The Visual Systems of Zebrafish.

The zebrafish visual system has become a paradigmatic preparation for behavioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 13 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tectum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographically organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated multistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior.

Brain-wide impacts of sedation on spontaneous activity and auditory processing in larval zebrafish.

Despite their widespread use, we have limited knowledge of the mechanisms by which sedatives mediate their effects on brain-wide networks. This is, in part, due to the technical challenge of observing activity across large populations of neurons in normal and sedated brains. In this study, we examined the effects of the sedative dexmedetomidine, and its antagonist atipamezole, on spontaneous brain dynamics and auditory processing in zebrafish larvae. Our brain-wide, cellular-resolution calcium imaging reveals, for the first time, the brain regions involved in these network-scale dynamics and the individual neurons that are affected within those regions. Further analysis reveals a variety of dynamic changes in the brain at baseline, including marked reductions in spontaneous activity, correlation, and variance. The reductions in activity and variance represent a "quieter" brain state during sedation, an effect that causes highly correlated evoked activity in the auditory system to stand out more than it does in un-sedated brains. We also observe a reduction in auditory response latencies across the brain during sedation, suggesting that the removal of spontaneous activity leaves the core auditory pathway free of impingement from other non-auditory information. Finally, we describe a less dynamic brain-wide network during sedation, with a higher energy barrier and a lower probability of brain state transitions during sedation. In total, our brain-wide, cellular-resolution analysis shows that sedation leads to quieter, more stable, and less dynamic brain, and that against this background, responses across the auditory processing pathway become sharper and more prominent. Significance Statement: Animals' brain states constantly fluctuate in response to their environment and context, leading to changes in perception and behavioral choices. Alterations in perception, sensorimotor gating, and behavioral selection are hallmarks of numerous neuropsychiatric disorders, but the circuit- and network-level underpinnings of these alterations are poorly understood.Pharmacological sedation alters perception and responsiveness and provides a controlled and repeatable manipulation for studying brain states and their underlying circuitry. Here, we show that sedation of larval zebrafish with dexmedetomidine reduces brain-wide spontaneous activity and locomotion but leaves portions of brain-wide auditory processing and behavior intact. We describe and computationally model changes at the levels of individual neurons, local circuits, and brain-wide networks that lead to altered brain states and sensory processing during sedation.

Auditory processing in rodent models of autism: a systematic review.

Autism is a complex condition with many traits, including differences in auditory sensitivity. Studies in human autism are plagued by the difficulty of controlling for aetiology, whereas studies in individual rodent models cannot represent the full spectrum of human autism. This systematic review compares results in auditory studies across a wide range of established rodent models of autism to mimic the wide range of aetiologies in the human population. A search was conducted in the PubMed and Web of Science databases to find primary research articles in mouse or rat models of autism which investigate central auditory processing. A total of 88 studies were included. These used non-invasive measures of auditory function, such as auditory brainstem response recordings, cortical event-related potentials, electroencephalography, and behavioural tests, which are translatable to human studies. They also included invasive measures, such as electrophysiology and histology, which shed insight on the origins of the phenotypes found in the non-invasive studies. The most consistent results across these studies were increased latency of the N1 peak of event-related potentials, decreased power and coherence of gamma activity in the auditory cortex, and increased auditory startle responses to high sound levels. Invasive studies indicated loss of subcortical inhibitory neurons, hyperactivity in the lateral superior olive and auditory thalamus, and reduced specificity of responses in the auditory cortex. This review compares the auditory phenotypes across rodent models and highlights those that mimic findings in human studies, providing a framework and avenues for future studies to inform understanding of the auditory system in autism.

Optical tweezers across scales in cell biology.

Optical tweezers (OT) provide a noninvasive approach for delivering minute physical forces to targeted objects. Controlling such forces in living cells or in vitro preparations allows for the measurement and manipulation of numerous processes relevant to the form and function of cells. As such, OT have made important contributions to our understanding of the structures of proteins and nucleic acids, the interactions that occur between microscopic structures within cells, the choreography of complex processes such as mitosis, and the ways in which cells interact with each other. In this review, we highlight recent contributions made to the field of cell biology using OT and provide basic descriptions of the physics, the methods, and the equipment that made these studies possible.

Brain-wide visual habituation networks in wild type and fmr1 zebrafish.

