Nonequivalent release sites govern synaptic depression.

Synaptic depression is prominent among synapses, but the underlying mechanisms remain uncertain. Here, we use paired patch clamp recording to study neuromuscular transmission between the caudal primary motor neuron and target skeletal muscle in zebrafish. This synapse has an unusually low number of release sites, all with high probabilities of release in response to low-frequency stimulation. During high-frequency stimulation, the synapse undergoes short-term depression and reaches steady-state levels of transmission that sustain the swimming behavior. To determine the release parameters underlying this steady state, we applied variance analysis. Our analysis revealed two functionally distinct subclasses of release sites differing by over 60-fold in rates of vesicle reloading. A slow reloading class requires seconds to recover and contributes to depression onset but not the steady-state transmission. By contrast, a fast reloading class recovers within tens of milliseconds and is solely responsible for steady-state transmission. Thus, in contrast to most current models that assign levels of steady-state depression to vesicle availability, our findings instead assign this function to nonuniform release site kinetics. The duality of active-site properties accounts for the highly nonlinear dependence of steady-state depression levels on frequency.

Reduced high-frequency motor neuron firing, EMG fractionation, and gait variability in awake walking ALS mice.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease prominently featuring motor neuron (MN) loss and paralysis. A recent study using whole-cell patch clamp recording of MNs in acute spinal cord slices from symptomatic adult ALS mice showed that the fastest firing MNs are preferentially lost. To measure the in vivo effects of such loss, awake symptomatic-stage ALS mice performing self-initiated walking on a wheel were studied. Both single-unit extracellular recordings within spinal cord MN pools for lower leg flexor and extensor muscles and the electromyograms (EMGs) of the corresponding muscles were recorded. In the ALS mice, we observed absent or truncated high-frequency firing of MNs at the appropriate time in the step cycle and step-to-step variability of the EMG, as well as flexor-extensor coactivation. In turn, kinematic analysis of walking showed step-to-step variability of gait. At the MN level, the higher frequencies absent from recordings from mutant mice corresponded with the upper range of frequencies observed for fast-firing MNs in earlier slice measurements. These results suggest that, in SOD1-linked ALS mice, symptoms are a product of abnormal MN firing due at least in part to loss of neurons that fire at high frequency, associated with altered EMG patterns and hindlimb kinematics during gait.

Sensation during Active Behaviors.

A substantial portion of our sensory experience happens during active behaviors such as walking around or paying attention. How do sensory systems work during such behaviors? Neural processing in sensory systems can be shaped by behavior in multiple ways ranging from a modulation of responsiveness or sharpening of tuning to a dynamic change of response properties or functional connectivity. Here, we review recent findings on the modulation of sensory processing during active behaviors in different systems: insect vision, rodent thalamus, and rodent sensory cortices. We discuss the circuit-level mechanisms that might lead to these modulations and their potential role in sensory function. Finally, we highlight the open questions and future perspectives of this exciting new field.

Hierarchical excitatory synaptic connectivity in mouse olfactory cortex.

Topological motifs in synaptic connectivity-such as the cortical column-are fundamental to processing of information in cortical structures. However, the mesoscale topology of cortical networks beyond columns remains largely unknown. In the olfactory cortex, which lacks an obvious columnar structure, sensory-evoked patterns of activity have failed to reveal organizational principles of the network and its structure has been considered to be random. We probed the excitatory network in the mouse olfactory cortex using variance analysis of paired whole-cell recording in olfactory cortex slices. On a given trial, triggered network-wide bursts in disinhibited slices had remarkably similar time courses in widely separated and randomly selected cell pairs of pyramidal neurons despite significant trial-to-trial variability within each neuron. Simulated excitatory network models with random topologies only partially reproduced the experimental burst-variance patterns. Network models with local (columnar) or distributed subnetworks, which have been predicted as the basis of encoding odor objects, were also inconsistent with the experimental data, showing greater variability between cells than across trials. Rather, network models with power-law and especially hierarchical connectivity showed the best fit. Our results suggest that distributed subnetworks are weak or absent in the olfactory cortex, whereas a hierarchical excitatory topology may predominate. A hierarchical excitatory network organization likely underlies burst generation in this epileptogenic region, and may also shape processing of sensory information in the olfactory cortex.

Triheteromeric NMDA receptors at hippocampal synapses.

