistency of evidence and meta-analyses