Habituation is a form of learning during which animals stop responding to repetitive stimuli, and deficits in habituation are characteristic of several psychiatric disorders. Due to technical challenges, the brain-wide networks mediating habituation are poorly understood. Here we report brain-wide calcium imaging during larval zebrafish habituation to repeated visual looming stimuli. We show that different functional categories of loom-sensitive neurons are located in characteristic locations throughout the brain, and that both the functional properties of their networks and the resulting behavior can be modulated by stimulus saliency and timing. Using graph theory, we identify a visual circuit that habituates minimally, a moderately habituating midbrain population proposed to mediate the sensorimotor transformation, and downstream circuit elements responsible for higher order representations and the delivery of behavior. Zebrafish larvae carrying a mutation in the fmr1 gene have a systematic shift toward sustained premotor activity in this network, and show slower behavioral habituation.

Contributions of Luminance and Motion to Visual Escape and Habituation in Larval Zebrafish.

Animals from insects to humans perform visual escape behavior in response to looming stimuli, and these responses habituate if looms are presented repeatedly without consequence. While the basic visual processing and motor pathways involved in this behavior have been described, many of the nuances of predator perception and sensorimotor gating have not. Here, we have performed both behavioral analyses and brain-wide cellular-resolution calcium imaging in larval zebrafish while presenting them with visual loom stimuli or stimuli that selectively deliver either the movement or the dimming properties of full loom stimuli. Behaviorally, we find that, while responses to repeated loom stimuli habituate, no such habituation occurs when repeated movement stimuli (in the absence of luminance changes) are presented. Dim stimuli seldom elicit escape responses, and therefore cannot habituate. Neither repeated movement stimuli nor repeated dimming stimuli habituate the responses to subsequent full loom stimuli, suggesting that full looms are required for habituation. Our calcium imaging reveals that motion-sensitive neurons are abundant in the brain, that dim-sensitive neurons are present but more rare, and that neurons responsive to both stimuli (and to full loom stimuli) are concentrated in the tectum. Neurons selective to full loom stimuli (but not to movement or dimming) were not evident. Finally, we explored whether movement- or dim-sensitive neurons have characteristic response profiles during habituation to full looms. Such functional links between baseline responsiveness and habituation rate could suggest a specific role in the brain-wide habituation network, but no such relationships were found in our data. Overall, our results suggest that, while both movement- and dim-sensitive neurons contribute to predator escape behavior, neither plays a specific role in brain-wide visual habituation networks or in behavioral habituation.

RNA-induced inflammation and migration of precursor neurons initiates neuronal circuit regeneration in zebrafish.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair.

The tectum/superior colliculus as the vertebrate solution for spatial sensory integration and action.

The superior colliculus, or tectum in the case of non-mammalian vertebrates, is a part of the brain that registers events in the surrounding space, often through vision and hearing, but also through electrosensation, infrared detection, and other sensory modalities in diverse vertebrate lineages. This information is used to form maps of the surrounding space and the positions of different salient stimuli in relation to the individual. The sensory maps are arranged in layers with visual input in the uppermost layer, other senses in deeper positions, and a spatially aligned motor map in the deepest layer. Here, we will review the organization and intrinsic function of the tectum/superior colliculus and the information that is processed within tectal circuits. We will also discuss tectal/superior colliculus outputs that are conveyed directly to downstream motor circuits or via the thalamus to cortical areas to control various aspects of behavior. The tectum/superior colliculus is evolutionarily conserved among all vertebrates, but tailored to the sensory specialties of each lineage, and its roles have shifted with the emergence of the cerebral cortex in mammals. We will illustrate both the conserved and divergent properties of the tectum/superior colliculus through vertebrate evolution by comparing tectal processing in lampreys belonging to the oldest group of extant vertebrates, larval zebrafish, rodents, and other vertebrates including primates.

Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system.

Most animals have complex auditory systems that identify salient features of the acoustic landscape to direct appropriate responses. In fish, these features include the volume, frequency, complexity, and temporal structure of acoustic stimuli transmitted through water. Larval fish have simple brains compared to adults but swim freely and depend on sophisticated sensory processing for survival.1-5 Zebrafish larvae, an important model for studying brain-wide neural networks, have thus far been found to possess a rudimentary auditory system, sensitive to a narrow range of frequencies and without evident sensitivity to acoustic features that are salient and ethologically important to adult fish.6,7 Here, we have combined a novel method for delivering water-borne sounds, a diverse assembly of acoustic stimuli, and whole-brain calcium imaging to describe the responses of individual auditory-responsive neurons across the brains of zebrafish larvae. Our results reveal responses to frequencies ranging from 100 Hz to 4 kHz, with evidence of frequency discrimination from 100 Hz to 2.5 kHz. Frequency-selective neurons are located in numerous regions of the brain, and neurons responsive to the same frequency are spatially grouped in some regions. Using functional clustering, we identified categories of neurons that are selective for a single pure-tone frequency, white noise, the sharp onset of acoustic stimuli, and stimuli involving a gradual crescendo. These results suggest a more nuanced auditory system than has previously been described in larval fish and provide insights into how a young animal's auditory system can both function acutely and serve as the scaffold for a more complex adult system.

