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Establishing a causal link between neural function and behavioral output has remained a challenging problem. Commonly used perturbation techniques enable unprecedented control over intrinsic activity patterns and can effectively identify crucial circuit elements important for specific behaviors. However, these approaches may severely disrupt activity, precluding an investigation into the behavioral relevance of moment-to-moment neural dynamics within a specified brain region. Here we discuss the application of mild focal cooling to slow down intrinsic neural circuit activity while preserving its overall structure. Using network modeling and examples from multiple species, we highlight the power and versatility of focal cooling for understanding how neural dynamics control behavior and argue for its wider adoption within the systems neuroscience community., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A vast array of motor skills can be maintained throughout life. Do these behaviors require stability of individual neuron tuning or can the output of a given circuit remain constant despite fluctuations in single cells? This question is difficult to address due to the variability inherent in most motor actions studied in the laboratory. A notable exception, however, is the courtship song of the adult zebra finch, which is a learned, highly precise motor act mediated by orderly dynamics within premotor neurons of the forebrain. By longitudinally tracking the activity of excitatory projection neurons during singing using two-photon calcium imaging, we find that both the number and the precise timing of song-related spiking events remain nearly identical over the span of several weeks to months. These findings demonstrate that learned, complex behaviors can be stabilized by maintaining precise and invariant tuning at the level of single neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The zebra finch brain features a set of clearly defined and hierarchically arranged motor nuclei that are selectively responsible for producing singing behavior. One of these regions, a critical forebrain structure called HVC, contains premotor neurons that are active at precise time points during song production. However, the neural representation of this behavior at a population level remains elusive. We used two-photon microscopy to monitor ensemble activity during singing, integrating across multiple trials by adopting a Bayesian inference approach to more precisely estimate burst timing. Additionally, we examined spiking and motor-related synaptic inputs using intracellular recordings during singing. With both experimental approaches, we find that premotor events do not occur preferentially at the onsets or offsets of song syllables or at specific subsyllabic motor landmarks. These results strongly support the notion that HVC projection neurons collectively exhibit a temporal sequence during singing that is uncoupled from ongoing movements., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the zebra finch, singing behavior is driven by a sequence of bursts within premotor neurons located in the forebrain nucleus HVC (proper name). In addition to these excitatory projection neurons, HVC also contains inhibitory interneurons with a role in premotor patterning that is unclear. Here, we used a range of electrophysiological and behavioral observations to test previously described models suggesting discrete functional roles for inhibitory interneurons in song production. We show that single HVC premotor neuron bursts are sufficient to drive structured activity within the interneuron network because of pervasive and facilitating synaptic connections. We characterize interneuron activity during singing and describe reliable pauses in the firing of those neurons. We then demonstrate that these gaps in inhibition are likely to be necessary for driving normal bursting behavior in HVC premotor neurons and suggest that structured inhibition and excitation may be a general mechanism enabling sequence generation in other circuits., (Copyright © 2015 the authors 0270-6474/15/351217-11$15.00/0.)

Sensory feedback is crucial for learning and performing many behaviors, but its role in the execution of complex motor sequences is poorly understood. To address this, we consider the forebrain nucleus HVC in the songbird, which contains the premotor circuitry for song production and receives multiple convergent sensory inputs. During singing, projection neurons within HVC exhibit precisely timed synaptic events that may represent the ongoing motor program or song-related sensory feedback. To distinguish between these possibilities, we recorded the membrane potential from identified HVC projection neurons in singing zebra finches. External auditory perturbations during song production did not affect synaptic inputs in these neurons. Furthermore, the systematic removal of three sensory feedback streams (auditory, proprioceptive, and vagal) did not alter the frequency or temporal precision of synaptic activity observed. These findings support a motor origin for song-related synaptic events and suggest an updated circuit model for generating behavioral sequences., (Copyright © 2015 the authors 0270-6474/15/350299-09$15.00/0.)