NMDA receptors are composed of two GluN1 (N1) and two GluN2 (N2) subunits. Constituent N2 subunits control the pharmacological and kinetic characteristics of the receptor. NMDA receptors in hippocampal or cortical neurons are often thought of as diheteromeric, meaning that they contain only one type of N2 subunit. However, triheteromeric receptors with more than one type of N2 subunit also have been reported, and the relative contribution of diheteromeric and triheteromeric NMDA receptors at synapses has been difficult to assess. Because wild-type hippocampal principal neurons express N1, N2A, and N2B, we used cultured hippocampal principal neurons from N2A and N2B knock-out mice as templates for diheteromeric synaptic receptors. However, summation of N1/N2B and N1/N2A EPSCs could not account for the deactivation kinetics of wild-type EPSCs. To make a quantitative estimate of NMDA receptor subtypes at wild-type synapses, we used the deactivation kinetics and the effects of the competitive antagonist NVP-AAM077. Our results indicate that three types of NMDA receptors contribute to wild-type EPSCs, with at least two-thirds being triheteromeric receptors. Functional isolation of synaptic triheteromeric receptors revealed deactivation kinetics and pharmacology that were distinct from either diheteromeric receptor subtype. Because of differences in open probability, synaptic triheteromeric receptors outnumbered N1/N2A receptors by 5.8 to 1 and N1/N2B receptors by 3.2 to 1. Our results suggest that triheteromeric NMDA receptors must either be preferentially assembled or preferentially localized at synapses.

Strategic stabilization of arousal boosts sustained attention.

Arousal and motivation interact to profoundly influence behavior. For example, experience tells us that we have some capacity to control our arousal when appropriately motivated, such as staying awake while driving a motor vehicle. However, little is known about how arousal and motivation jointly influence decision computations, including if and how animals, such as rodents, adapt their arousal state to their needs. Here, we developed and show results from an auditory, feature-based, sustained-attention task with intermittently shifting task utility. We use pupil size to estimate arousal across a wide range of states and apply tailored signal-detection theoretic, hazard function, and accumulation-to-bound modeling approaches in a large cohort of mice. We find that pupil-linked arousal and task utility both have major impacts on multiple aspects of task performance. Although substantial arousal fluctuations persist across utility conditions, mice partially stabilize their arousal near an intermediate and optimal level when task utility is high. Behavioral analyses show that multiple elements of behavior improve during high task utility and that arousal influences some, but not all, of them. Specifically, arousal influences the likelihood and timescale of sensory evidence accumulation but not the quantity of evidence accumulated per time step while attending. In sum, the results establish specific decision-computational signatures of arousal, motivation, and their interaction in attention. So doing, we provide an experimental and analysis framework for studying arousal self-regulation in neurotypical brains and in diseases such as attention-deficit/hyperactivity disorder.

Cortical acetylcholine dynamics are predicted by cholinergic axon activity and behavior state.

Even under spontaneous conditions and in the absence of changing environmental demands, awake animals alternate between increased or decreased periods of alertness. These changes in brain state can occur rapidly, on a timescale of seconds, and neuromodulators such as acetylcholine (ACh) are thought to play an important role in driving these spontaneous state transitions. Here, we perform the first simultaneous imaging of ACh sensors and GCaMP-expressing axons in vivo, to examine the spatiotemporal properties of cortical ACh activity and release during spontaneous changes in behavioral state. We observed a high correlation between simultaneously recorded basal forebrain axon activity and neuromodulator sensor fluorescence around periods of locomotion and pupil dilation. Consistent with volume transmission of ACh, increases in axon activity were accompanied by increases in local ACh levels that fell off with the distance from the nearest axon. GRAB-ACh fluorescence could be accurately predicted from axonal activity alone, providing the first validation that neuromodulator axon activity is a reliable proxy for nearby neuromodulator levels. Deconvolution of fluorescence traces allowed us to account for the kinetics of the GRAB-ACh sensor and emphasized the rapid clearance of ACh for smaller transients outside of running periods. Finally, we trained a predictive model of ACh fluctuations from the combination of pupil size and running speed; this model performed better than using either variable alone, and generalized well to unseen data. Overall, these results contribute to a growing understanding of the precise timing and spatial characteristics of cortical ACh during fast brain state transitions.

Generating synchrony from the asynchronous: compensation for cochlear traveling wave delays by the dendrites of individual brainstem neurons.

Broadband transient sounds, such as clicks and consonants, activate a traveling wave in the cochlea. This wave evokes firing in auditory nerve fibers that are tuned to high frequencies several milliseconds earlier than in fibers tuned to low frequencies. Despite this substantial traveling wave delay, octopus cells in the brainstem receive broadband input and respond to clicks with submillisecond temporal precision. The dendrites of octopus cells lie perpendicular to the tonotopically organized array of auditory nerve fibers, placing the earliest arriving inputs most distally and the latest arriving closest to the soma. Here, we test the hypothesis that the topographic arrangement of synaptic inputs on dendrites of octopus cells allows octopus cells to compensate the traveling wave delay. We show that in mice the full cochlear traveling wave delay is 1.6 ms. Because the dendrites of each octopus cell spread across approximately one-third of the tonotopic axis, a click evokes a soma-directed sweep of synaptic input lasting 0.5 ms in individual octopus cells. Morphologically and biophysically realistic, computational models of octopus cells show that soma-directed sweeps with durations matching in vivo measurements result in the largest and sharpest somatic EPSPs. A low input resistance and activation of a low-voltage-activated potassium conductance that are characteristic of octopus cells are important determinants of sweep sensitivity. We conclude that octopus cells have dendritic morphologies and biophysics tailored to accomplish the precise encoding of broadband transient sounds.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction.