Optical Tweezers Exploring Neuroscience.

Over the past decade, optical tweezers (OT) have been increasingly used in neuroscience for studies of molecules and neuronal dynamics, as well as for the study of model organisms as a whole. Compared to other areas of biology, it has taken much longer for OT to become an established tool in neuroscience. This is, in part, due to the complexity of the brain and the inherent difficulties in trapping individual molecules or manipulating cells located deep within biological tissue. Recent advances in OT, as well as parallel developments in imaging and adaptive optics, have significantly extended the capabilities of OT. In this review, we describe how OT became an established tool in neuroscience and we elaborate on possible future directions for the field. Rather than covering all applications of OT to neurons or related proteins and molecules, we focus our discussions on studies that provide crucial information to neuroscience, such as neuron dynamics, growth, and communication, as these studies have revealed meaningful information and provide direction for the field into the future.

Sound generation in zebrafish with Bio-Opto-Acoustics.

Hearing is a crucial sense in underwater environments for communication, hunting, attracting mates, and detecting predators. However, the tools currently used to study hearing are limited, as they cannot controllably stimulate specific parts of the auditory system. To date, the contributions of hearing organs have been identified through lesion experiments that inactivate an organ, making it difficult to gauge the specific stimuli to which each organ is sensitive, or the ways in which inputs from multiple organs are combined during perception. Here, we introduce Bio-Opto-Acoustic (BOA) stimulation, using optical forces to generate localized vibrations in vivo, and demonstrate stimulation of the auditory system of zebrafish larvae with precise control. We use a rapidly oscillated optical trap to generate vibrations in individual otolith organs that are perceived as sound, while adjacent otoliths are either left unstimulated or similarly stimulated with a second optical laser trap. The resulting brain-wide neural activity is characterized using fluorescent calcium indicators, thus linking each otolith organ to its individual neuronal network in a way that would be impossible using traditional sound delivery methods. The results reveal integration and cooperation of the utricular and saccular otoliths, which were previously described as having separate biological functions, during hearing.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Altered brain-wide auditory networks in a zebrafish model of fragile X syndrome.

BACKGROUND: Loss or disrupted expression of the FMR1 gene causes fragile X syndrome (FXS), the most common monogenetic form of autism in humans. Although disruptions in sensory processing are core traits of FXS and autism, the neural underpinnings of these phenotypes are poorly understood. Using calcium imaging to record from the entire brain at cellular resolution, we investigated neuronal responses to visual and auditory stimuli in larval zebrafish, using fmr1 mutants to model FXS. The purpose of this study was to model the alterations of sensory networks, brain-wide and at cellular resolution, that underlie the sensory aspects of FXS and autism. RESULTS: Combining functional analyses with the neurons' anatomical positions, we found that fmr1-/- animals have normal responses to visual motion. However, there were several alterations in the auditory processing of fmr1-/- animals. Auditory responses were more plentiful in hindbrain structures and in the thalamus. The thalamus, torus semicircularis, and tegmentum had clusters of neurons that responded more strongly to auditory stimuli in fmr1-/- animals. Functional connectivity networks showed more inter-regional connectivity at lower sound intensities (a - 3 to - 6 dB shift) in fmr1-/- larvae compared to wild type. Finally, the decoding capacities of specific components of the ascending auditory pathway were altered: the octavolateralis nucleus within the hindbrain had significantly stronger decoding of auditory amplitude while the telencephalon had weaker decoding in fmr1-/- mutants. CONCLUSIONS: We demonstrated that fmr1-/- larvae are hypersensitive to sound, with a 3-6 dB shift in sensitivity, and identified four sub-cortical brain regions with more plentiful responses and/or greater response strengths to auditory stimuli. We also constructed an experimentally supported model of how auditory information may be processed brain-wide in fmr1-/- larvae. Our model suggests that the early ascending auditory pathway transmits more auditory information, with less filtering and modulation, in this model of FXS.

Brain-Wide Mapping of Water Flow Perception in Zebrafish.