Where are the 'prime movers' that control behavior? Which circuits in the brain control the order in which individual motor gestures of a learned behavior are generated, and the speed at which they progress? Here we describe two techniques recently applied to localizing and characterizing the circuitry underlying the generation of vocal sequences in the songbird. The first utilizes small, localized, temperature changes in the brain to perturb the speed of neural dynamics. The second utilizes intracellular manipulation of membrane potential in the freely behaving animal to perturb the dynamics within a single neuron. Both of these techniques are broadly applicable in behaving animals to test hypotheses about the biophysical and circuit dynamics that allow neural circuits to march from one state to the next., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In songbirds, the remarkable temporal precision of song is generated by a sparse sequence of bursts in the premotor nucleus HVC. To distinguish between two possible classes of models of neural sequence generation, we carried out intracellular recordings of HVC neurons in singing zebra finches (Taeniopygia guttata). We found that the subthreshold membrane potential is characterized by a large, rapid depolarization 5-10 ms before burst onset, consistent with a synaptically connected chain of neurons in HVC. We found no evidence for the slow membrane potential modulation predicted by models in which burst timing is controlled by subthreshold dynamics. Furthermore, bursts ride on an underlying depolarization of ∼10-ms duration, probably the result of a regenerative calcium spike within HVC neurons that could facilitate the propagation of activity through a chain network with high temporal precision. Our results provide insight into the fundamental mechanisms by which neural circuits can generate complex sequential behaviours.

We introduce a novel wireless, low-power neural stimulation system for use in freely behaving animals. The system consists of an external transmitter and a miniature, implantable wireless receiver-stimulator. The implant uses a custom integrated chip to deliver biphasic current pulses to four addressable bipolar electrodes at 32 selectable current levels (10 microA to 1 mA). To achieve maximal battery life, the chip enters a sleep mode when not needed and can be awakened remotely when required. To test our device, we implanted bipolar stimulating electrodes into the songbird motor nucleus HVC (formerly called the high vocal center) of zebra finches. Single-neuron recordings revealed that wireless stimulation of HVC led to a strong increase of spiking activity in its downstream target, the robust nucleus of the arcopallium. When we used this device to deliver biphasic pulses of current randomly during singing, singing activity was prematurely terminated in all birds tested. Thus our device is highly effective for remotely modulating a neural circuit and its corresponding behavior in an untethered, freely behaving animal.

In the suprachiasmatic nucleus (SCN), the master circadian pacemaker, neurons show circadian variations in firing frequency. There is also considerable synchrony of spiking across SCN neurons on a scale of milliseconds, but the mechanisms are poorly understood. Using paired whole-cell recordings, we have found that many neurons in the rat SCN communicate via electrical synapses. Spontaneous spiking was often synchronized in pairs of electrically coupled neurons, and the degree of this synchrony could be predicted from the magnitude of coupling. In wild-type mice, as in rats, the SCN contained electrical synapses, but electrical synapses were absent in connexin36-knockout mice. The knockout mice also showed dampened circadian activity rhythms and a delayed onset of activity during transition to constant darkness. We suggest that electrical synapses in the SCN help to synchronize its spiking activity, and that such synchrony is necessary for normal circadian behavior.

The inhibitory neurons of the thalamic reticular nucleus (TRN) contribute to the generation of widespread oscillations in the thalamocortical system. Some TRN neurons are interconnected by electrical synapses, and here we tested the possibility that electrical synapses mediate rhythmic synchrony in juvenile rats. Both the incidence and strength of electrical coupling between pairs of TRN neurons were a steep function of intersomatic distance, and coupling was absent at distances >40 microm. Presynaptic spike bursts evoked much larger electrical postsynaptic potentials than did single presynaptic spikes. Activation of metabotropic glutamate receptors (mGluRs) with a bath-applied agonist or an endogenous ligand released during tetanic stimulation induced robust rhythms of the subthreshold membrane potential, with a mean frequency of approximately 10 Hz. In the absence of fast chemical synaptic transmission, subthreshold rhythms and the action potentials that they evoked were well synchronized between closely spaced, electrically coupled pairs; rhythms in noncoupled cells were not synchronized. The results suggest that electrical synapses can coordinate spindle-frequency rhythms among small clusters of mGluR-activated TRN cells.

Many neurons in the mammalian central nervous system communicate through electrical synapses, defined here as gap junction-mediated connections. Electrical synapses are reciprocal pathways for ionic current and small organic molecules. They are often strong enough to mediate close synchronization of subthreshold and spiking activity among clusters of neurons. The most thoroughly studied electrical synapses occur between excitatory projection neurons of the inferior olivary nucleus and between inhibitory interneurons of the neocortex, hippocampus, and thalamus. All these synapses require the gap junction protein connexin36 (Cx36) for robust electrical coupling. Cx36 appears to interconnect neurons exclusively, and it is expressed widely along the mammalian neuraxis, implying that there are undiscovered electrical synapses throughout the central nervous system. Some central neurons may be electrically coupled by other connexin types or by pannexins, a newly described family of gap junction proteins. Electrical synapses are a ubiquitous yet underappreciated feature of neural circuits in the mammalian brain.

