A robust role for motor cortex.

The role of motor cortex in non-primate mammals remains unclear. More than a century of stimulation, anatomical and electrophysiological studies has implicated neural activity in this region with all kinds of movement. However, following the removal of motor cortex, rats retain most of their adaptive behaviors, including previously learned skilled movements. Here we revisit these two conflicting views of motor cortex and present a new behavior assay, challenging animals to respond to unexpected situations while navigating a dynamic obstacle course. Surprisingly, rats with motor cortical lesions show clear impairments facing an unexpected collapse of the obstacles, while showing no impairment with repeated trials in many motor and cognitive metrics of performance. We propose a new role for motor cortex: extending the robustness of sub-cortical movement systems, specifically to unexpected situations demanding rapid motor responses adapted to environmental context. The implications of this idea for current and future research are discussed.

An experimental method for evoking and characterizing dynamic color patterning of cuttlefish during prey capture.

Cuttlefish are active carnivores that possess a wide repertoire of body patterns that can be changed within milliseconds for many types of camouflage and communication. The forms and functions of many body patterns are well known from ethological studies in the field and laboratory. Yet one aspect has not been reported in detail: the category of rapid, brief and high-contrast changes in body coloration ("Tentacle Shot Patterns" or TSPs) that always occur with the ejection of two ballistic tentacles to strike live moving prey ("Tentacles Go Ballistic" or TGB moment). We designed and tested a mechanical device that presented prey in a controlled manner, taking advantage of a key stimulus for feeding: motion of the prey. High-speed video recordings show a rapid transition into TSPs starting 114 ms before TGB (N = 114). TSPs are then suppressed as early as 470-500 ms after TGB (P < 0.05) in unsuccessful hunts, while persisting for at least 3 s after TGB in successful hunts. A granularity analysis revealed significant differences in the large-scale high-contrast body patterning present in TSPs compared to the camouflage body pattern deployed beforehand. TSPs best fit the category of secondary defense called deimatic displaying, meant to briefly startle predators and interrupt their attack sequence while cuttlefish are distracted by striking prey. We characterize TSPs as a pattern category for which the main distinguishing feature is a high-contrast signaling pattern with aspects of Acute Conflict Mottle or Acute Disruptive Pattern. The data and methodology presented here open opportunities for quantifying the rapid neural responses in this visual sensorimotor set of behaviors.

Does Impedance Matter When Recording Spikes With Polytrodes?

Extracellular microelectrodes have been widely used to measure brain activity, yet there are still basic questions about the requirements for a good extracellular microelectrode. One common source of confusion is how much an electrode's impedance affects the amplitude of extracellular spikes and background noise. Here we quantify the effect of an electrode's impedance on data quality in extracellular recordings, which is crucial for both the detection of spikes and their assignment to the correct neurons. This study employs commercial polytrodes containing 32 electrodes (177 µm2) arranged in a dense array. This allowed us to directly compare, side-by-side, the same extracellular signals measured by modified low impedance (∼100 kΩ) microelectrodes with unmodified high impedance (∼1 MΩ) microelectrodes. We begin with an evaluation of existing protocols to lower the impedance of the electrodes. The poly (3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT-PSS) electrodeposition protocol is a simple, stable, and reliable method for decreasing the impedance of a microelectrode up to 10-fold. We next record in vivo using polytrodes that are modified in a 'chess board' pattern, such that the signal of one neuron is detected by multiple coated and non-coated electrodes. The performance of the coated and non-coated electrodes is then compared on measures of background noise and amplitude of the detected action potentials. If the proper recording system is used, then the impedance of a microelectrode within the range of standard polytrodes (∼0.1 to 2 MΩ) does not greatly affect data quality and spike sorting. This study should encourage neuroscientists to stop worrying about one more unknown.

t-SNE Visualization of Large-Scale Neural Recordings.