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Comprehensive analysis of cellular specializations that initiate parallel auditory processing pathways in mice.

The cochlear nuclear complex (CN) is the starting point for all central auditory processing and comprises a suite of neuronal cell types that are highly specialized for neural coding of acoustic signals. To examine how their striking functional specializations are determined at the molecular level, we performed single-nucleus RNA sequencing of the mouse CN to molecularly define all constituent cell types and related them to morphologically- and electrophysiologically-defined neurons using Patch-seq. We reveal an expanded set of molecular cell types encompassing all previously described major types and discover new subtypes both in terms of topographic and cell-physiologic properties. Our results define a complete cell-type taxonomy in CN that reconciles anatomical position, morphological, physiological, and molecular criteria. This high-resolution account of cellular heterogeneity and specializations from the molecular to the circuit level illustrates molecular underpinnings of functional specializations and enables genetic dissection of auditory processing and hearing disorders with unprecedented specificity.

Experience-dependent maturation of the glomerular microcircuit.

Spontaneous and patterned activity, largely attributed to chemical transmission, shape the development of virtually all neural circuits. However, electrical transmission also has an important role in coordinated activity in the brain. In the olfactory bulb, gap junctions between apical dendrites of mitral cells increase excitability and synchronize firing within each glomerulus. We report here that the development of the glomerular microcircuit requires both sensory experience and connexin (Cx)36-mediated gap junctions. Coupling coefficients, which measure electrical coupling between mitral cell dendrites, were high in young mice, but decreased after postnatal day (P)10 because of a maturational increase in membrane conductance. Sensory deprivation, induced by unilateral naris occlusion at birth, slowed the morphological development of mitral cells and arrested the maturational changes in membrane conductance and coupling coefficients. As the coupling coefficients decreased in normal mice, a glutamate-mediated excitatory postsynaptic current (EPSC) between mitral cells emerged by P30. Although mitral-mitral EPSCs were generally unidirectional, they were not present in young adult Cx36(-/-) mice, suggesting that gap junctions are required for the development and/or function of the mature circuit. The experience-dependent transition from electrical transmission to combined chemical and electrical transmission provides a previously unappreciated mechanism that may tune the response properties of the glomerular microcircuit.

Why study mechanisms of brain stimulation therapies? To modulate the right neurons, in the right way, at the right time.

Clinical applications of vagus nerve stimulation (VNS) are burgeoning, but mechanistic work lags behind. In this issue of Neuron, Bowles and colleagues show that VNS timed with positive reinforcement improves motor learning and cortical function by a cholinergic mechanism.

Loss of olfactory cell adhesion molecule reduces the synchrony of mitral cell activity in olfactory glomeruli.

Odours generate activity in olfactory receptor neurons, whose axons contact the dendritic tufts of mitral cells within olfactory bulb glomeruli. These axodendritic synapses are anatomically separated from dendrodendritic synapses within each glomerulus. Mitral cells within a glomerulus show highly synchronized activity as assessed with whole-cell recording from pairs of mitral cells. We examined glomerular activity in mice lacking the olfactory cell adhesion molecule (OCAM). Glomeruli in mice lacking OCAM show a redistribution of synaptic subcompartments, but the total area occupied by axonal inputs was similar to wild-type mice. Stimulation of olfactory nerve bundles showed that excitatory synaptic input to mitral cells as well as dendrodendritic inhibition was unaffected in the knockout. However, correlated spiking in mitral cells was significantly reduced, as was electrical coupling between apical dendrites. To analyse slow network dynamics we induced slow oscillations with a glutamate uptake blocker. Evoked and spontaneous slow oscillations in mitral cells and external tufted cells were broader and had multiple peaks in OCAM knockout mice, indicating that synchrony of slow glomerular activity was also reduced. To assess the degree of shared activity between mitral cells under physiological conditions, we analysed spontaneous sub-threshold voltage oscillations using coherence analysis. Coherent activity was markedly reduced in cells from OCAM knockout mice across a broad range of frequencies consistent with a decrease in tightly time-locked activity. We suggest that synchronous activity within each glomerulus is dependent on segregation of synaptic subcompartments.

Membrane and synaptic properties of pyramidal neurons in the anterior olfactory nucleus.

