Induction of Short-Term Sensitization by an Aversive Chemical Stimulus in Zebrafish Larvae.

Larval zebrafish possess a number of molecular and genetic advantages for rigorous biological analyses of learning and memory. These advantages have motivated the search for novel forms of memory in these animals that can be exploited for understanding the cellular and molecular bases of vertebrate memory formation and consolidation. Here, we report a new form of behavioral sensitization in zebrafish larvae that is elicited by an aversive chemical stimulus [allyl isothiocyanate (AITC)] and that persists for ≥30 min. This form of sensitization is expressed as enhanced locomotion and thigmotaxis, as well as elevated heart rate. To characterize the neural basis of this nonassociative memory, we used transgenic zebrafish expressing the fluorescent calcium indicator GCaMP6 (Chen et al., 2013); because of the transparency of larval zebrafish, we could optically monitor neural activity in the brain of intact transgenic zebrafish before and after the induction of sensitization. We found a distinct brain area, previously linked to locomotion, that exhibited persistently enhanced neural activity following washout of AITC; this enhanced neural activity correlated with the behavioral sensitization. These results establish a novel form of memory in larval zebrafish and begin to unravel the neural basis of this memory.

Multifocal fluorescence microscope for fast optical recordings of neuronal action potentials.

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera's native frame rate. We demonstrate that this approach is capable of recording Ca(2+) transients resulting from APs in neurons labeled with the Ca(2+) sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations.

Multisensory control of multimodal behavior: do the legs know what the tongue is doing?

Understanding of adaptive behavior requires the precisely controlled presentation of multisensory stimuli combined with simultaneous measurement of multiple behavioral modalities. Hence, we developed a virtual reality apparatus that allows for simultaneous measurement of reward checking, a commonly used measure in associative learning paradigms, and navigational behavior, along with precisely controlled presentation of visual, auditory and reward stimuli. Rats performed a virtual spatial navigation task analogous to the Morris maze where only distal visual or auditory cues provided spatial information. Spatial navigation and reward checking maps showed experience-dependent learning and were in register for distal visual cues. However, they showed a dissociation, whereby distal auditory cues failed to support spatial navigation but did support spatially localized reward checking. These findings indicate that rats can navigate in virtual space with only distal visual cues, without significant vestibular or other sensory inputs. Furthermore, they reveal the simultaneous dissociation between two reward-driven behaviors.

Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing.

In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.

High-throughput single-molecule fluorescence spectroscopy using parallel detection.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors.

Hybrid photodetector for single-molecule spectroscopy and microscopy.

We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications.

Development of an ultrafast single photon counting imager for single molecule imaging.

We have begun developing an innovative ultra-fast single-photon counting imager which comprises a mega-pixel CMOS array and a newly-designed Image Intensifier. It is expected to have single photon sensitivity with 100 psec time resolution, operational at a total counting rate exceeding 1MHz. The readout is based on dead-time-free flash ADC, running at 1-2GS/s, followed by a FPGA for real-time parallel data processing. Such a device has not been realized before and is expected to revolutionize time-resolved fluorescence imaging and spectroscopy from a single-molecule to whole animal level. To evaluate the design principle, an Image Intensifier with a GaAsP photocathode (>40% quantum efficiency at 400-600 nm) followed by double MCP was evaluated together with an existing CMOS camera. In our future design, the image from CMOS Camera will be combined with the MCP output, followed by a set of FPGA and CPU for real time data processing. This stream line method will allow ultra fast single-photon counting with 100 psec time resolution and 20 µm position resolution (1M pixel imaging). In this paper, we present the design principle and preliminary results on its performance. Our future plan and the design goals are also described.