Electrophysiology is entering the era of big data. Multiple probes, each with hundreds to thousands of individual electrodes, are now capable of simultaneously recording from many brain regions. The major challenge confronting these new technologies is transforming the raw data into physiologically meaningful signals, that is, single unit spikes. Sorting the spike events of individual neurons from a spatiotemporally dense sampling of the extracellular electric field is a problem that has attracted much attention (Rey, Pedreira, & Quian Quiroga, 2015 ; Rossant et al., 2016 ) but is still far from solved. Current methods still rely on human input and thus become unfeasible as the size of the data sets grows exponentially. Here we introduce the [Formula: see text]-student stochastic neighbor embedding (t-SNE) dimensionality reduction method (Van der Maaten & Hinton, 2008 ) as a visualization tool in the spike sorting process. t-SNE embeds the [Formula: see text]-dimensional extracellular spikes ([Formula: see text] = number of features by which each spike is decomposed) into a low- (usually two-) dimensional space. We show that such embeddings, even starting from different feature spaces, form obvious clusters of spikes that can be easily visualized and manually delineated with a high degree of precision. We propose that these clusters represent single units and test this assertion by applying our algorithm on labeled data sets from both hybrid (Rossant et al., 2016 ) and paired juxtacellular/extracellular recordings (Neto et al., 2016 ). We have released a graphical user interface (GUI) written in Python as a tool for the manual clustering of the t-SNE embedded spikes and as a tool for an informed overview and fast manual curation of results from different clustering algorithms. Furthermore, the generated visualizations offer evidence in favor of the use of probes with higher density and smaller electrodes. They also graphically demonstrate the diverse nature of the sorting problem when spikes are recorded with different methods and arise from regions with different background spiking statistics.

Validating silicon polytrodes with paired juxtacellular recordings: method and dataset.

Cross-validating new methods for recording neural activity is necessary to accurately interpret and compare the signals they measure. Here we describe a procedure for precisely aligning two probes for in vivo "paired-recordings" such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micropipette. Our new method allows for efficient, reliable, and automated guidance of both probes to the same neural structure with micrometer resolution. We also describe a new dataset of paired-recordings, which is available online. We propose that our novel targeting system, and ever expanding cross-validation dataset, will be vital to the development of new algorithms for automatically detecting/sorting single-units, characterizing new electrode materials/designs, and resolving nagging questions regarding the origin and nature of extracellular neural signals.

Development of social behavior in young zebrafish.

Adult zebrafish are robustly social animals whereas larva is not. We designed an assay to determine at what stage of development zebrafish begin to interact with and prefer other fish. One week old zebrafish do not show significant social preference whereas most 3 weeks old zebrafish strongly prefer to remain in a compartment where they can view conspecifics. However, for some individuals, the presence of conspecifics drives avoidance instead of attraction. Social preference is dependent on vision and requires viewing fish of a similar age/size. In addition, over the same 1-3 weeks period larval zebrafish increasingly tend to coordinate their movements, a simple form of social interaction. Finally, social preference and coupled interactions are differentially modified by an NMDAR antagonist and acute exposure to ethanol, both of which are known to alter social behavior in adult zebrafish.

Bonsai: an event-based framework for processing and controlling data streams.

The design of modern scientific experiments requires the control and monitoring of many different data streams. However, the serial execution of programming instructions in a computer makes it a challenge to develop software that can deal with the asynchronous, parallel nature of scientific data. Here we present Bonsai, a modular, high-performance, open-source visual programming framework for the acquisition and online processing of data streams. We describe Bonsai's core principles and architecture and demonstrate how it allows for the rapid and flexible prototyping of integrated experimental designs in neuroscience. We specifically highlight some applications that require the combination of many different hardware and software components, including video tracking of behavior, electrophysiology and closed-loop control of stimulation.

Motor cortex is required for learning but not for executing a motor skill.

Motor cortex is widely believed to underlie the acquisition and execution of motor skills, but its contributions to these processes are not fully understood. One reason is that studies on motor skills often conflate motor cortex's established role in dexterous control with roles in learning and producing task-specific motor sequences. To dissociate these aspects, we developed a motor task for rats that trains spatiotemporally precise movement patterns without requirements for dexterity. Remarkably, motor cortex lesions had no discernible effect on the acquired skills, which were expressed in their distinct pre-lesion forms on the very first day of post-lesion training. Motor cortex lesions prior to training, however, rendered rats unable to acquire the stereotyped motor sequences required for the task. These results suggest a remarkable capacity of subcortical motor circuits to execute learned skills and a previously unappreciated role for motor cortex in "tutoring" these circuits during learning.

