Peixin Zhu

Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system

The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

Circuit Neuroscience in Zebrafish

A central goal of modern neuroscience is to obtain a mechanistic understanding of higher brain functions under healthy and diseased conditions. Addressing this challenge requires rigorous experimental and theoretical analysis of neuronal circuits. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of synaptic wiring diagrams have created new opportunities to achieve this goal. To fully harness these methods, model organisms should allow for a combination of genetic and neurophysiological approaches in vivo. Moreover, the brain should be small in terms of neuron numbers and physical size. A promising vertebrate organism is the zebrafish because it is small, it is transparent at larval stages and it offers a wide range of genetic tools and advantages for neurophysiological approaches. Recent studies have highlighted the potential of zebrafish for exhaustive measurements of neuronal activity patterns, for manipulations of defined cell types in vivo and for studies of causal relationships between circuit function and behavior. In this article, we summarize background information on the zebrafish as a model in modern systems neuroscience and discuss recent results.

High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device

Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications.

Neuronal filtering of multiplexed odour representations

Neuronal activity patterns contain information in their temporal structure, indicating that information transfer between neurons may be optimized by temporal filtering. In the zebrafish olfactory bulb, subsets of output neurons (mitral cells) engage in synchronized oscillations during odour responses, but information about odour identity is contained mostly in non-oscillatory firing rate patterns. Using optogenetic manipulations and odour stimulation, we found that firing rate responses of neurons in the posterior zone of the dorsal telencephalon (Dp), a target area homologous to olfactory cortex, were largely insensitive to oscillatory synchrony of mitral cells because passive membrane properties and synaptic currents act as low-pass filters. Nevertheless, synchrony influenced spike timing. Moreover, Dp neurons responded primarily during the decorrelated steady state of mitral cell activity patterns. Temporal filtering therefore tunes Dp neurons to components of mitral cell activity patterns that are particularly informative about precise odour identity. These results demonstrate how temporal filtering can extract specific information from multiplexed neuronal codes.

Equalization of odor representations by a network of electrically coupled inhibitory interneurons

Robustness of neuronal activity patterns against variations in input intensity is critical for neuronal computations. We found that odor representations in the olfactory bulb were stabilized by interneurons that were densely coupled to the output neurons by electrical and GABAergic synapses. This interneuron network modulated responses of output neurons as a function of stimulus intensity in two ways: it globally boosted responses to weak odors, but attenuated responses to strong odors, and it increased the sensitivity of some output neurons, but decreased the sensitivity of others. These effects are closely related to strategies used in engineering to increase dynamic range. Together, they maintained not only the mean, but also the distribution, of activity across the population of output neurons within narrow limits, which is important for pattern classification. Neuronal circuits in the olfactory bulb therefore stabilize combinatorial sensory representations against variations in stimulus intensity by generic mechanisms.

Dopaminergic Modulation of Mitral Cells and Odor Responses in the Zebrafish Olfactory Bulb

In the olfactory bulb, the modulatory neurotransmitter dopamine (DA) is coexpressed with GABA by local interneurons, but its role in odor processing remains obscure. We examined functions of DA mediated by D2-like receptors in the olfactory bulb of adult zebrafish by pharmacology, whole-cell recordings, calcium imaging, and optogenetics. Bath application of DA had no detectable effect on odorant-evoked sensory input. DA directly hyperpolarized mitral cells (MCs) via D2-like receptors and slightly increased their response gain. Consistent with this effect on input–output functions of MCs, small odorant responses were suppressed, whereas strong responses were enhanced in the presence of DA. These effects increased the root-mean-square contrast of population activity patterns but did not reduce their correlations. Optical stimulation of interneurons expressing channelrhodopsin-2 evoked fast GABAergic inhibitory currents in mitral cells but failed to activate D2 receptor-mediated currents when stimuli were short. Prolonged stimulus trains, however, activated a slow hyperpolarizing current that was blocked by an antagonist of D2-like receptors. GABA and DA are therefore both released from interneurons by electrical activity and hyperpolarize MCs, but D2-dependent dopaminergic effects occur on slower timescales. Additional effects of DA may be mediated by D1-like receptors. These results indicate that DA acts on D2-like receptors via asynchronous release and/or volume transmission and implicate DA in the slow adaptation of circuit function. The shift of the membrane potential away from spike threshold could adapt mitral cells to background input without compromising their sensitivity.

Control of a specific motor program by a small brain area in zebrafish

Complex motor behaviors are thought to be coordinated by networks of brain nuclei that may control different elementary motor programs. Transparent zebrafish larvae offer the opportunity to analyze the functional organization of motor control networks by optical manipulations of neuronal activity during behavior. We examined motor behavior in transgenic larvae expressing channelrhodopsin-2 throughout many neurons in the brain. Wide-field optical stimulation triggered backward and rotating movements caused by the repeated execution of J-turns, a specific motor program that normally occurs during prey capture. Although optically-evoked activity was widespread, behavioral responses were highly coordinated and lateralized. 3-D mapping of behavioral responses to local optical stimuli revealed that J-turns can be triggered specifically in the anterior-ventral optic tectum (avOT) and/or the adjacent pretectum. These results suggest that the execution of J-turns is controlled by a small group of neurons in the midbrain that may act as a command center. The identification of a brain area controlling a defined motor program involved in prey capture is a step toward a comprehensive analysis of neuronal circuits mediating sensorimotor behaviors of zebrafish.