Neurons of the thalamic reticular nucleus (TRN) provide inhibitory input to thalamic relay cells and generate synchronized activity during sleep and seizures. It is widely assumed that TRN cells interact only via chemical synaptic connections. However, we show that many neighboring pairs of TRN neurons in rats and mice are electrically coupled. In paired-cell recordings, electrical synapses were able to mediate close correlations between action potentials when the coupling was strong; they could modulate burst-firing states even when the coupling strength was more modest. Electrical synapses between TRN neurons were absent in mice with a null mutation for the connexin36 (Cx36) gene. Surprisingly, inhibitory chemical synaptic connections between pairs of neurons were not observed, although strong extracellular stimuli could evoke inhibition in single TRN neurons. We conclude that Cx36-dependent gap junctions play an important role in the regulation of neural firing patterns within the TRN. When combined with recent observations from the cerebral cortex, our results imply that electrical synapses are a common mechanism for generating synchrony within networks of inhibitory neurons in the mammalian forebrain.

In this issue of Neuron , Chen et al. (2017) examine premotor activity representing motor planning, Allen et al. (2017) observe the global representation of goal-directed movement on the cortical network, and Makino et al. (2017) track changes in such dynamics throughout learning. [ABSTRACT FROM AUTHOR]

Electrophysiological recordings from behaving animals provide an unparalleled view into the functional role of individual neurons. Intracellular approaches can be especially revealing as they provide information about a neuron''s inputs and intrinsic cellular properties, which together determine its spiking output. Recent technical developments have made intracellular recording possible during an ever-increasing range of behaviors in both head-fixed and freely moving animals. These recordings have yielded fundamental insights into the cellular and circuit mechanisms underlying neural activity during natural behaviors in such areas as sensory perception, motor sequence generation, and spatial navigation, forging a direct link between cellular and systems neuroscience. [Copyright &y& Elsevier]

Synchronous activity is common in the neocortex, although its significance, mechanisms, and development are poorly understood. Previous work showed that networks of electrically coupled inhibitory interneurons called low-threshold spiking (LTS) cells can fire synchronously when stimulated by metabotropic glutamate receptors. Here we found that the coordinated inhibition emerging from an activated LTS network could induce correlated spiking patterns among neighboring excitatory cells. Synchronous activity among LTS cells was absent at postnatal day 12 (P12) but appeared abruptly over the next few days. The rapid development of the LTS-synchronizing system coincided with the maturation of the inhibitory outputs and intrinsic membrane properties of the neurons. In contrast, the incidence and magnitude of electrical synapses remained constant between P8 and P15. The developmental transformation of LTS interneurons into a synchronous, oscillatory network overlaps with the onset of active somatosensory exploration, suggesting a potential role for this synchronizing system in sensory processing. [ABSTRACT FROM AUTHOR]

The sequential activation of neurons has been observed in various areas of the brain, but in no case is the underlying network structure well understood. Here we examined the circuit anatomy of zebra finch HVC, a cortical region that generates sequences underlying the temporal progression of the song. We combined serial block-face electron microscopy with light microscopy to determine the cell types targeted by HVC(RA) neurons, which control song timing. Close to their soma, axons almost exclusively targeted inhibitory interneurons, consistent with what had been found with electrical recordings from pairs of cells. Conversely, far from the soma the targets were mostly other excitatory neurons, about half of these being other HVC(RA) cells. Both observations are consistent with the notion that the neural sequences that pace the song are generated by global synaptic chains in HVC embedded within local inhibitory networks. [ABSTRACT FROM AUTHOR]

Vocal imitation involves incorporating instructive auditory information into relevant motor circuits through processes that are poorly understood. In zebra finches, we found that exposure to a tutor's song drives spiking activity within premotor neurons in the juvenile, whereas inhibition suppresses such responses upon learning in adulthood. We measured inhibitory currents evoked by the tutor song throughout development while simultaneously quantifying each bird's learning trajectory. Surprisingly, we found that the maturation of synaptic inhibition onto premotor neurons is correlated with learning but not age. We used synthetic tutoring to demonstrate that inhibition is selective for specific song elements that have already been learned and not those still in refinement. Our results suggest that structured inhibition plays a crucial role during song acquisition, enabling a piece-by-piece mastery of complex tasks. [ABSTRACT FROM AUTHOR]