Synaptic Connectivity of Sensorimotor Circuits for Vocal Imitation in the Songbird
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Curated by eLife
eLife Assessment
The songbird vocal motor nucleus HVC contains cells that project to the basal ganglia, the auditory system, or downstream vocal motor structures. In this fundamental study, the authors conduct optogenetic circuit mapping to clarify how four distinct inputs to HVC act on these distinct HVC cell types. They provide compelling evidence that all long-range projections engage inhibitory circuits in HVC and can also exhibit cell-type specific preferences in monosynaptic input strength. Understanding the HVC microcircuit at this microcircuit level is critical for informing models of song learning and production.
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Abstract
Sensorimotor computations for learning and behavior rely on precise patterns of synaptic connectivity. Yet, we typically lack the synaptic wiring diagrams for long-range connections between sensory and motor circuits in the brain. Here we provide the synaptic wiring diagram for sensorimotor circuits involved in learning and production of male zebra finch song, a natural and ethologically relevant behavior. We examined the functional synaptic connectivity from the 4 main sensory afferent pathways onto the 3 known classes of projection neurons of the song premotor cortical region HVC. Recordings from hundreds of identified projection neurons reveal rules for monosynaptic connectivity and the existence of polysynaptic ensembles of excitatory and inhibitory neuronal populations in HVC. Circuit tracing further identifies novel connections between HVC’s presynaptic partners. Our results indicate a modular organization of ensemble-like networks for integrating long-range input with local circuits, providing important context for information flow and computations for learned vocal behavior.
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eLife Assessment
The songbird vocal motor nucleus HVC contains cells that project to the basal ganglia, the auditory system, or downstream vocal motor structures. In this fundamental study, the authors conduct optogenetic circuit mapping to clarify how four distinct inputs to HVC act on these distinct HVC cell types. They provide compelling evidence that all long-range projections engage inhibitory circuits in HVC and can also exhibit cell-type specific preferences in monosynaptic input strength. Understanding the HVC microcircuit at this microcircuit level is critical for informing models of song learning and production.
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Reviewer #1 (Public review):
Summary:
This work tried to map the synaptic connectivity between the inputs and outputs of the song premotor nucleus, HVC in zebra finches to understand how sensory (auditory) to motor circuits interact to coordinate song production and learning. The authors optimized the optogenetic technique via AAV to manipulate auditory inputs from a specific auditory area one-by-one and recorded synaptic activity from a neuron with whole-cell recording from slice preparation with identification of the projection area by retrograde neuronal tracing. This thorough and detailed analysis provides compelling evidence of synaptic connections between 4 major auditory inputs (3 forebrain and 1 thalamic region) within three projection neurons in the HVC; all areas give monosynaptic excitatory inputs and polysynaptic inhibitory …
Reviewer #1 (Public review):
Summary:
This work tried to map the synaptic connectivity between the inputs and outputs of the song premotor nucleus, HVC in zebra finches to understand how sensory (auditory) to motor circuits interact to coordinate song production and learning. The authors optimized the optogenetic technique via AAV to manipulate auditory inputs from a specific auditory area one-by-one and recorded synaptic activity from a neuron with whole-cell recording from slice preparation with identification of the projection area by retrograde neuronal tracing. This thorough and detailed analysis provides compelling evidence of synaptic connections between 4 major auditory inputs (3 forebrain and 1 thalamic region) within three projection neurons in the HVC; all areas give monosynaptic excitatory inputs and polysynaptic inhibitory inputs, but proportions of projection to each projection neuron varied. They also find specific reciprocal connections between mMAN and Av. Taken together the authors provide the map of the synaptic connection between intercortical sensory to motor areas which is suggested to be involved in zebra finch song production and learning.
Strengths:
The authors optimized optogenetic tools with eGtACR1 by using AAV which allow them to manipulate synaptic inputs in a projection-specific manner in zebra finches. They also identify HVC cell types based on projection area. With their technical advance and thorough experiments, they provided detailed map synaptic connections.
Weaknesses:
As it is the study in brain slice, the functional implication of synaptic connectivity is limited. Especially as all the experiments were done in the adult preparation, there could be a gap in discussing the functions of developmental song learning.
