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Electrical stimulation is the state-of-the-art method to restore hearing in deaf patients. However, the method offers limited frequency resolution due to the unavoidable electric field spread in the neural tissue. To overcome this limitation, we performed precise optogenetic stimulation in the murine inferior colliculus (IC) using a tapered optical fiber. Tapered fibers permit switching of light output which enables stimulation at various depths in the IC. Both the light-sensitive pump, channelrhodopsin-2 (ChR2) and archaerhodopsin-3 for neuronal activation and inhibition, respectively were co-expressed in the murine IC. We demonstrate that ChR2 stimulation at spatially separated regions can elicit distinct changes in the local field potential recorded at the auditory cortex. Furthermore, inhibition of neuronal activity by optical perturbation of Arch-T neurons between the stimulated regions increases the sensitivity of the IC to the ChR2 activation.
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Holographic multimode fibre endoscopes have recently shown their promising potential as a minimally invasive imaging tool, particularly in the field of neurobiology. Currently, their resolution is limited by diffraction, constrained by the relatively low numerical aperture of the multimode fibres used (typically less than 0.4). Overcoming the diffraction-limit barrier would open new possibilities for detailed observation of dendritic spines and their motility in-vivo.
In this presentation we demonstrate pulsed STED microscopy delivered through a holographic multimode fibre endoscope. We show resolution improvements over 3-fold of the diffraction limit. Moreover, we discuss and showcase its applicability to bio-imaging.
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We present a new approach for imaging intracellular neurotransmitter molecules with stimulated Raman scattering microscopy. We leverage the isolated vibrational peaks of carbon-deuterium bonds to observe these neurotransmitters directly and quantitatively. By using deuterated versions of neurotransmitters, we minimize perturbation to neurochemical activity with respect to previously demonstrated fluorescence-based methods. We show direct imaging of deuterated dopamine and GABA uptake and release dynamics in PC12 chromafin cells, and in primary hippocampal neurons, respectively. Specifically, we show that stimulation of neurotransmitter release results in a 20-50% intracellular neurotransmitter concentration reduction, with the ability to observe inter- and intracellular variation in vesicular neurotransmitter release. Taken together, our data suggest that neurotransmitter isotopologues can serve as a generic, commercially-available, non-perturbative, and biocompatible method to image neurotransmitters that are chemically homologous to their native counterparts.
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Just like the earth, the brain is not flat. Visualizing brain-wide neuronal activities at single-cell resolution in living brains requires not only expanding the view of conventional flat-field microscopy but also considerations that the brain is not flat at this scale. Here, i present both previously published works and ongoing, unpublished works that all address this simple issue of geometry underlying the growing need and expectations of neuroscience.
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Cells use molecules to exchange information. A very prominent example is neurotransmitter signaling between neuronal cells. The neurotransmitter dopamine is released from discrete axonal structures called varicosities. Its release is essential in behaviour and is critically implicated in prevalent neuropsychiatric diseases but existing dopamine detection methods are not able to image dopamine release events from multiple locations. Here, we develop a near infrared (NIR) fluorescent (980 nm) dopamine nanosensor ‘paint’ (AndromeDA) and show that action potential-evoked dopamine release is highly heterogeneous across release sites. The sensors are based on single-walled carbon nanotubes (SWCNTs) that emit fluorescence in the highly beneficial NIR tissue transparency window. We visualize dopamine release at up to 100 dopaminergic varicosities simultaneously within a single imaging field with high temporal resolution (15 images/s). We find that ‘hotspots’ of dopamine release are highly heterogeneous and are detected at only ~17% of all varicosities. In summary, we demonstrate NIR imaging of neurotransmitters and provide insights into the spatiotemporal organization of dopamine release.
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Emerging Trends in Monitoring and Stimulating Brain Function
Diffuse Correlation Spectroscopy (DCS) allows the optical and label-free investigation of microvascular dynamics. Commonly, DCS is implemented with highly sensitive and ultra fast single-photon avalanche diodes (SPAD) for blood flow measurements from around 1-1.5cm deep inside tissue (source detector separation of 2.5-3 cm). In parallelized DCS (pDCS), we use arrays of multiple SPADs to boost the signal-to-noise ratio by averaging many independent DCS measurements. In this study, we explored the capabilities of an innovative, massively parallelized SPAD array with 500x500 single pixels for DCS for up to 250,000 parallel DCS measurements. We can show that this massively parallelized array enables viable blood flow measurements at 2cm depth (4cm source detector separation) in human subjects. Furthermore, we applied a dual detection strategy, where a secondary SPAD array probes the superficial blood flow simultaneously as a build-in reference measurement. In addition to our main results, we test and discuss methods to correct the deep flow measurement, by including simultaneously measured flow dynamics deep and superficial tissue layers via our novel dual-SPAD array measurement setup.
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Investigating the processes that underly memory formation in the brain can be a difficult task. Conventional in vivo experiments involve millions of different cells whose processes are difficult to be isolated and singularly studied. This is where in vitro techniques can offer a controlled environment to examine the mechanisms involved in the operation of the basic circuits involved in memory (engrams).
We developed a technique that combines digital light processing (DLP) with Optogenetics to achieve precise control over neuronal activity. We were able to generate small, discrete modules of engram circuits in vitro that adhere to Hebb's postulate. By utilizing the Synactive labeling technique alongside our strategy, we were able to identify and track strengthened spines between active neurons. This enabled us to examine how a pattern of activity between neurons is established within the engram circuits, which are also known as synaptic engrams. This method can be used to create more precise experimental models of memory storage and retrieval, opening the doors to a new understanding of the processes underlying brain activity.
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A wearable and distributed functional near-infrared spectroscopy (fNIRS) system has been developed to facilitate hyperscanning of real-world interactions. This innovative system allows for synchronous measurement, wireless master-slave connections, and reduced power consumption.
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