The anterior olfactory nucleus (AON) is positioned to coordinate activity between the piriform cortex and olfactory bulbs, yet the physiology of AON principal neurons has been little explored. Here, we examined the membrane properties and excitatory synapses of AON principal neurons in brain slices of PND22-28 mice and compared their properties to principal cells in other olfactory cortical areas. AON principal neurons had firing rates, spike rate adaptation, spike widths, and I-V relationships that were generally similar to pyramidal neurons in piriform cortex, and typical of cerebral cortex, consistent with a role for AON in cortical processing. Principal neurons in AON had more hyperpolarized action potential thresholds, smaller afterhyperpolarizations, and tended to fire doublets of action potentials on depolarization compared with ventral anterior piriform cortex and the adjacent epileptogenic region preendopiriform nucleus (pEN). Thus, AON pyramidal neurons have enhanced membrane excitability compared with surrounding subregions. Interestingly, principal neurons in pEN were the least excitable, as measured by a larger input conductance, lower firing rates, and more inward rectification. Afferent and recurrent excitatory synapses onto AON pyramidal neurons had small amplitudes, paired pulse facilitation at afferent synapses, and GABA(B) modulation at recurrent synapses, a pattern similar to piriform cortex. The enhanced membrane excitability and recurrent synaptic excitation within the AON, together with its widespread outputs, suggest that the AON can boost and distribute activity in feedforward and feedback circuits throughout the olfactory system.

Sensory Neurobiology: A New Crossroads Deep in the Cortex.

Self-generated movements elicit concomitant sensory responses that are beneficial to ignore. Combining state-of-the-art physiology, behavior, and anatomy, a new study discovers a neural crossroads in the deepest layer of auditory cortex where pre-lick ramping activity suppresses peri-lick sound responses across layers.

Brain States: Sensory Modulations All the Way Down.

From wakefulness to sleep, and from moment to moment, the arousal state of the brain is a powerful internal context that shapes our perception and actions. Using cutting-edge imaging methods, two new studies show that arousal already sculpts visual information as it first enters the brain.

Pupil-linked phasic arousal predicts a reduction of choice bias across species and decision domains.

Decisions are often made by accumulating ambiguous evidence over time. The brain's arousal systems are activated during such decisions. In previous work in humans, we found that evoked responses of arousal systems during decisions are reported by rapid dilations of the pupil and track a suppression of biases in the accumulation of decision-relevant evidence (de Gee et al., 2017). Here, we show that this arousal-related suppression in decision bias acts on both conservative and liberal biases, and generalizes from humans to mice, and from perceptual to memory-based decisions. In challenging sound-detection tasks, the impact of spontaneous or experimentally induced choice biases was reduced under high phasic arousal. Similar bias suppression occurred when evidence was drawn from memory. All of these behavioral effects were explained by reduced evidence accumulation biases. Our results point to a general principle of interplay between phasic arousal and decision-making.

Rate thresholds determine the precision of temporal integration in principal cells of the ventral cochlear nucleus.

The three types of principal cells of the ventral cochlear nucleus (VCN), bushy, octopus, and T stellate, differ in the detection of coincidence among synaptic inputs. To explore the role of the action-potential-generation mechanism in the detection of coincident inputs, we examined responses to depolarizing currents that increased at varying rates. To fire an action potential, bushy cells, likely of the globular subtype, had to be depolarized faster than 4.8+/-2.8 mV/ms, octopus cells faster than 9.5+/-3.6 mV/ms, and T stellate cells fired irrespective of the rate of depolarization. The threshold rate of depolarization permitted definition of a time window over which depolarization could contribute to generating action potentials. This integration window differed between cell types. It was 5.3+/-1.8 ms for bushy cells and 1.4+/-0.3 ms for octopus cells. T Stellate cells fired action potentials in response to even slow depolarizations, showing that their integration window was unlimited so that temporal summation in these cells is limited by the time course of synaptic potentials. The rate of depolarization threshold in octopus and bushy cells was decreased by alpha-dendrotoxin while T stellate cells were largely insensitive to alpha-dendrotoxin indicating that low-voltage-activated K+ conductances (gKL) are important determinants of the integration window.

Knockout of Foxp2 disrupts vocal development in mice.

The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control.

Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex.

Rapid variations in cortical state during wakefulness have a strong influence on neural and behavioural responses and are tightly coupled to changes in pupil size across species. However, the physiological processes linking cortical state and pupil variations are largely unknown. Here we demonstrate that these rapid variations, during both quiet waking and locomotion, are highly correlated with fluctuations in the activity of corticopetal noradrenergic and cholinergic projections. Rapid dilations of the pupil are tightly associated with phasic activity in noradrenergic axons, whereas longer-lasting dilations of the pupil, such as during locomotion, are accompanied by sustained activity in cholinergic axons. Thus, the pupil can be used to sensitively track the activity in multiple neuromodulatory transmitter systems as they control the state of the waking brain.