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Reviewer #2 (Public review):
Summary:
The manuscript describes synaptic connectivity in the Songbird cortex's four main classes of sensory neuron afferents onto three known classes of projection neurons of the pre-motor cortical region HVC. HVC is a region associated with the generation of learned bird songs. Investigators here use all male zebra finches to examine the functional anatomy of this region using patch clamp methods combined with optogenetic activation of select neuronal groups.
Strengths:
The quality of the recordings is extremely high and the quantity of data is on a very significant scale, this will certainly aid the field.
Weaknesses:
The authors could make the figures a little easier to navigate. Most of the figures use actual anatomical images but it would be nice to have this linked with a zebra finch atlas in more of …
Reviewer #2 (Public review):
Summary:
The manuscript describes synaptic connectivity in the Songbird cortex's four main classes of sensory neuron afferents onto three known classes of projection neurons of the pre-motor cortical region HVC. HVC is a region associated with the generation of learned bird songs. Investigators here use all male zebra finches to examine the functional anatomy of this region using patch clamp methods combined with optogenetic activation of select neuronal groups.
Strengths:
The quality of the recordings is extremely high and the quantity of data is on a very significant scale, this will certainly aid the field.
Weaknesses:
The authors could make the figures a little easier to navigate. Most of the figures use actual anatomical images but it would be nice to have this linked with a zebra finch atlas in more of a cartoon format that accompanied each fluro image. Additionally, for the most part, figures showing the labeling lack scale bar values (in um). These should be added not just shown in the legends.
The authors could make it clear in the abstract that this is all male zebra finches - perhaps this is obvious given the bird song focus, but it should be stated. The number of recordings from each neuron class and the overall number of birds employed should be clearly stated in the methods (this is in the figures, but it should say n=birds or cells as appropriate).
The authors should consider sharing the actual electrophysiology records as data.
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Reviewer #3 (Public review):
Nucleus HVC is critical both for song production as well as learning and arguably, sitting at the top of the song control system, is the most critical node in this circuit receiving a multitude of inputs and sending precisely timed commands that determine the temporal structure of song. The complexity of this structure and its underlying organization seem to become more apparent with each experimental manipulation, and yet our understanding of the underlying circuit organization remains relatively poorly understood. In this study, Trusel and Roberts use classic whole-cell patch clamp techniques in brain slices coupled with optogenetic stimulation of select inputs to provide a careful characterization and quantification of synaptic inputs into HVC. By identifying individual projection neurons using retrograde …
Reviewer #3 (Public review):
Nucleus HVC is critical both for song production as well as learning and arguably, sitting at the top of the song control system, is the most critical node in this circuit receiving a multitude of inputs and sending precisely timed commands that determine the temporal structure of song. The complexity of this structure and its underlying organization seem to become more apparent with each experimental manipulation, and yet our understanding of the underlying circuit organization remains relatively poorly understood. In this study, Trusel and Roberts use classic whole-cell patch clamp techniques in brain slices coupled with optogenetic stimulation of select inputs to provide a careful characterization and quantification of synaptic inputs into HVC. By identifying individual projection neurons using retrograde tracer injections combined with pharmacological manipulations, they classify monosynaptic inputs onto each of the three main classes of glutamatergic projection neurons in HVC (RA-, Area X- and Av-projecting neurons). This study is remarkable in the amount of information that it generates, and the tremendous labor involved for each experiment, from the expression of opsins in each of the target inputs (Uva, NIf, mMAN, and Av), the retrograde labelling of each type of projection neuron, and ultimately the optical stimulation of infected axons while recording from identified projection neurons. Taken together, this study makes an important contribution to increasing our identification, and ultimately understanding, of the basic synaptic elements that make up the circuit organization of HVC, and how external inputs, which we know to be critical for song production and learning, contribute to the intrinsic computations within this critic circuit.
This study is impressive in its scope, rigorous in its implementation, and thoughtful regarding its limitations. The manuscript is well-written, and I appreciate the clarity with which the authors use our latest understanding of the evolutionary origins of this circuit to place these studies within a larger context and their relevance to the study of vocal control, including human speech. My comments are minor and primarily about legibility, clarification of certain manipulations, and organization of some of the summary figures.
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