Sensory regions of the brain integrate environmental cues with copies of motor-related signals important for imminent and ongoing movements. In mammals, signals propagating from the motor cortex to the auditory cortex are thought to play a critical role in normal hearing and behavior, yet the synaptic and circuit mechanisms by which these motor-related signals influence auditory cortical activity remain poorly understood. Using in vivo intracellular recordings in behaving mice, we find that excitatory neurons in the auditory cortex are suppressed prior to and during movement, due in part to increased activity of local parvalbumin-positive (PV+) interneurons. Electrophysiology and optogenetic gain- and loss-of-function experiments reveal that motor-related changes in auditory cortical dynamics are driven by a subset of neurons in the secondary motor cortex that innervate the auditory cortex and are active during movement. These findings provide a synaptic and circuit basis for the motor-related corollary discharge hypothesized to facilitate hearing and auditory-guided behaviors.

Brain mechanisms for communication must establish a correspondence between sensory and motor codes used to represent the signal. One idea is that this correspondence is established at the level of single neurons that are active when the individual performs a particular gesture or observes a similar gesture performed by another individual. Although neurons that display a precise auditory-vocal correspondence could facilitate vocal communication, they have yet to be identified. Here we report that a certain class of neurons in the swamp sparrow forebrain displays a precise auditory-vocal correspondence. We show that these neurons respond in a temporally precise fashion to auditory presentation of certain note sequences in this songbird's repertoire and to similar note sequences in other birds' songs. These neurons display nearly identical patterns of activity when the bird sings the same sequence, and disrupting auditory feedback does not alter this singing-related activity, indicating it is motor in nature. Furthermore, these neurons innervate striatal structures important for song learning, raising the possibility that singing-related activity in these cells is compared to auditory feedback to guide vocal learning.

Songbirds learn and maintain their songs via auditory experience. Neurons in many telencephalic nuclei important to song production and development are song selective, firing more to forward auditory playback of the bird's own song (BOS) than to reverse BOS or conspecific songs. Elucidating circuits that generate these responses can localize where auditory experience influences vocalization, bridging cellular and systems analyses of song learning. Song-selective responses in many song nuclei, including the vocal premotor nucleus robustus archistriatalis (RA) and the basal ganglia homolog area X, are thought to originate in nucleus HVc (used as a proper name), which contains interneurons and relay cells that innervate either RA or area X. Previous studies indicated that only X-projecting neurons have auditory responses, leaving open the source of RA's auditory input and the degree to which song selectivity may be refined in HVc. Here, in vivo intracellular recordings from morphologically and electrophysiologically identified HVc neurons revealed that both relay cell types fire song-selectively. However, their firing arises via markedly different subthreshold processes, and only X-projecting neurons appear to be sites for auditory refinement. RA-projecting neurons exhibited purely depolarizing subthreshold responses that were highly song selective and that were excitatory. In contrast, subthreshold responses of X-projecting neurons included less-selective depolarizing and highly selective hyperpolarizing components. Within individual birds, these BOS-evoked hyperpolarizations closely matched interneuronal firing, suggesting that HVc interneurons make restricted inputs onto X-projecting neurons. Because of the two relay cell types' subthreshold differences, factors affecting their resting membrane potentials could enable them to transmit distinct song representations to their targets.

Behavioural learning depends on the brain's capacity to respond to instructive experience and is often enhanced during a juvenile sensitive period. How instructive experience acts on the juvenile brain to trigger behavioural learning remains unknown. In vitro studies show that forms of synaptic strengthening thought to underlie learning are accompanied by increased stability, number and size of dendritic spines, the major site of excitatory synaptic transmission in the vertebrate brain1-7. In vivo imaging studies in sensory cortical regions reveal that these structural features can be affected by disrupting sensory experience and that spine turnover is elevated during sensitive periods for sensory map formation8-12. These observations support two hypotheses: 1) the increased capacity for behavioural learning during a sensitive period is associated with enhanced spine dynamics on sensorimotor neurons important to the learned behaviour; 2) instructive experience rapidly stabilizes and strengthens these dynamic spines. Here we tested these hypotheses using two-photon in vivo imaging to measure spine dynamics in zebra finches, which learn to sing by imitating a tutor song during a juvenile sensitive period13,14. Spine dynamics were measured in the forebrain nucleus HVC, the proximal site where auditory information merges with an explicit song motor representation15-19, immediately before and after juvenile finches first experienced tutor song20. Higher levels of spine turnover prior to tutoring correlated with a greater capacity for subsequent song imitation. In juveniles with high levels of spine turnover, hearing a tutor song led to the rapid (~24h) stabilization, accumulation and enlargement of dendritic spines in HVC. Moreover, in vivo intracellular recordings made immediately before and after the first day of tutoring revealed robust enhancement of synaptic activity in HVC. These findings suggest behavioural learning results when instructive experience is able to rapidly stabilize and strengthen synapses on sensorimotor neurons important to the control of the learned behaviour.Investigating structural correlates of song learning requires repeated imaging of dendritic structure as a juvenile bird learns to sing. We used lentivirus-GFP constructs to fluorescently Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence and requests for materials should be addressed to R.M. (mooney@neuro.duke.edu). Supplementary Information accompanies the paper Author ContributionsT.F.R. and R.M. designed the study and wrote the manuscript. T.F.R. and K.A.T. collected and analyzed the imaging and behavioural data. T.F.R. and M.E.K. designed the lentiviral construct and M.E.K. made the lentivirus. T.F.R and R.M. collected the electrophysiological data. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAu...

