Amperometric Monitoring of Sensory-Evoked Dopamine Release in Awake Larval Zebrafish.

Dopamine plays crucial roles in a broad spectrum of brain functions, and neural circuit mechanisms underlying dopaminergic regulation have been intensively studied in the past decade. As larval zebrafish have relatively simple and highly conserved dopaminergic systems, it can serve as an ideal vertebrate animal model to tackle this issue at a whole-brain scale. For this purpose, it is important to develop methods for monitoring endogenous dopamine release in intact larval zebrafish. Here, we developed a real-time method to monitor dopamine release at high spatiotemporal resolution in the brain of awake larval zebrafish using carbon fiber microelectrodes. As an example for application, we combined this method with genetic tools and in vivo calcium imaging and found that food extract can activate pretectal dopaminergic neurons, which in turn release dopamine at the visual center through their projection, providing a dopaminergic circuit mechanism for olfactory modulation of visual functions. Thus, our study demonstrates, for the first time, the utility of carbon fiber microelectrodes for monitoring sensory-evoked dopamine release in the brain of an awake small organism. SIGNIFICANCE STATEMENT: With carbon fiber microelectrodes, we have succeeded in monitoring sensory-evoked dopamine release in the brain of an awake small organism for the first time. By elucidating the circuitry origin of the dopamine release, we illustrated the potential application of this method in dissection of the neural circuitry mechanisms underlying dopaminergic neuromodulation.

Real-time analysis of large-scale neuronal imaging enables closed-loop investigation of neural dynamics.

Large-scale imaging of neuronal activities is crucial for understanding brain functions. However, it is challenging to analyze large-scale imaging data in real time, preventing closed-loop investigation of neural circuitry. Here we develop a real-time analysis system with a field programmable gate array-graphics processing unit design for an up to 500-megabyte-per-second image stream. Adapted to whole-brain imaging of awake larval zebrafish, the system timely extracts activity from up to 100,000 neurons and enables closed-loop perturbations of neural dynamics.

Periodic stimulation induces long-range modulation of cortical responses and visual perception.

Periodic sensory stimuli are prevalent in natural environment and may signal events of particular importance. However, whether periodic and aperiodic stimuli are differentially processed by neural circuits remains unclear. Here we show that periodic stimuli exert influences over longer distances than aperiodic stimuli at both neuronal and perceptual levels. Whole-cell recording from rat visual cortex showed that periodic visual stimulation (1-11 Hz) outside the neuronal receptive field evoked robust membrane potential oscillation at the stimulation frequency, while the same stimulus applied aperiodically had little effect. Human psychophysical experiments showed that periodic luminance changes in the distant surround also exert a stronger modulation of perceived brightness of a centre test stimulus than that exerted by aperiodic changes. Furthermore, both perceptual and neuronal modulation increased with the number of stimulation cycles for periodic surround stimuli. Thus, periodic stimuli can modulate both cortical responses and visual perception over larger spatial scales, generating more global impact on cortical information processing.

All-optical imaging and manipulation of whole-brain neuronal activities in behaving larval zebrafish.

All-optical interrogation of population neuron activity is a promising approach to deciphering the neural circuit mechanisms supporting brain functions. However, this interrogation is currently limited to local brain areas. Here, we incorporate patterned photo-stimulation into light-sheet microscopy, allowing simultaneous targeted optogenetic manipulation and brain-wide monitoring of the neuronal activities of head-restrained behaving larval zebrafish. Using this system, we photo-stimulate arbitrarily selected neurons (regions as small as ~10-20 neurons in 3D) in zebrafish larvae with pan-neuronal expression of a spectrally separated calcium indicator, GCaMP6f, and an activity actuator, ChrimsonR, and observe downstream neural circuit activation and behavior generation. This approach allows us to dissect the causal role of neural circuits in brain functions and behavior generation.

Calcium influx through hyperpolarization-activated cation channels (I(h) channels) contributes to activity-evoked neuronal secretion.

The hyperpolarization-activated cation channels (I(h)) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca(2+) can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and I(h) channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca(2+) imaging, we found that there is a Ca(2+) influx in proportion to I(h) induced by hyperpolarization in HEK293 cells. The I(h) channel blockers Cs(+) and ZD7288 inhibit both HCN current and Ca(2+) influx. Measurements of the fractional Ca(2+) current showed that it constitutes 0.60 +/- 0.02% of the net inward current through HCN4 at -120 mV. This fractional current is similar to that of the low Ca(2+)-permeable AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of I(h) for 30 s also resulted in a Ca(2+) influx and an elevated action potential-induced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for I(h) channels in modulating neuronal secretion by permitting Ca(2+) influx at negative membrane potentials.