Information about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, neuronal responses involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to head-to-tail (h-t) flow, tail-to-head (t-h) flow, or both. Early in this pathway, in the lateral line ganglia, neurons respond almost exclusively to the simple presence of h-t or t-h flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated displacement of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to h-t flow detection, and this is reinforced by our finding that details like flow velocity and the total accumulated flow are only encoded for the h-t direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.SIGNIFICANCE STATEMENT In aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find neurons that respond to the simple presence of water flow, and others attuned to the direction, speed, and duration of flow, or the accumulated displacement of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating head-to-tail motion but rudimentary in processing flow in the tail-to-head direction.

Calcium Imaging and the Curse of Negativity.

The imaging of neuronal activity using calcium indicators has become a staple of modern neuroscience. However, without ground truths, there is a real risk of missing a significant portion of the real responses. Here, we show that a common assumption, the non-negativity of the neuronal responses as detected by calcium indicators, biases all levels of the frequently used analytical methods for these data. From the extraction of meaningful fluorescence changes to spike inference and the analysis of inferred spikes, each step risks missing real responses because of the assumption of non-negativity. We first show that negative deviations from baseline can exist in calcium imaging of neuronal activity. Then, we use simulated data to test three popular algorithms for image analysis, CaImAn, suite2p, and CellSort, finding that suite2p may be the best suited to large datasets. We also tested the spike inference algorithms included in CaImAn, suite2p, and Cellsort, as well as the dedicated inference algorithms MLspike and CASCADE, and found each to have limitations in dealing with inhibited neurons. Among these spike inference algorithms, FOOPSI, from CaImAn, performed the best on inhibited neurons, but even this algorithm inferred spurious spikes upon the return of the fluorescence signal to baseline. As such, new approaches will be needed before spikes can be sensitively and accurately inferred from calcium data in inhibited neurons. We further suggest avoiding data analysis approaches that, by assuming non-negativity, ignore inhibited responses. Instead, we suggest a first exploratory step, using k-means or PCA for example, to detect whether meaningful negative deviations are present. Taking these steps will ensure that inhibition, as well as excitation, is detected in calcium imaging datasets.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Cellular-Resolution Imaging of Vestibular Processing across the Larval Zebrafish Brain.

The vestibular system, which reports on motion and gravity, is essential to postural control, balance, and egocentric representations of movement and space. The motion needed to stimulate the vestibular system complicates studying its circuitry, so we previously developed a method for fictive vestibular stimulation in zebrafish, using optical trapping to apply physical forces to the otoliths. Here, we combine this approach with whole-brain calcium imaging at cellular resolution, delivering a comprehensive map of the brain regions and cellular responses involved in basic vestibular processing. We find responses broadly distributed across the brain, with unique profiles of cellular responses and topography in each region. The most widespread and abundant responses involve excitation that is graded to the stimulus strength. Other responses, localized to the telencephalon and habenulae, show excitation that is only weakly correlated to stimulus strength and that is sensitive to weak stimuli. Finally, numerous brain regions contain neurons that are inhibited by vestibular stimuli, and these neurons are often tightly localized spatially within their regions. By exerting separate control over the left and right otoliths, we explore the laterality of brain-wide vestibular processing, distinguishing between neurons with unilateral and bilateral vestibular sensitivity and revealing patterns whereby conflicting signals from the ears mutually cancel. Our results confirm previously identified vestibular responses in specific regions of the larval zebrafish brain while revealing a broader and more extensive network of vestibular responsive neurons than has previously been described. This provides a departure point for more targeted studies of the underlying functional circuits.

Luminance Changes Drive Directional Startle through a Thalamic Pathway.

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations.

Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light-sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element.

Integrative whole-brain neuroscience in larval zebrafish.

Due to their small size and transparency, zebrafish larvae are amenable to a range of fluorescence microscopy techniques. With the development of sensitive genetically encoded calcium indicators, this has extended to the whole-brain imaging of neural activity with cellular resolution. This technique has been used to study brain-wide population dynamics accompanying sensory processing and sensorimotor transformations, and has spurred the development of innovative closed-loop behavioral paradigms in which stimulus-response relationships can be studied. More recently, microscopes have been developed that allow whole-brain calcium imaging in freely swimming and behaving larvae. In this review, we highlight the technologies underlying whole-brain functional imaging in zebrafish, provide examples of the sensory and motor processes that have been studied with this technique, and discuss the need to merge data from whole-brain functional imaging studies with neurochemical and anatomical information to develop holistic models of functional neural circuits.