Synaptic interactions between telencephalic neurons innervating descending motor or basal ganglia pathways are essential in the learning, planning, and execution of complex movements. Synaptic interactions within the songbird telencephalic nucleus HVC are implicated in motor and auditory activity associated with learned vocalizations. HVC contains projection neurons (PNs) (HVC RA ) that innervate song premotor areas, other PNs (HVC X ) that innervate a basal ganglia pathway necessary for vocal plasticity, and interneurons (HVC INT ). During singing, HVC RA fire in temporally sparse bursts, possibly because of HVC INT -HVC RA interactions, and a corollary discharge can be detected in the basal ganglia pathway, likely because of synaptic transmission from HVC RA to HVC X cells. During song playback, local interactions, including inhibition onto HVC X cells, shape highly selective responses that distinguish HVC from its auditory afferents. To better understand the synaptic substrate for the motor and auditory properties of HVC, we made intracellular recordings from pairs of HVC neurons in adult male zebra finch brain slices and used spike-triggered averages to assess synaptic connectivity. A major synaptic interaction between the PNs was a disynaptic inhibition from HVC RA to HVC X , which could link song motor signals in the two outputs of HVC and account for some of the song playback-evoked inhibition in HVC X cells. Furthermore, single interneurons made divergent connections onto PNs of both types, and either PN type could form reciprocal connections with interneurons. In these two regards, the synaptic architecture of HVC resembles that described in some pattern-generating networks, underscoring features likely to be important to singing and song learning.

Summary Cholinergic inputs to the auditory cortex from the basal forebrain (BF) are important to auditory processing and plasticity, but little is known about the organization of these synapses onto different auditory cortical neuron types, how they influence auditory responsiveness, and their activity patterns during various behaviors. Using intersectional tracing, optogenetic circuit mapping, and in vivo calcium imaging, we found that cholinergic axons arising from the caudal BF target major excitatory and inhibitory auditory cortical cell types, rapidly modulate auditory cortical tuning, and display fast movement-related activity. Furthermore, the BF and the motor cortex – another source of movement-related activity – provide convergent input onto some of the same auditory cortical neurons. Cholinergic and motor cortical afferents to the auditory cortex display distinct activity patterns and presynaptic partners, indicating that the auditory cortex integrates bottom-up cholinergic signals related to ongoing movements and arousal with top-down information concerning impending movements and motor planning.

The zebra finch forebrain song control nucleus RA (robust nucleus of the archistriatum) generates a phasic and temporally precise neural signal that drives vocal and respiratory motoneurons during singing. RA's output during singing predicts individual notes, even though afferent drive to RA from the song nucleus HVc is more tonic, and predicts song syllables, independent of the particular notes that comprise the syllable. Therefore RA's intrinsic circuitry transforms neural activity from HVc into a highly precise premotor output. To understand how RA's intrinsic circuitry effects this transformation, we characterized RA interneurons and projection neurons using intracellular recordings in brain slices. RA interneurons fired fast action potentials with steep current-frequency relationships and had small somata with thin aspinous processes that extended throughout large portions of the nucleus; the similarity of their fine processes to those labeled with a glutamic acid decarboxylase (GAD) antibody strongly suggests that these interneurons are GABAergic. Electrical stimulation revealed that RA interneurons receive excitatory inputs from RA's afferents, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and HVc, and from local axon collaterals of RA projection neurons. To map the functional connections that RA interneurons make onto RA projection neurons, we focally uncaged glutamate, revealing long-range inhibitory connections in RA. Thus these interneurons provide fast feed-forward and feedback inhibition to RA projection neurons and could help create the phasic pattern of bursts and pauses that characterizes RA output during singing. Furthermore, selectively activating the inhibitory network phase locks the firing of otherwise unconnected pairs of projection neurons, suggesting that local inhibition could coordinate RA output during singing.

SUMMARY Rodents begin to use bilaterally coordinated, rhythmic sweeping of their vibrissae (“whisking”) for environmental exploration around two weeks after birth. Whether and how vibrissal control circuitry changes after birth is unknown, and relevant premotor circuitry remains poorly characterized. Using a modified rabies virus transsynaptic tracing strategy, we labeled neurons synapsing directly onto vibrissa facial motor neurons (vFMNs). Sources of potential excitatory, inhibitory, and modulatory vFMN premotor neurons, and differences between the premotor circuitry for vFMNs innervating intrinsic versus extrinsic vibrissal muscles, were systematically characterized. The emergence of whisking is accompanied by the addition of “new” sets of bilateral excitatory inputs to vFMNs from neurons in the lateral paragigantocellularis (LPGi). Furthermore, descending axons from the motor cortex directly innervate LPGi premotor neurons. Thus, neural modules well suited to facilitate the bilateral coordination and cortical control of whisking are added to premotor circuitry in parallel with the emergence of this exploratory behavior.