Cortical control: learning from the lamprey.

The function of the motor cortex has been a persistent mystery. A recent study has found striking correspondence between the descending projections of lamprey pallium and mammalian motor cortex, encouraging comparative studies of the origin (and role) of forebrain motor control.

Big behavioral data: psychology, ethology and the foundations of neuroscience.

Behavior is a unifying organismal process where genes, neural function, anatomy and environment converge and interrelate. Here we review the current state and discuss the future effect of accelerating advances in technology for behavioral studies, focusing on rodents as an example. We frame our perspective in three dimensions: the degree of experimental constraint, dimensionality of data and level of description. We argue that 'big behavioral data' presents challenges proportionate to its promise and describe how these challenges might be met through opportunities afforded by the two rival conceptual legacies of twentieth century behavioral science, ethology and psychology. We conclude that, although 'more is not necessarily better', copious, quantitative and open behavioral data has the potential to transform and unify these two disciplines and to solidify the foundations of others, including neuroscience, but only if the development of new theoretical frameworks and improved experimental designs matches the technological progress.

Prey capture behavior evoked by simple visual stimuli in larval zebrafish.

Understanding how the nervous system recognizes salient stimuli in the environment and selects and executes the appropriate behavioral responses is a fundamental question in systems neuroscience. To facilitate the neuroethological study of visually guided behavior in larval zebrafish, we developed "virtual reality" assays in which precisely controlled visual cues can be presented to larvae whilst their behavior is automatically monitored using machine vision algorithms. Freely swimming larvae responded to moving stimuli in a size-dependent manner: they directed multiple low amplitude orienting turns (∼20°) toward small moving spots (1°) but reacted to larger spots (10°) with high-amplitude aversive turns (∼60°). The tracking of small spots led us to examine how larvae respond to prey during hunting routines. By analyzing movie sequences of larvae hunting paramecia, we discovered that all prey capture routines commence with eye convergence and larvae maintain their eyes in a highly converged position for the duration of the prey-tracking and capture swim phases. We adapted our virtual reality assay to deliver artificial visual cues to partially restrained larvae and found that small moving spots evoked convergent eye movements and J-turns of the tail, which are defining features of natural hunting. We propose that eye convergence represents the engagement of a predatory mode of behavior in larval fish and serves to increase the region of binocular visual space to enable stereoscopic targeting of prey.

Monitoring neural activity with bioluminescence during natural behavior.

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of approximately 20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.

Control of visually guided behavior by distinct populations of spinal projection neurons.

A basic question in the field of motor control is how different actions are represented by activity in spinal projection neurons. We used a new behavioral assay to identify visual stimuli that specifically drive basic motor patterns in zebrafish. These stimuli evoked consistent patterns of neural activity in the neurons projecting to the spinal cord, which we could map throughout the entire population using in vivo two-photon calcium imaging. We found that stimuli that drive distinct behaviors activated distinct subsets of projection neurons, consisting, in some cases, of just a few cells. This stands in contrast to the distributed activation seen for more complex behaviors. Furthermore, targeted cell by cell ablations of the neurons associated with evoked turns abolished the corresponding behavioral response. This description of the functional organization of the zebrafish motor system provides a framework for identifying the complete circuit underlying a vertebrate behavior.

Spatiotemporal specificity of neuronal activity directs the modification of receptive fields in the developing retinotectal system.

The precise temporal relation between pre- and postsynaptic activity modulates the strength of synaptic connections. Despite its extensive characterization in vivo and in vitro, the degree to which spike timing-dependent plasticity can shape receptive field properties is unclear. We use in vivo patch-clamp recordings of tectal neurons in developing Xenopus tadpoles to control the precise timing of action potentials with respect to the arrival of a subset of visual inputs evoked by local light stimulation on the retina. The pattern of visual inputs onto a tectal neuron was tracked over time by rapid reverse correlation mapping of receptive fields. Spike timing-dependent potentiation or depression of a subset of synapses reliably shifts the spatial receptive fields toward or away from the trained subregion of visual space, respectively. These results demonstrate that natural patterns of activity evoked by sensory stimuli play an instructive role in the developing nervous system.